Most recent papers:

• Leptin acts in the carotid bodies to increase minute ventilation during wakefulness and sleep and augment the hypoxic ventilatory response.
  Candela Caballero‐Eraso, Mi‐Kyung Shin, Huy Pho, Lenise J Kim, Luis E. Pichard, Zhi‐Juan Wu, Chenjuan Gu, Slava Berger, Luu Pham, Ho‐Yee (Bonnie) Yeung, Machiko Shirahata, Alan R. Schwartz, Wan‐Yee (Winnie) Tang, James S. K. Sham, Vsevolod Y. Polotsky.
  The Journal of Physiology. November 29, 2018
  --- - |2+ Key points Leptin is a potent respiratory stimulant. A long functional isoform of leptin receptor, LepRb, was detected in the carotid body (CB), a key peripheral hypoxia sensor. However, the effect of leptin on minute ventilation (VE) and the hypoxic ventilatory response (HVR) has not been sufficiently studied. We report that LepRb is present in approximately 74% of the CB glomus cells. Leptin increased carotid sinus nerve activity at baseline and in response to hypoxia in vivo. Subcutaneous infusion of leptin increased VE and HVR in C57BL/6J mice and this effect was abolished by CB denervation. Expression of LepRb in the carotid bodies of LepRb deficient obese db/db mice increased VE during wakefulness and sleep and augmented the HVR. We conclude that leptin acts on LepRb in the CBs to stimulate breathing and HVR, which may protect against sleep disordered breathing in obesity. Abstract Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb, but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb‐deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and on HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 mL/min/g during normoxia (P < 0.01) and from 3.6 to 4.7 mL/min/g during hypoxia (P < 0.001), augmenting HVR from 0.23 to 0.31 mL/min/g/Δ (P < 0.001). The effects of leptin on VE and HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 mL/min/g during normoxia (P < 0.05) and from 2.8 to 3.2 mL/min/g during hypoxia (P < 0.02), increasing HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout non‐rapid eye movement (20.1 vs. −27.7 mL/min respectively, P < 0.05) and rapid eye movement sleep (16.5 vs 23.4 mL/min, P < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity. - 'The Journal of Physiology, EarlyView. '

  November 29, 2018   doi: 10.1113/JP276900   open full text
• ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain.
  M. Bulla, G. Gyimesi, J.H. Kim, R. Bhardwaj, M.A. Hediger, M. Frieden, N. Demaurex.
  The Journal of Physiology. November 28, 2018
  --- - |2+ Key points Gain‐of‐function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM. Abstract Skeletal muscle differentiation relies on store‐operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+‐permeable ORAI channels. Gain‐of‐function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK‐293T cells or human primary myoblasts, the mutated channels increased basal and store‐operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK‐7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1‐V107M constitutive activity and revealed larger STIM1‐gated V107M‐ and T184M‐mediated currents with conserved fast and slow Ca2+‐dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1−/−/Stim2−/− mouse embryonic fibroblasts expressing the binding‐deficient STIM1‐F394H mutant, indicating a higher sensitivity for STIM1‐mediated gating, with ORAI1‐T184M gain‐of‐function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain‐of‐function mutations in ORAI1. - 'The Journal of Physiology, EarlyView. '

  November 28, 2018   doi: 10.1113/JP277079   open full text
• Anticipation of food intake induces phosphorylation switch to regulate basolateral amino acid transporter LAT4 (SLC43A2) function.
  Lalita Oparija, Anuradha Rajendran, Nadège Poncet, François Verrey.
  The Journal of Physiology. November 28, 2018
  --- - |2+ Key points Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non‐essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site‐specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. Abstract The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site‐specific antibodies and a time‐restricted diet. In Xenopus oocytes, mimicking non‐phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild‐type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food‐entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time. - 'The Journal of Physiology, EarlyView. '

  November 28, 2018   doi: 10.1113/JP276714   open full text
• Inhibition of non‐receptor tyrosine kinase Src induces phosphoserine 256‐independent aquaporin‐2 membrane accumulation.
  Pui W. Cheung, Abby Terlouw, Sam Antoon Janssen, Dennis Brown, Richard Bouley.
  The Journal of Physiology. November 28, 2018
  --- - |2+ Key Points Aquaporin‐2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA, and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 c‐terminus. It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues. We found that Src inhibition causes serine 256 independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independent of serine 256. This targeted phosphorylation of serine 269 is important for Src inhibition induce AQP2 membrane accumulation. Without serine 269, Src inhibition exerts no effect on AQP2 trafficking. This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signaling is defective. Abstract Aquaporin‐2 (AQP2) is essential for water homeostasis. Upon stimulation by vasopressin, AQP2 is phosphorylated at serine 256 (S256), S264 and S269, and dephosphorylated at S261. It is thought that S256 is the master regulator of AQP2 trafficking and membrane accumulation, and that its phosphorylation has to precede phosphorylation of other serine residues. In this study, we found that VP reduces Src kinase phosphorylation: by suppressing Src using the inhibitor dasatinib and siRNA, we could increase AQP2 membrane accumulation in cultured AQP2‐expressing cells and in kidney collecting duct principal cells. Src inhibition increased exocytosis and inhibited clathrin‐mediated endocytosis of AQP2, but exerted its effect in a cAMP, PKA and S256 phosphorylation (pS256) independent manner. Despite the lack of S256 phosphorylation, dasatinib increased phosphorylation of S269, even in S256A mutant cells in which S256 phosphorylation cannot occur. To confirm the importance of pS269 in AQP2 re‐distribution, we expressed an AQP2 S269A mutant in LLC‐PK1 cells, and found that dasatinib no longer induced AQP2 membrane accumulation. In conclusion, Src inhibition causes phosphorylation of S269 independently of pS256, and induces AQP2 membrane accumulation by inhibiting clathrin‐mediated endocytosis and increasing exocytosis. We conclude that S269 can be phosphorylated without pS256, and pS269 alone is important for AQP2 apical membrane accumulation under some conditions. These data increase our understanding of the independent pathways that can phosphorylate different residues in the AQP2 c‐terminus, and suggest new strategies to target distinct AQP2 serine residues to induce membrane expression of this water channel when VP signaling is defective. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 28, 2018   doi: 10.1113/JP277024   open full text
• Increased endothelial shear stress improves insulin‐stimulated vasodilatation in skeletal muscle.
  Lauren K. Walsh, Thaysa Ghiarone, T. Dylan Olver, Areli Medina‐Hernandez, Jenna C. Edwards, Pamela K. Thorne, Craig A. Emter, Jonathan R. Lindner, Camila Manrique‐Acevedo, Luis A. Martinez‐Lemus, Jaume Padilla.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points It has been postulated that increased blood flow‐associated shear stress on endothelial cells is an underlying mechanism by which physical activity enhances insulin‐stimulated vasodilatation. This report provides evidence supporting the hypothesis that increased shear stress exerts insulin‐sensitizing effects in the vasculature and this evidence is based on experiments in vitro in endothelial cells, ex vivo in isolated arterioles and in vivo in humans. Given the recognition that vascular insulin signalling, and associated enhanced microvascular perfusion, contributes to glycaemic control and maintenance of vascular health, strategies that stimulate an increase in limb blood flow and shear stress have the potential to have profound metabolic and vascular benefits mediated by improvements in endothelial insulin sensitivity. Abstract The vasodilator actions of insulin contribute to glucose uptake by skeletal muscle, and previous studies have demonstrated that acute and chronic physical activity improves insulin‐stimulated vasodilatation and glucose uptake. Because this effect of exercise primarily manifests in vascular beds highly perfused during exercise, it has been postulated that increased blood flow‐associated shear stress on endothelial cells is an underlying mechanism by which physical activity enhances insulin‐stimulated vasodilatation. Accordingly, herein we tested the hypothesis that increased shear stress, in the absence of muscle contraction, can acutely render the vascular endothelium more insulin‐responsive. To test this hypothesis, complementary experiments were conducted using (1) cultured endothelial cells, (2) isolated and pressurized skeletal muscle arterioles from swine, and (3) humans. In cultured endothelial cells, 1 h of increased shear stress from 3 to 20 dynes cm−2 caused a significant shift in insulin signalling characterized by greater activation of eNOS relative to MAPK. Similarly, isolated arterioles exposed to 1 h of intraluminal shear stress (20 dynes cm−2) subsequently exhibited greater insulin‐induced vasodilatation compared to arterioles kept under no‐flow conditions. Finally, we found in humans that increased leg blood flow induced by unilateral limb heating for 1 h subsequently augmented insulin‐stimulated popliteal artery blood flow and muscle perfusion. In aggregate, these findings across models (cells, isolated arterioles and humans) support the hypothesis that elevated shear stress causes the vascular endothelium to become more insulin‐responsive and thus are consistent with the notion that shear stress may be a principal mechanism by which physical activity enhances insulin‐stimulated vasodilatation. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP277050   open full text
• Sleep disordered breathing in children disrupts the maturation of autonomic control of heart rate and its association with cerebral oxygenation.
  Lisa M Walter, Knarik Tamanyan, Aidan J Weichard, Margot J Davey, Gillian M Nixon, Rosemary SC Horne.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key Point Summary Sleep disordered breathing (SDB) affects 4 ‐ 11% of children and is associated with adverse neurocognitive, behavioural and cardiovascular outcomes, including reduced autonomic control. The relationship between heart rate variability (HRV; a measure of autonomic control), and age found in non‐snoring control children was absent during sleep in children with SDB. Age significantly predicted increasing cerebral oxygenation during wake in non‐snoring control children, whereas during sleep, HRV significantly predicted decreasing cerebral oxygenation. Cerebral oxygenation was not associated with either age or HRV in children with SDB during both wake and sleep. SDB significantly disrupts the normal maturation of autonomic control and the positive association between autonomic control and cerebral oxygenation found in non‐snoring children, and we speculate that the dampened autonomic control exhibited by children with SDB may have an attenuating effect on cerebral autoregulation via the moderating influence of HRV on cerebral blood flow. Abstract Background The repetitive episodes of hypoxia that are features of sleep disordered breathing (SDB) in children are associated with alterations in autonomic control of heart rate in an age dependent manner. We aimed to relate heart rate variability (HRV) parameters to age and measures of cerebral oxygenation in children (3‐12 y) with SDB and non‐snoring controls. Methods Children (SDB, n = 117; controls, n = 42; 3–12 y) underwent overnight polysomnography. Total (TP), low (LF) and high frequency (HF) power, tissue oxygenation index (TOI) and fractional tissue oxygen extraction (FTOE) were analysed during wake and sleep. Pearson's correlations determined the association between age and HRV parameters, and multiple linear regressions between HRV, age and cerebral oxygenation parameters. Results During wake, age had a positive association with LF power reflecting increased parasympathetic and sympathetic activity with increasing age for both control and SDB groups. This association was also evident during sleep in controls, but was absent in children with SDB. In controls, during wake TOI had a positive, and FTOE a negative association with age. During sleep, TP, LF and HF power were significant, negative determinants of TOI and positive determinants of FTOE. These associations were not seen in children with SDB during wake or sleep. Conclusion SDB disrupts the normal maturation of the autonomic control of heart rate and the association between HRV and cerebral oxygenation exhibited by non‐snoring control children of primary school age. These results highlight the impact SDB has on cardiovascular control and the potential impact on adverse cardiovascular outcomes. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 24, 2018   doi: 10.1113/JP276933   open full text
• The Na+/H+ exchanger NHE1 localizes as clusters to cryptic lamellipodia and accelerates collective epithelial cell migration.
  Helene H. Jensen, Gitte A. Pedersen, Jeanette J. Morgen, Maddy Parsons, Stine F. Pedersen, Lene N. Nejsum.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points summary Exogenous NHE1 expression stimulated collective migration of epithelial cell sheets Stimulation with epidermal growth factor (EGF), a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows NHE1 expression disrupted the morphology of epithelial cell sheets and 3D cysts Abstract Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na+/H+ Exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, yet its possible role in collective migration of normal epithelial cells has remained unresolved. Here, we show that NHE1 expression in MDCK‐II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor (EGF), a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and EGF. Finally, NHE1 expression resulted in disorganized development of MDCK‐II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 24, 2018   doi: 10.1113/JP277383   open full text
• Ih contributes to increased motoneuron excitability in restless legs syndrome.
  Dirk Czesnik, James Howells, Michael Bartl, Elisabeth Veiz, Rebecca Ketzler, Olga Kemmet, Arthur S. Walters, Claudia Trenkwalder, David Burke, Walter Paulus.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points Restless legs patients complain about sensory and motor symptoms leading to sleep disturbances. Symptoms include painful sensations, an urge to move and involuntary leg movements. The responsible mechanisms of restless legs syndrome are still not known, although current studies indicate an increased neuronal network excitability. Reflex studies indicate the involvement of spinal structures. Peripheral mechanisms have not been investigated so far. In the present study, we provide evidence of increased hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel‐mediated inward rectification in motor axons. The excitability of sensory axons was not changed. We conclude that, in restless legs syndrome, an increased HCN current in motoneurons may play a pathophysiological role, such that these channels could represent a valuable target for pharmaceutical intervention. Abstract Restless legs syndrome is a sensorimotor network disorder. So far, the responsible pathophysiological mechanisms are poorly understood. In the present study, we provide evidence that the excitability of peripheral motoneurons contributes to the pathophysiology of restless legs syndrome. In vivo excitability studies on motor and sensory axons of the median nerve were performed on patients with idiopathic restless legs syndrome (iRLS) who were not currently on treatment. The iRLS patients had greater accommodation in motor but not sensory axons to long‐lasting hyperpolarization compared to age‐matched healthy subjects, indicating greater inward rectification in iRLS. The most reasonable explanation is that hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels open at less hyperpolarized membrane potentials, a view supported by mathematical modelling. The half‐activation potential for HCN channels (Bq) was the single best parameter that accounted for the difference between normal controls and iRLS data. A 6 mV depolarization of Bq reduced the discrepancy between the normal control model and the iRLS data by 92.1%. Taken together, our results suggest an increase in the excitability of motor units in iRLS that could enhance the likelihood of leg movements. The abnormal axonal properties are consistent with other findings indicating that the peripheral system is part of the network involved in iRLS. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP275341   open full text
• Characterization and regulation of wild‐type and mutant TASK‐1 two pore domain potassium channels indicated in pulmonary arterial hypertension.
  Kevin P. Cunningham, Robyn G. Holden, Pilar M. Escribano‐Subias, Angel Cogolludo, Emma L. Veale, Alistair Mathie.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points The TASK‐1 channel gene (KCNK3) has been identified as a possible disease‐causing gene in heritable pulmonary arterial hypertension (PAH). In the present study, we show that novel mutated TASK‐1 channels, seen in PAH patients, have a substantially reduced current compared to wild‐type TASK‐1 channels. These mutated TASK‐1 channels are located at the plasma membrane to the same degree as wild‐type TASK‐1 channels. ONO‐RS‐082 and alkaline pH 8.4 both activate TASK‐1 channels but do not recover current through mutant TASK‐1 channels. We show that the guanylate cyclase activator, riociguat, a novel treatment for PAH, enhances current through TASK‐1 channels but does not recover current through mutant TASK‐1 channels. Abstract Pulmonary arterial hypertension (PAH) affects ∼15–50 people per million. KCNK3, the gene that encodes the two pore domain potassium channel TASK‐1 (K2P3.1), has been identified as a possible disease‐causing gene in heritable PAH. Recently, two new mutations have been identified in KCNK3 in PAH patients: G106R and L214R. The present study aimed to characterize the functional properties and regulation of wild‐type (WT) and mutated TASK‐1 channels and determine how these might contribute to PAH and its treatment. Currents through WT and mutated human TASK‐1 channels transiently expressed in tsA201 cells were measured using whole‐cell patch clamp electrophysiology. Localization of fluorescence‐tagged channels was visualized using confocal microscopy and quantified with in‐cell and on‐cell westerns. G106R or L214R mutated channels were located at the plasma membrane to the same degree as WT channels; however, their current was markedly reduced compared to WT TASK‐1 channels. Functional current through these mutated channels could not be restored using activators of WT TASK‐1 channels (pH 8.4, ONO‐RS‐082). The guanylate cyclase activator, riociguat, enhanced current through WT TASK‐1 channels; however, similar to the other activators investigated, riociguat did not have any effect on current through mutated TASK‐1 channels. Thus, novel mutations in TASK‐1 seen in PAH substantially alter the functional properties of these channels. Current through these channels could not be restored by activators of TASK‐1 channels. Riociguat enhancement of current through TASK‐1 channels could contribute to its therapeutic benefit in the treatment of PAH. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP277275   open full text
• Exaggerated systemic oxidative‐inflammatory‐nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression.
  Damian M. Bailey, Julien V. Brugniaux, Teresa Filipponi, Christopher J. Marley, Benjamin Stacey, Rodrigo Soria, Stefano F. Rimoldi, David Cerny, Emrush Rexhaj, Lorenza Pratali, Carlos Salinas Salmòn, Carla Murillo Jáuregui, Mercedes Villena, Jonathan D. Smirl, Ogoh Shigehiko, Sylvia Pietri, Urs Scherrer, Claudio Sartori.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points Chronic mountain sickness (CMS) is a maladaptation syndrome encountered at high altitude (HA) characterised by severe hypoxaemia that carries a higher risk of stroke and migraine and is associated with increased morbidity and mortality. We examined if exaggerated oxidative‐inflammatory‐nitrosative stress (OXINOS) and corresponding decrease in vascular nitric oxide bioavailability in patients with CMS (CMS+) is associated with impaired cerebrovascular function and adverse neurological outcome. Systemic OXINOS was markedly elevated in CMS+ compared to healthy HA (CMS−) and low‐altitude controls. OXINOS was associated with blunted cerebral perfusion and vasoreactivity to hypercapnia, impaired cognition and, in CMS+, symptoms of depression. These findings are the first to suggest that a physiological continuum exists for hypoxaemia‐induced systemic OXINOS in HA dwellers that when excessive is associated with accelerated cognitive decline and depression, helping identify those in need of more specialist neurological assessment and targeted support. Abstract Chronic mountain sickness (CMS) is a maladaptation syndrome encountered at high altitude (HA) characterised by severe hypoxaemia that carries a higher risk of stroke and migraine and is associated with increased morbidity and mortality. The present cross‐sectional study examined to what extent exaggerated systemic oxidative‐inflammatory‐nitrosative stress (OXINOS), defined by an increase in free radical formation and corresponding decrease in vascular nitric oxide (NO) bioavailability, is associated with impaired cerebrovascular function, accelerated cognitive decline and depression in CMS. Venous blood was obtained from healthy male lowlanders (80 m, n = 17), and age‐ and gender‐matched HA dwellers born and bred in La Paz, Bolivia (3600 m) with (CMS+, n = 23) and without (CMS−, n = 14) CMS. We sampled blood for oxidative (electron paramagnetic resonance spectroscopy, HPLC), nitrosative (ozone‐based chemiluminescence) and inflammatory (fluorescence) biomarkers. We employed transcranial Doppler ultrasound to measure cerebral blood flow (CBF) and reactivity. We utilised psychometric tests and validated questionnaires to assess cognition and depression. Highlanders exhibited elevated systemic OXINOS (P < 0.05 vs. lowlanders) that was especially exaggerated in the more hypoxaemic CMS+ patients (P < 0.05 vs. CMS−). OXINOS was associated with blunted cerebral perfusion and vasoreactivity to hypercapnia, impaired cognition and, in CMS+, symptoms of depression. Collectively, these findings are the first to suggest that a physiological continuum exists for hypoxaemia‐induced OXINOS in HA dwellers that when excessive is associated with accelerated cognitive decline and depression, helping identify those in need of specialist neurological assessment and support. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP276898   open full text
• Interneuronal NMDA receptors regulate long‐term depression and motor learning in the cerebellum.
  Maya Kono, Wataru Kakegawa, Kazunari Yoshida, Michisuke Yuzaki.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points NMDA receptors (NMDARs) are required for long‐term depression (LTD) at parallel fibre–Purkinje cell synapses, but their cellular localization and physiological functions in vivo are unclear. NMDARs in molecular‐layer interneurons (MLIs), but not granule cells or Purkinje cells, are required for LTD, but not long‐term potentiation induced by low‐frequency stimulation of parallel fibres. Nitric oxide produced by NMDAR activation in MLIs probably mediates LTD induction. NMDARs in granule cells or Purkinje cells are dispensable for motor learning during adaptation of horizontal optokinetic responses. Abstract Long‐term potentiation (LTP) and depression (LTD), which serve as cellular synaptic plasticity models for learning and memory, are crucially regulated by N‐methyl‐d‐aspartate receptors (NMDARs) in various brain regions. In the cerebellum, LTP and LTD at parallel fibre (PF)–Purkinje cell (PC) synapses are thought to mediate certain forms of motor learning. However, while NMDARs are essential for LTD in vitro, their cellular localization remains controversial. In addition, whether and how NMDARs mediate motor learning in vivo remains unclear. Here, we examined the contribution of NMDARs expressed in granule cells (GCs), PCs and molecular‐layer interneurons (MLIs) to LTD/LTP and motor learning by generating GC‐, PC‐ and MLI/PC‐specific knockouts of Grin1, a gene encoding an obligatory GluN1 subunit of NMDARs. While robust LTD and LTP were induced at PF–PC synapses in GC‐ and PC‐specific Grin1 (GC‐Grin1 and PC‐Grin1, respectively) conditional knockout (cKO) mice, only LTD was impaired in MLI/PC‐specific Grin1 (MLI/PC‐Grin1) cKO mice. Application of diethylamine nitric oxide (NO) sodium, a potent NO donor, to the cerebellar slices restored LTD in MLI/PC‐Grin1 cKO mice, suggesting that NO is probably downstream to NMDARs. Furthermore, the adaptation of horizontal optokinetic responses (hOKR), a cerebellar motor learning task, was normally observed in GC‐Grin1 cKO and PC‐Grin1 cKO mice, but not in MLI/PC‐Grin1 cKO mice. These results indicate that it is the NMDARs expressed in MLIs, but not in PCs or GCs, that play important roles in LTD in vitro and motor learning in vivo. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP276794   open full text
• Prolonged exercise training improves the acute type II muscle fibre satellite cell response in healthy older men.
  Tim Snijders, Joshua P. Nederveen, Kirsten E. Bell, Sean W. Lau, Nicole Mazara, Dinesh A. Kumbhare, Stuart M. Phillips, Gianni Parise.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points Skeletal muscle stem cells, termed satellite cells, play a crucial role in repair and remodelling of muscle in response to exercise An age‐related decline in satellite cell number and/or function has been hypothesized to be a key factor in the development of sarcopenia and/or the blunted muscle fibre adaptive response to prolonged exercise training in older persons We report that performing prolonged exercise training improves the acute type II muscle fibre satellite cell response following a single bout of resistance exercise in older men. The observed improvement in muscle satellite function is associated with an increase in muscle fibre capillarization following exercise training suggesting a possible functional link between capillarization and satellite cell function. Abstract Age‐related type II muscle fibre atrophy is accompanied by a fibre type‐specific decline in satellite cell number and function. Exercise training restores satellite cell quantity in older adults; however, whether it can restore the impaired satellite cell response to exercise in older adults remains unknown. Therefore we assessed the acute satellite cell response to a single exercise session before and after prolonged exercise training in older men. Fourteen older men (74 ± 8 years) participated in a 12‐week exercise training programme (resistance exercise performed twice per week, high intensity interval training once per week). Before and after training, percutaneous biopsies from the vastus lateralis muscle were taken prior to and following 24 and 48 h of post‐exercise recovery. Muscle fibre characteristics were evaluated by immunohistochemistry and mRNA expression by RT‐PCR. Whereas no changes were observed in type II muscle fibres, type I muscle fibre satellite cell content increased significantly at 24 and 48 h after a single bout of resistance exercise before the exercise training programme (P < 0.01). Following the exercise training programme, both type I and type II muscle fibre satellite cell content increased significantly at 24 and 48 h after a single bout of resistance exercise (P < 0.05). The greater acute increase in type II muscle fibre satellite cell content at 24 h post‐exercise recovery after training was correlated with an increase in type II muscle fibre capillarization (r = 0.671, P = 0.012). We show that the acute muscle satellite cell response following exercise can be improved by prolonged exercise training in older men. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP276260   open full text
• Developmental maturation of activity‐induced K+ and pH transients and the associated extracellular space dynamics in the rat hippocampus.
  Brian Roland Larsen, Anca Stoica, Nanna MacAulay.
  The Journal of Physiology. November 24, 2018
  --- - |2+ Key points Neuronal activity induces fluctuation in extracellular space volume, [K+]o and pHo, the management of which influences neuronal function The neighbour astrocytes buffer the K+ and pH and swell during the process, causing shrinkage of the extracellular space In the present study, we report the developmental rise of the homeostatic control of the extracellular space dynamics, for which regulation becomes tighter with maturation and thus is proposed to ensure efficient synaptic transmission in the mature animals The extracellular space dynamics of volume, [K+]o and pHo evolve independently with developmental maturation and, although all of them are inextricably tied to neuronal activity, they do not couple directly. Abstract Neuronal activity in the mammalian central nervous system associates with transient extracellular space (ECS) dynamics involving elevated K+ and pH and shrinkage of the ECS. These ECS properties affect membrane potentials, neurotransmitter concentrations and protein function and are thus anticipated to be under tight regulatory control. It remains unresolved to what extent these ECS dynamics are developmentally regulated as synaptic precision arises and whether they are directly or indirectly coupled. To resolve the development of homeostatic control of [K+]o, pH, and ECS and their interaction, we utilized ion‐sensitive microelectrodes in electrically stimulated rat hippocampal slices from rats of different developmental stages (postnatal days 3–28). With the employed stimulation paradigm, the stimulus‐evoked peak [K+]o and pHo transients were stable across age groups, until normalized to neuronal activity (field potential amplitude), in which case the K+ and pH shifted significantly more in the younger animals. By contrast, ECS dynamics increased with age until normalized to the field potential, and thus correlated with neuronal activity. With age, the animals not only managed the peak [K+]o better, but also displayed swifter post‐stimulus removal of [K+]o, in correlation with the increased expression of the α1‐3 isoforms of the Na+/K+‐ATPase, and a swifter return of ECS volume. The different ECS dynamics approached a near‐identical temporal pattern in the more mature animals. In conclusion, although these phenomena are inextricably tied to neuronal activity, our data suggest that they do not couple directly. - 'The Journal of Physiology, EarlyView. '

  November 24, 2018   doi: 10.1113/JP276768   open full text
• Oxytocin can regulate myometrial smooth muscle excitability by inhibiting the Na+‐activated K+ channel, Slo2.1.
  Juan J. Ferreira, Alice Butler, Richard Stewart, Ana Laura Gonzalez‐Cota, Pascale Lybaert, Chinwendu Amazu, Erin L. Reinl, Monali Wakle‐Prabagaran, Lawrence Salkoff, Sarah K. England, Celia M. Santi.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points At the end of pregnancy, the uterus transitions from a quiescent state to a highly contractile state. This transition requires that the uterine (myometrial) smooth muscle cells increase their excitability, although how this occurs is not fully understood. We identified SLO2.1, a potassium channel previously unknown in uterine smooth muscle, as a potential significant contributor to the electrical excitability of myometrial smooth muscle cells. We found that activity of the SLO2.1 channel is negatively regulated by oxytocin via Gαq‐protein‐coupled receptor activation of protein kinase C. This results in depolarization of the uterine smooth muscle cells and calcium entry, which may contribute to uterine contraction. These findings provide novel insights into a previously unknown mechanism by which oxytocin may act to modulate myometrial smooth muscle cell excitability. Our findings also reveal a new potential pharmacological target for modulating uterine excitability. Abstract During pregnancy, the uterus transitions from a quiescent state to a more excitable contractile state. This is considered to be at least partly a result of changes in the myometrial smooth muscle cell (MSMC) resting membrane potential. However, the ion channels controlling the myometrial resting membrane potential and the mechanism of transition to a more excitable state have not been fully clarified. In the present study, we show that the sodium‐activated, high‐conductance, potassium leak channel, SLO2.1, is expressed and active at the resting membrane potential in MSMCs. Additionally, we report that SLO2.1 is inhibited by oxytocin binding to the oxytocin receptor. Inhibition of SLO2.1 leads to membrane depolarization and activation of voltage‐dependent calcium channels, resulting in calcium influx. The results of the present study reveal that oxytocin may modulate MSMC electrical activity by inhibiting SLO2.1 potassium channels. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276806   open full text
• Intercellular Ca2+ signalling in the adult mouse cochlea.
  Piotr Sirko, Jonathan E Gale, Jonathan F Ashmore.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points Intercellular Ca2+ waves are increases in cytoplasmic Ca2+ levels that propagate between cells. Periodic Ca2+ waves have been linked to gene regulation and are thought to play a crucial role in the development of our hearing epithelium, the organ of Corti and the acquisition of hearing. We observed regular periodic intercellular Ca2+ waves in supporting cells of an ex vivo preparation of the adult mouse organ of Corti, and these waves were found to propagate independently of extracellular ATP and were inhibited by the gap junction blockers 1‐octanol and carbenoxolone. Our results establish that the existence of periodic Ca2+ waves in the organ of Corti is not restricted to the prehearing period. Abstract We have investigated wave‐like cytoplasmic calcium (Ca2+) signalling in an ex vivo preparation of the adult mouse organ of Corti. Two types of intercellular Ca2+ waves that differ in propagation distance and speed were observed. One type was observed to travel up to 100 μm with an average velocity of 7 μm/s. Such waves were initiated by local tissue damage in the outer hair cell region. The propagation distance was decreased when the purinergic receptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS; 50 μm) or suramin (150 μm) were added to the extracellular buffer. Immunocytochemical analysis and experiments with calcium indicator dyes showed that both P2X and P2Y receptors were present in supporting cells. A second class of waves identified to travel longitudinally along the organ of Corti propagated at a lower velocity of 1–3 μm/s. These ‘slow’ Ca2+ waves were particularly evident in the inner sulcus and Deiters’ cells. They travelled for distances of up to 500 μm. The slow Ca2+ signalling varied periodically (approximately one wave every 10 min) and was maintained for more than 3 h. The slow waves were not affected by apyrase, or by the P2 receptor agonists suramin (150 μm) or PPADS (50 μm) but were blocked by the connexin channel blockers octanol (1 mm) and carbenoxolone (100 μm). It is proposed that the observed Ca2+ waves might be a physiological response to a change in extracellular environment and may be involved in critical gene regulation activities in the supporting cells of the cochlea. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276400   open full text
• Exercise training reduces the insulin‐sensitizing effect of a single bout of exercise in human skeletal muscle.
  Dorte E. Steenberg, Nichlas B. Jørgensen, Jesper B. Birk, Kim A. Sjøberg, Bente Kiens, Erik A. Richter, Jørgen F.P. Wojtaszewski.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points A single bout of exercise is capable of increasing insulin sensitivity in human skeletal muscle. Whether this ability is affected by training status is not clear. Studies in mice suggest that the AMPK‐TBC1D4 signalling axis is important for the increased insulin‐stimulated glucose uptake after a single bout of exercise. The present study is the first longitudinal intervention study to show that, although exercise training increases insulin‐stimulated glucose uptake in skeletal muscle at rest, it diminishes the ability of a single bout of exercise to enhance muscle insulin‐stimulated glucose uptake. The present study provides novel data indicating that AMPK in human skeletal muscle is important for the insulin‐sensitizing effect of a single bout of exercise. Abstract Not only chronic exercise training, but also a single bout of exercise, increases insulin‐stimulated glucose uptake in skeletal muscle. However, it is not well described how adaptations to exercise training affect the ability of a single bout of exercise to increase insulin sensitivity. Rodent studies suggest that the insulin‐sensitizing effect of a single bout of exercise is AMPK‐dependent (presumably via the α2β2γ3 AMPK complex). Whether this is also the case in humans is unknown. Previous studies have shown that exercise training decreases the expression of the α2β2γ3 AMPK complex and diminishes the activation of this complex during exercise. Thus, we hypothesized that exercise training diminishes the ability of a single bout of exercise to enhance muscle insulin sensitivity. We investigated nine healthy male subjects who performed one‐legged knee‐extensor exercise at the same relative intensity before and after 12 weeks of exercise training. Training increased and expression of mitochondrial proteins in muscle, whereas the expression of AMPKγ3 was decreased. Training also increased whole body and muscle insulin sensitivity. Interestingly, insulin‐stimulated glucose uptake in the acutely exercised leg was not enhanced further by training. Thus, the increase in insulin‐stimulated glucose uptake following a single bout of one‐legged exercise was lower in the trained vs. untrained state. This was associated with reduced signalling via confirmed α2β2γ3 AMPK downstream targets (ACC and TBC1D4). These results suggest that the insulin‐sensitizing effect of a single bout of exercise is also AMPK‐dependent in human skeletal muscle. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276735   open full text
• On exercise thermoregulation in females: interaction of endogenous and exogenous ovarian hormones.
  Tze‐Huan Lei, James D. Cotter, Zachary J. Schlader, Stephen R. Stannard, Blake G. Perry, Matthew J. Barnes, Toby Mündel.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points One in two female athletes chronically take a combined, monophasic oral contraceptive pill (OCP). Previous thermoregulatory investigations proposed that an endogenous rhythm of the menstrual cycle still occurs with OCP usage. Forthcoming large international sporting events will expose female athletes to hot environments differing in their thermal profile, yet few data exist on how trained women will respond from both a thermoregulatory and performance stand‐point. In the present study, we have demonstrated that a small endogenous rhythm of the menstrual cycle still affects Tcore and also that chronic OCP use attenuates the sweating response, whereas behavioural thermoregulation is maintained. Furthermore, humid heat affects both performance and thermoregulatory responses to a greater extent than OCP usage and the menstrual cycle does. Abstract We studied thermoregulatory responses of ten well‐trained (, 57 ± 7 mL min−1 kg−1) women taking a combined, monophasic oral contraceptive pill (OCP) (≥12 months) during exercise in dry and humid heat, across their active OCP cycle. They completed four trials, each of resting and cycling at fixed intensities (125 and 150 W), aiming to assess autonomic regulation, and then a self‐paced intensity (30‐min work trial) to assess behavioural regulation. Trials were conducted in quasi‐follicular (qF) and quasi‐luteal (qL) phases in dry (DRY) and humid (HUM) heat matched for wet bulb globe temperature (WBGT) (27°C). During rest and exercise at 125 W, rectal temperature was 0.15°C higher in qL than qF (P = 0.05) independent of environment (P = 0.17). The onset threshold and thermosensitivity of local sweat rate and forearm blood flow relative to mean body temperature was unaffected by the OCP cycle (both P > 0.30). Exercise performance did not differ between quasi‐phases (qF: 268 ± 31 kJ, qL: 263 ± 26 kJ, P = 0.31) but was 5 ± 7% higher during DRY than during HUM (273 ± 29 kJ, 258 ± 28 kJ; P = 0.03). Compared to matched eumenorrhoeic athletes, chronic OCP use impaired the sweating onset threshold and thermosensitivity (both P < 0.01). In well‐trained, OCP‐using women exercising in the heat: (i) a performance‐thermoregulatory trade‐off occurred that required behavioural adjustment; (ii) humidity impaired performance as a result of reduced evaporative power despite matched WBGT; and (iii) the sudomotor but not behavioural thermoregulatory responses were impaired compared to matched eumenorrhoeic athletes. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276233   open full text
• Glycinergic neurotransmission in the rostral ventrolateral medulla controls the time course of baroreflex‐mediated sympathoinhibition.
  Hong Gao, Willian S. Korim, Song T. Yao, Cheryl M. Heesch, Andrei V. Derbenev.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points To maintain appropriate blood flow to various tissues of the body under a variety of physiological states, autonomic nervous system reflexes regulate regional sympathetic nerve activity and arterial blood pressure. Our data obtained in anaesthetized rats revealed that glycine released in the rostral ventrolateral medulla (RVLM) plays a critical role in maintaining arterial baroreflex sympathoinhibition. Manipulation of brainstem nuclei with known inputs to the RVLM (nucleus tractus solitarius and caudal VLM) unmasked tonic glycinergic inhibition in the RVLM. Whole‐cell, patch clamp recordings demonstrate that both GABA and glycine inhibit RVLM neurons. Potentiation of neurotransmitter release from the active synaptic inputs in the RVLM produced saturation of GABAergic inhibition and emergence of glycinergic inhibition. Our data suggest that GABA controls threshold excitability, wherreas glycine increases the strength of inhibition under conditions of increased synaptic activity within the RVLM. Abstract The arterial baroreflex is a rapid negative‐feedback system that compensates changes in blood pressure by adjusting the output of presympathetic neurons in the rostral ventrolateral medulla (RVLM). GABAergic projections from the caudal VLM (CVLM) provide a primary inhibitory input to presympathetic RVLM neurons. Although glycine‐dependent regulation of RVLM neurons has been proposed, its role in determining RVLM excitability is ill‐defined. The present study aimed to determine the physiological role of glycinergic neurotransmission in baroreflex function, identify the mechanisms for glycine release, and evaluate co‐inhibition of RVLM neurons by GABA and glycine. Microinjection of the glycine receptor antagonist strychnine (4 mm, 100 nL) into the RVLM decreased the duration of baroreflex‐mediated inhibition of renal sympathetic nerve activity (control = 12 ± 1 min; RVLM‐strychnine = 5.1 ± 1 min), suggesting that RVLM glycine plays a critical role in regulating the time course of sympathoinhibition. Blockade of output from the nucleus tractus solitarius and/or disinhibition of the CVLM unmasked tonic glycinergic inhibition of the RVLM. To evaluate cellular mechanisms, RVLM neurons were retrogradely labelled (prior injection of pseudorabies virus PRV‐152) and whole‐cell, patch clamp recordings were obtained in brainstem slices. Under steady‐state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of the overall inhibition. By contrast, stimulation of synaptic inputs in the RVLM decreased GABAergic inhibition to 53%; and increased glycinergic inhibition to 47%. Thus, under conditions of increased synaptic activity in the RVLM, glycinergic inhibition is recruited to strengthen sympathoinhibition. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276467   open full text
• Four weeks of exercise early in life reprograms adult skeletal muscle insulin resistance caused by a paternal high‐fat diet.
  Filippe Falcão‐Tebas, Jujiao Kuang, Chelsea Arceri, Jarrod P. Kerris, Sofianos Andrikopoulos, Evelyn C. Marin, Glenn K. McConell.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points A paternal high‐fat diet/obesity before mating can negatively influence the metabolism of offspring. Exercise only early in life has a remarkable effect with respect to reprogramming adult rat offspring exposed to detrimental insults before conception. Exercise only early in life normalized adult whole body and muscle insulin resistance as a result of having a high‐fat fed/obese father. Unlike the effects on the muscle, early exercise did not normalize the reduced adult pancreatic beta cell mass as a result of having a high‐fat fed/obese father. Early‐life exercise training may be able to reprogram an individual whose father was obese, inducing long‐lasting beneficial effects on health. Abstract A paternal high‐fat diet (HFD) impairs female rat offspring glucose tolerance, pancreatic morphology and insulin secretion. We examined whether only 4 weeks of exercise early in life could reprogram these negative effects. Male Sprague–Dawley rats consumed a HFD for 10 weeks before mating with chow‐fed dams. Female offspring remained sedentary or performed moderate intensity treadmill exercise (5 days week−1, 60 min day−1, 20 m min−1) from 5 to 9 weeks of age. Paternal HFD impaired (P < 0.05) adult offspring whole body insulin sensitivity (i.p. insulin sensitivity test), as well as skeletal muscle ex vivo insulin sensitivity and TBC1D4 phosphorylation. It also lowered β‐cell mass and reduced in vivo insulin secretion in response to an i.p. glucose tolerance test. Early‐life exercise in offspring reprogrammed the negative effects of a paternal HFD on whole body insulin sensitivity, skeletal muscle ex vivo insulin‐stimulated glucose uptake and TBC1D4 phosphorylation and also increased glucose transporter 4 protein. However, early exercise did not normalize the reduced pancreatic β‐cell mass or insulin secretion. In conclusion, only 4 weeks of exercise early in life in female rat offspring reprograms reductions in insulin sensitivity in adulthood caused by a paternal HFD without affecting pancreatic β‐cell mass or insulin secretion. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276386   open full text
• The impact of 2 years of high‐intensity exercise training on a model of integrated cardiovascular regulation.
  Michinari Hieda, Erin J. Howden, Satyam Sarma, William Cornwell, Justin S. Lawley, Takashi Tarumi, Dean Palmer, Mitchel Samels, Braden Everding, Sheryl Livingston, Qi Fu, Rong Zhang, Benjamin D. Levine.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key points Heart rate variability, a common and easily measured index of cardiovascular dynamics, is the output variable of complicated cardiovascular and respiratory control systems. Both neural and non‐neural control mechanisms may contribute to changes in heart rate variability. We previously developed an innovative method using transfer function analysis to assess the effect of prolonged exercise training on integrated cardiovascular regulation. In the present study, we modified and applied this to investigate the effect of 2 years of high‐intensity training on circulatory components to tease out the primary effects of training. Our method incorporated the dynamic Starling mechanism, dynamic arterial elastance and arterial–cardiac baroreflex function. The dynamic Starling mechanism gain and arterial–cardiac baroreflex gain were significantly increased in the exercise group. These parameters remained unchanged in the controls. Conversely, neither group experienced a change in dynamic arterial elastance. The integrated cardiovascular regulation gain in the exercise group was 1.34‐fold larger than that in the control group after the intervention. In these previously sedentary, otherwise healthy, middle‐aged adults, 2 years of high‐intensity exercise training improved integrated cardiovascular regulation by enhancing the dynamic Starling mechanism and arterial–cardiac baroreflex sensitivity. Abstract Assessing the effects of exercise training on cardiovascular variability is challenging because of the complexity of multiple mechanisms. In a prospective, parallel‐group, randomized controlled study, we examined the effect of 2 years of high‐intensity exercise training on integrated cardiovascular function, which incorporates the dynamic Starling mechanism, dynamic arterial elastance and arterial–cardiac baroreflex function. Sixty‐one healthy participants (48% male, aged 53 years, range 52–54 years) were randomized to either 2 years of exercise training (exercise group: n = 34) or control/yoga group (controls: n = 27). Before and after 2 years, subjects underwent a 6 min recording of beat‐by‐beat pulmonary artery diastolic pressure (PAD), stroke volume index (SV index), systolic blood pressure (sBP) and RR interval measurements with controlled respiration at 0.2 Hz. The dynamic Starling mechanism, dynamic arterial elastance and arterial–cardiac baroreflex function were calculated by transfer function gain between PAD and SV index; SV index and sBP; and sBP and RR interval, respectively. Fifty‐three participants (controls: n = 25; exercise group: n = 28) completed the intervention. After 2 years, the dynamic Starling mechanism gain (Group × Time interaction: P = 0.008) and the arterial–cardiac baroreflex gain (P = 0.005) were significantly increased in the exercise group but remained unchanged in the controls. There was no change in dynamic arterial elastance in either of the two groups. The integrated cardiovascular function gain in the exercise group increased 1.34‐fold, whereas there was no change in the controls (P = 0.02). In these previously sedentary, otherwise healthy middle‐aged adults, a 2 year programme of high‐intensity exercise training improved integrated cardiovascular regulation by enhancing the dynamic Starling mechanism and arterial–cardiac baroreflex sensitivity, without changing dynamic arterial elastance. - 'The Journal of Physiology, EarlyView. '

  November 22, 2018   doi: 10.1113/JP276676   open full text
• Induced‐in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness.
  Michael D. Tarpey, Ana P. Valencia, Kathryn C. Jackson, Adam J. Amorese, Nicholas P. Balestrieri, Randall H. Renegar, Stephen J. P. Pratt, Terence E. Ryan, Joseph M. McClung, Richard M. Lovering, Espen E. Spangenburg.
  The Journal of Physiology. November 22, 2018
  --- - |2+ Key Points Summary Breast cancer 1, early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA (mtDNA) mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mtDNA mutations may represent a potential underlying mechanism. Abstract Recent evidence suggests that the breast cancer 1, early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The purpose of this study was to determine if induced‐loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in skeletal muscle of adult mice to generate skeletal muscle‐specific Brca1 homozygote knockout (Brca1KOsmi) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles when compared to age‐matched wildtype (WT) mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fiber type and, in parallel, increased myofiber size and reduced capillary numbers. Surprisingly, myofiber bundle mitochondrial respiration was reduced while contraction‐induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mtDNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and which were more susceptible to swelling. In summary, skeletal muscle‐specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 22, 2018   doi: 10.1113/JP276863   open full text
• Metabolic remodeling of glucose, fatty acid and redox pathways in the heart of type 2 diabetic mice.
  Sonia Cortassa, Viviane Caceres, Carlo G. Tocchetti, Michel Bernier, Rafael Cabo, Nazareno Paolocci, Steven J. Sollott, Miguel A. Aon.
  The Journal of Physiology. November 21, 2018
  --- - |2+ Key points Hearts from type 2 diabetic animals display perturbations in excitation‐contraction coupling, impairing myocyte contractility and delaying relaxation, along with altered substrate consumption patterns. Under high glucose and β‐adrenergic stimulation conditions, palmitate can, at least in part, offset left ventricle (LV) dysfunction in hearts from diabetic mice improving contractility and relaxation while restoring coronary perfusion pressure. Fluxome calculations of central catabolism in diabetic hearts show that, in presence of palmitate, there is a metabolic remodeling involving tricarboxylic acid cycle, polyol and pentose phosphate pathways, leading to improved redox balance in cytoplasmic and mitochondrial compartments. Under high glucose and increased energy demand, the metabolic/fluxomic re‐direction leading to restored redox balance imparted by palmitate helps explain maintained LV function and may contribute to design novel therapeutic approaches to prevent cardiac dysfunction in diabetic patients. Abstract Type‐2 diabetes (T2DM) leads to reduced myocardial performance, and eventually heart failure. Excessive accumulation of lipids and glucose are central to T2DM cardiomyopathy. Previous data showed that palmitate (Palm) or glutathione preserved heart mitochondrial energy/redox balance under excess glucose rescuing β‐adrenergic‐stimulated cardiac excitation‐contraction coupling. However, the mechanisms underlying the accompanying improved contractile performance have been largely ignored. Herein we explore in intact heart under substrate excess the metabolic remodeling associated with cardiac function in diabetic db/db mice subjected to stress given by β‐adrenergic stimulation with isoproterenol and high‐glucose compared to their nondiabetic controls (+/+, WT) under euglycemic conditions. When perfused with Palm, T2DM hearts exhibit improved contractility/relaxation compared to WT, accompanied by extensive metabolic remodeling as demonstrated by metabolomics‐fluxomics combined with bioinformatics and computational modeling. The T2DM heart metabolome showed significant differences in the abundance of metabolites in pathways related to glucose, lipids, and redox metabolism. Using a validated computational model of heart's central catabolism, comprising glucose and fatty acid (FA) oxidation in cytoplasmic and mitochondrial compartments, we estimated that fluxes through glucose degradation pathways are ∼2‐fold lower in heart from T2DM vs. WT under all conditions studied. Palm addition elicits improvement of the redox status via enhanced β‐oxidation and decreased glucose uptake, leading to flux‐redirection away from redox‐consuming pathways (e.g., polyol) while maintaining the flux through redox‐generating pathways together with glucose‐FA “shared fueling” of oxidative phosphorylation. Thus, available FAs such as Palm may help improve function via enhanced redox balance in T2DM hearts during peaks of hyperglycemia and increased workload. Sonia Cortassa has a PhD in Chemical Sciences from the Universidad Nacional de Córdoba, Argentina, country where she held research and teaching positions at Universidad Nacional de Tucumán and Consejo Nacional Investigaciones Científicas y Técnicas (CONICET). In the United States of America, she continued her research at the Johns Hopkins University and, at present, at the Laboratory of Cardiovascular Sciences/National Institute on Aging/NIH. Her field of research is Physiology, Bioenergetics, with expertise in Computational modeling of metabolic networks. She believes that quantitative Systems Biology approaches represent a real opportunity to contribute to the understanding of human body function in health and disease. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 21, 2018   doi: 10.1113/JP276824   open full text
• Simulation of P2X‐mediated calcium signaling in microglia.
  Byeongjae Chun, Bradley D. Stewart, Darin D. Vaughan, Adam D. Bachstetter, Peter M. Kekenes‐Huskey.
  The Journal of Physiology. November 21, 2018
  --- - "\n\nKey Points Summary\n\n•\tA computational model of P2X channel activation in microglia was developed that includes downfield Ca2+ dependent signaling pathways.\n•\tThis model provides quantitative insights into how diverse signaling pathways in microglia converge to control microglial function.\n\n\n\nAbstract\nMicroglia function is orchestrated through highly‐coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adeno‐ sine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P2 × 4 and P2 × 7, with the graded production of a gene product, namely the tumor necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia‐specific processes including ATP‐dependent P2 × 4 and P2 × 7 activation, activation of nuclear factor of activated T‐cells (NFAT) transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our results suggest that pulsatile stimulation of P2 × 4 via micromolar ATP may be sufficient to promote TNFα production, whereas high amplitude ATP exposure is necessary for production via P2 × 7. Further, under conditions that increase P2 × 4 expression, for instance the activation by pathogen associated molecular factors, P2 × 4‐associated TNFα production is greatly enhanced. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.\nThis article is protected by copyright. All rights reserved\n\n" - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 21, 2018   doi: 10.1113/JP277377   open full text
• Lower body negative pressure to safely reduce intracranial pressure.
  Lonnie G. Petersen, Justin S. Lawley, Alexander Lilja‐Cyron, Johan C. G. Petersen, Erin J. Howden, Satyam Sarma, William K. Cornwell, Rong Zhang, Louis A. Whitworth, Michael A. Williams, Marianne Juhler, Benjamin D. Levine.
  The Journal of Physiology. November 20, 2018
  --- - |2+ Key points During long‐term missions, some astronauts experience structural and functional changes of the eyes and brain which resemble signs/symptoms experienced by patients with intracranial hypertension. Weightlessness prevents the normal cerebral volume and pressure ‘unloading’ associated with upright postures on Earth, which may be part of the cerebral and ocular pathophysiology. By placing the lower body in a negative pressure device (LBNP) that pulls fluid away from cranial compartments, we simulated effects of gravity and significantly lowered pressure within the brain parenchyma and ventricle compartments. Application of incremental LBNP demonstrated a non‐linear dose–response curve, suggesting 20 mmHg LBNP as the optimal level for reducing pressure in the brain without impairing cerebral perfusion pressure. This non‐invasive method of reducing pressure in the brain holds potential as a countermeasure in space as well as having treatment potential for patients on Earth with traumatic brain injury or other pathology leading to intracranial hypertension. Abstract Patients with elevated intracranial pressure (ICP) exhibit neuro‐ocular symptoms including headache, papilloedema and loss of vision. Some of these symptoms are also present in astronauts during and after prolonged space‐flight where lack of gravitational stress prevents daily lowering of ICP associated with upright posture. Lower body negative pressure (LBNP) simulates the effects of gravity by displacing fluid caudally and we hypothesized that LBNP would lower ICP without compromising cerebral perfusion. Ten cerebrally intact volunteers were included: six ambulatory neurosurgical patients with parenchymal ICP‐sensors and four former cancer patients with Ommaya‐reservoirs to the frontal horn of a lateral ventricle. We applied LBNP while recording ICP and blood pressure while supine, and during simulated intracranial hypertension by 15° head‐down tilt. LBNP from 0 to 50 mmHg at increments of 10 mmHg lowered ICP in a non‐linear dose‐dependent fashion; when supine (n = 10), ICP was decreased from 15 ± 2 mmHg to 14 ± 4, 12 ± 5, 11 ± 4, 10 ± 3 and 9 ± 4 mmHg, respectively (P < 0.0001). Cerebral perfusion pressure (CPP), calculated as mean arterial blood pressure at midbrain level minus ICP, was unchanged (from 70 ± 12 mmHg to 67 ± 9, 69 ± 10, 70 ± 12, 72 ± 13 and 74 ± 15 mmHg; P = 0.02). A 15° head‐down tilt (n = 6) increased ICP to 26 ± 4 mmHg, while application of LBNP lowered ICP (to 21 ± 4, 20 ± 4, 18 ± 4, 17 ± 4 and 17 ± 4 mmHg; P < 0.0001) and increased CPP (P < 0.01). An LBNP of 20 mmHg may be the optimal level to lower ICP without impairing CPP to counteract spaceflight‐associated neuro‐ocular syndrome in astronauts. Furthermore, LBNP holds clinical potential as a safe, non‐invasive method for lowering ICP and improving CPP for patients with pathologically elevated ICP on Earth. - 'The Journal of Physiology, EarlyView. '

  November 20, 2018   doi: 10.1113/JP276557   open full text
• Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: a preclinical animal study.
  Kerstin Hoppe, Sunisa Chaiklieng, Frank Lehmann‐Horn, Karin Jurkat‐Rott, Scott Wearing, Werner Klingler.
  The Journal of Physiology. November 20, 2018
  --- - |2+ Key points During myotonia congenita, reduced chloride (Cl−) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called ‘warm up’. Pharmacological inhibition of low Cl− channels by anthracene‐9‐carboxylic acid in muscle from mice and ADR (‘arrested development of righting response’) muscle from mice showed a relaxation deficit under physiological conditions compared to wild‐type muscle. At increased osmolarity up to 400 mosmol L–1, the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti‐myotonic effects of hypertonicity were at least partly antagonized by the application of bumetanide. Abstract Low chloride‐conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl−) channel gene type 1 (CLCN1). Reduced Cl− conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called ‘warm up’. To gain further insight into the patho‐mechanism of impaired muscle stiffness and the warm‐up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Functional force and membrane potential measurements were performed on muscle specimens of ADR (‘arrested development of righting response’) mice (an animal model for low gCl– conductance myotonia) and pharmacologically‐induced myotonia. Specimens were exposed to solutions of increasing osmolarity at the same time as force and membrane potentials were monitored. In the second set of experiments, ADR muscle and pharmacologically‐induced myotonic muscle were exposed to an antagonist of NKCC1. Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control wild‐type muscle. High osmolarity diminished myotonia and facilitated the warm‐up phenomenon as depicted by a faster muscle relaxation time (T90/10). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the anti‐myotonic effect of high osmolarity. Increased osmolarity decreased signs of myotonia and facilitated the warm‐up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm‐up and reduces the number of contractions required to achieve normal relaxation kinetics. - 'The Journal of Physiology, EarlyView. '

  November 20, 2018   doi: 10.1113/JP276528   open full text
• The postnatal development of ultrasonic vocalization‐associated breathing is altered in glycine transporter 2‐deficient mice.
  Swen Hülsmann, Yoshihiko Oke, Guillaume Mesuret, A. Tobias Latal, Michal G. Fortuna, Marcus Niebert, Johannes Hirrlinger, Julia Fischer, Kurt Hammerschmidt.
  The Journal of Physiology. November 20, 2018
  --- - |2+ Key points Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2‐knockout mice do not acquire adult ultrasonic vocalization‐associated breathing patterns. Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read‐out in translational research, these data are highly relevant for a broad range of research fields. Abstract Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post‐inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2‐knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2‐KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild‐type mice acquire different USV‐associated breathing patterns that depend on post‐inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2‐KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization. - 'The Journal of Physiology, EarlyView. '

  November 20, 2018   doi: 10.1113/JP276976   open full text
• Rapid saline infusion and/or drinking enhance skin sympathetic nerve activity components reduced by hypovolaemia and hyperosmolality in hyperthermia.
  Yoshi‐ichiro Kamijo, Kazunobu Okazaki, Shigeki Ikegawa, Yoshiyuki Okada, Hiroshi Nose.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points In hyperthermia, plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating responses and this suppression is removed by oropharyngeal stimulation such as drinking. Hypovolaemia suppresses only cutaneous vasodilatation, which is enhanced by rapid infusion in hyperthermia. Our recent studies suggested that skin sympathetic nerve activity (SSNA) involves components synchronized and non‐synchronized with the cardiac cycle, which are associated with an active vasodilator and a sudomotor, respectively. In the present study, plasma hyperosmolality suppressed both components; drinking removed the hyperosmolality‐induced suppressions, simultaneously with increases in cutaneous vasodilatation and sweating, while not altering plasma volume and osmolality. Furthermore, a rapid saline infusion increased the synchronized component and cutaneous vasodilatation in hypovolaemic and hyperthermic humans. The results support our idea that SSNA involves an active cutaneous vasodilator and a sudomotor, and that a site where osmolality signals are projected to control thermoregulation is located more superior than the medulla where signals from baroreceptors are projected. Abstract We reported that skin sympathetic nerve activity (SSNA) involved components synchronized and non‐synchronized with the cardiac cycle; both components increased in hyperthermia and our results suggested that the components are associated with an active vasodilator and a sudomotor, respectively. In the present study, we examined whether the increases in the components in hyperthermia would be suppressed by plasma hyperosmolality simultaneously with suppression of cutaneous vasodilatation and sweating and whether this suppression was released by oropharyngeal stimulation (drinking). Also, effects of a rapid saline infusion on both components and responses of cutaneous vasodilatation and sweating were tested in hypovolaemic and hyperthermic subjects. We found that (1) plasma hyperosmolality suppressed both components in hyperthermia, (2) the suppression was released by drinking 200 mL of water simultaneously with enhanced cutaneous vasodilatation and sweating responses, and (3) a rapid infusion at 1.0 and 0.2 ml min−1 kg−1 for the first 10 min and the following 20 min, respectively, increased the synchronized component and cutaneous vasodilatation in diuretic‐induced hypovolaemia greater than those in a time control; at 0.1 ml min−1 kg−1 for 30 min no greater increases in the non‐synchronized component and sweating responses were observed during rapid infusion than in the time control. The results support the idea that SSNA involves components synchronized and non‐synchronized with the cardiac cycle, associated with the active cutaneous vasodilator and sudomotor, and a site of osmolality‐induced modulation for thermoregulation is located superior to the medulla where signals from baroreceptors are projected. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5443-5459, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276633   open full text
• Commissural communication allows mouse intergeniculate leaflet and ventral lateral geniculate neurons to encode interocular differences in irradiance.
  A. Pienaar, L. Walmsley, E. Hayter, M. Howarth, T. M. Brown.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points Unlike other visual thalamic regions, the intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/vLGN) possess extensive reciprocal commissural connections, the functions of which are unknown. Using electrophysiological approaches, it is shown that commissural projecting IGL/vLGN cells are primarily activated by light increments to the contralateral eye while cells receiving commissural input typically exhibit antagonistic binocular responses. Across antagonistic cells, the nature of the commissural input (excitatory or inhibitory) corresponds to the presence of ipsilateral ON or OFF visual responses and in both cases antagonistic responses disappear following inactivation of the contralateral thalamus. The steady state firing rates of antagonistic cells uniquely encode interocular differences in irradiance. There is a pivotal role for IGL/vLGN commissural signalling in generating new sensory properties that are potentially useful for the proposed contributions of these nuclei to visuomotor/vestibular and circadian control. Abstract The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/vLGN) are portions of the visual thalamus implicated in circadian and visuomotor/vestibular control. A defining feature of IGL/vLGN organisation is the presence of extensive reciprocal commissural connections, the functions of which are at present unknown. Here we use a combination of multielectrode recording, electrical microstimulation, thalamic inactivation and a range of visual stimuli in mice to address this deficit. Our data indicate that, like most IGL/vLGN cells, those that project commissurally primarily convey contralateral ON visual signals while most IGL/vLGN neurons that receive this input exhibit antagonistic binocular responses (i.e. excitatory responses driven by one eye and inhibitory responses driven by the other), enabling them to encode interocular differences in irradiance. We also confirm that this property derives from commissural input since, following inactivation of the contralateral visual thalamus, these cells instead display monocular contralateral‐driven ON responses. Our data thereby reveal a fundamental role for commissural signalling in generating new visual response properties at the level of the visual thalamus. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5461-5481, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276917   open full text
• UBC‐Nepal expedition: peripheral fatigue recovers faster in Sherpa than lowlanders at high altitude.
  Luca Ruggiero, Ryan L. Hoiland, Alexander B. Hansen, Philip N. Ainslie, Chris J. McNeil.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points The reduced oxygen tension of high altitude compromises performance in lowlanders. In this environment, Sherpa display superior performance, but little is known on this issue. Sherpa present unique genotypic and phenotypic characteristics at the muscular level, which may enhance resistance to peripheral fatigue at high altitude compared to lowlanders. We studied the impact of gradual ascent and exposure to high altitude (5050 m) on peripheral fatigue in age‐matched lowlanders and Sherpa, using intermittent electrically‐evoked contractions of the knee extensors. Peripheral fatigue (force loss) was lower in Sherpa during the first part of the protocol. Post‐protocol, the rate of force development and contractile impulse recovered faster in Sherpa than in lowlanders. At any time, indices of muscle oxygenation were not different between groups. Muscle contractile properties in Sherpa, independent of muscle oxygenation, were less perturbed by non‐volitional fatigue. Hence, elements within the contractile machinery contribute to the superior physical performance of Sherpa at high altitude. Abstract Altitude‐related acclimatisation is characterised by marked muscular adaptations. Lowlanders and Sherpa differ in their muscular genotypic and phenotypic characteristics, which may influence peripheral fatigability at altitude. After gradual ascent to 5050 m, 12 lowlanders and 10 age‐matched Sherpa (32 ± 10 vs. 31 ± 11 years, respectively) underwent three bouts (separated by 15 s rest) of 75 intermittent electrically‐evoked contractions (12 pulses at 15 Hz, 1.6 s between train onsets) of the dominant leg quadriceps, at the intensity which initially evoked 30% of maximal voluntary force. Trains were also delivered at minutes 1, 2 and 3 after the protocol to measure recovery. Tissue oxygenation index (TOI) and total haemoglobin (tHb) were quantified by a near‐infrared spectroscopy probe secured over rectus femoris. Superficial femoral artery blood flow was recorded using ultrasonography, and delivery of oxygen was estimated (eDO2). At the end of bout 1, peak force was greater in Sherpa than in lowlanders (91.5% vs. 84.5% baseline, respectively; P < 0.05). Peak rate of force development (pRFD), the first 200 ms of the contractile impulse (CI200), and half‐relaxation time (HRT) recovered faster in Sherpa than in lowlanders (percentage of baseline at 1 min: pRFD: 89% vs. 74%; CI200: 91% vs. 80%; HRT: 113% vs. 123%, respectively; P < 0.05). Vascular measures were pooled for lowlanders and Sherpa as they did not differ during fatigue or recovery (P < 0.05). Mid bout 3, TOI was decreased (90% baseline) whereas tHb was increased (109% baseline). After bout 3, eDO2 was markedly increased (1266% baseline). The skeletal muscle of Sherpa seemingly favours repeated force production at altitude for similar oxygen delivery compared to lowlanders. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5365-5377, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276599   open full text
• Nicotine modulates human brain plasticity via calcium‐dependent mechanisms.
  Jessica Grundey, Jerick Barlay, Giorgi Batsikadze, Min‐Fang Kuo, Walter Paulus, Michael Nitsche.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points Nicotine (NIC) modulates cognition and memory function by targeting the nicotinic ACh receptor and releasing different transmitter systems postsynaptically. With both NIC‐generated mechanisms, calcium influx and calcium permeability can be regulated, which is a key requirement for the induction of long‐term potentiation, comprising the physiological basis of learning and memory function. We attempt to unmask the underlying mechanism of nicotinic effects on anodal transcranial direct current stimulation (tDCS)‐induced long‐term potentiation‐like plasticity based on the hypothesis of calcium‐dependency. Abolished tDCS‐induced neuroplasticity as a result of NIC administration is reversed by calcium channel blockade with flunarizine in a dose‐dependent manner. The results of the present study suggest that there is a dose determination of NIC/NIC agonists in therapeutical settings when treating cognitive dysfunction, which partially explains the heterogeneous results on cognition observed in subjects in different experimental settings. Abstract Nicotine (NIC) modulates neuroplasticity and improves cognitive performance in animals and humans mainly by increased calcium permeability and modulation of diverse transmitter systems. NIC administration impairs calcium‐dependent plasticity induced by non‐invasive brain stimulation with transcranial direct current stimulation (tDCS) in non‐smoking participants probably as a result of intracellular calcium overflow. To test this hypothesis, we analysed the effect of calcium channel blockade with flunarizine (FLU) on anodal tDCS‐induced cortical excitability changes in healthy non‐smokers under NIC. We applied anodal tDCS combined with NIC patch and FLU at three different doses (2.5, 5 and 10 mg) or with placebo medication. NIC abolished anodal tDCS‐induced neuroplasticity. Under medium dosage (but not under low and high dosage) of FLU combined with NIC, plasticity was re‐established. For FLU alone, the lowest dosage weakened long‐term potentiation (LTP)‐like plasticity, whereas the highest dosage again abolished tDCS‐induced plasticity. The medium dosage turned LTP‐like plasticity in long‐term depression‐like plasticity. The results of the present study suggest a key role of calcium influx and calcium levels in nicotinic effects on LTP‐like plasticity in humans. This knowledge might be relevant for the development of new therapeutic strategies in cognitive dysfunction. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5429-5441, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276502   open full text
• Ventilatory and integrated physiological responses to chronic hypercapnia in goats.
  Nicholas J. Burgraff, Suzanne E. Neumueller, Kirstyn Buchholz, Thomas M. Langer, Matthew R. Hodges, Lawrence Pan, Hubert V. Forster.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points Chronic hypercapnia per se has distinct effects on the mechanisms regulating steady‐state ventilation and the CO2/H+ chemoreflex. Chronic hypercapnia leads to sustained hyperpnoea that exceeds predicted ventilation based upon the CO2/H+ chemoreflex. There is an integrative ventilatory, cardiovascular and metabolic physiological response to chronic hypercapnia. Chronic hypercapnia leads to deterioration of cognitive function. Abstract Respiratory diseases such as chronic obstructive pulmonary disease (COPD) often lead to chronic hypercapnia which may exacerbate progression of the disease, increase risk of mortality and contribute to comorbidities such as cognitive dysfunction. Determining the contribution of hypercapnia per se to adaptations in ventilation and cognitive dysfunction within this patient population is complicated by the presence of multiple comorbidities. Herein, we sought to determine the role of chronic hypercapnia per se on the temporal pattern of ventilation and the ventilatory CO2/H+ chemoreflex by exposing healthy goats to either room air or an elevated inspired CO2 (InCO2) of 6% for 30 days. A second objective was to determine whether chronic hypercapnia per se contributes to cognitive dysfunction. During 30 days of exposure to 6% InCO2, steady‐state (SS) ventilation (I) initially increased to 335% of control, and then within 1–5 days decreased and stabilized at ∼230% of control. There was an initial respiratory acidosis that was partially mitigated over time due to increased arterial [HCO3−]. There was a transient decrease in the ventilatory CO2/H+ chemoreflex, followed by return to pre‐exposure levels. The SS I during chronic hypercapnia was greater than predicted from the acute CO2/H+ chemoreflex, suggesting separate mechanisms regulating SS I and the chemoreflex. Finally, as assessed by a shape discrimination test, we found a sustained decrease in cognitive function during chronic hypercapnia. We conclude that chronic hypercapnia per se results in: (1) a disconnect between SS I and the CO2/H+ chemoreflex, and (2) deterioration of cognitive function. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5343-5363, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276666   open full text
• Growth hormone secretagogue receptor constitutive activity impairs voltage‐gated calcium channel‐dependent inhibitory neurotransmission in hippocampal neurons.
  Valentina Martínez Damonte, Silvia Susana Rodríguez, Jesica Raingo.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points Presynaptic CaV2 voltage‐gated calcium channels link action potentials arriving at the presynaptic terminal to neurotransmitter release. Hence, their regulation is essential to fine tune brain circuitry. CaV2 channels are highly sensitive to G protein‐coupled receptor (GPCR) modulation. Our previous data indicated that growth hormone secretagogue receptor (GHSR) constitutive activity impairs CaV2 channels by decreasing their surface density. We present compelling support for the impact of CaV2.2 channel inhibition by agonist‐independent GHSR activity exclusively on GABA release in hippocampal cultures. We found that this selectivity arises from a high reliance of GABA release on CaV2.2 rather than on CaV2.1 channels. Our data provide new information on the effects of the ghrelin–GHSR system on synaptic transmission, suggesting a putative physiological role of the constitutive signalling of a GPCR that is expressed at high levels in brain areas with restricted access to its natural agonist. Abstract Growth hormone secretagogue receptor (GHSR) displays high constitutive activity, independent of its endogenous ligand, ghrelin. Unlike ghrelin‐induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic CaV2 voltage‐gated calcium channels. Here we postulate that GHSR constitutive activity‐mediated modulation of CaV2 channels could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole‐cell patch‐clamp in hippocampal primary cultures from E16‐ to E18‐day‐old C57BL6 wild‐type and GHSR‐deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs CaV2.1 and CaV2.2 native calcium currents and that CaV2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher CaV2.2 over CaV2.1 contribution to GABA release (∼40% for CaV2.2 in wild‐type vs. ∼20% in wild‐type GHSR‐overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra‐granule cell inhibitory postsynaptic current (IPSC) size ∼−67 pA in wild‐type vs. ∼−100 pA in GHSR‐deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a CaV2‐dependent mechanism without affecting glutamatergic transmission. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5415-5428, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276256   open full text
• Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission.
  C. A. Cobine, M. McKechnie, R. J. Brookfield, K. I. Hannigan, K. D. Keef.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+/VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. Abstract Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)‐dependent contractile inhibition in both muscles whereas P2Y1‐dependent purinergic NMT was restricted to rectum. An additional NOS‐independent, α‐chymotrypsin‐sensitive component was identified in the IAS consistent with vasoactive intestinal peptide‐ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS‐dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1‐dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+/SK3+ cells were closely aligned with nNOS+/VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2‐14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5319-5341, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP275437   open full text
• Sodium and potassium conductances in principal neurons of the mouse piriform cortex: a quantitative description.
  Kaori Ikeda, Norimitsu Suzuki, John M. Bekkers.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points The primary olfactory (or piriform) cortex is a promising model system for understanding how the cerebral cortex processes sensory information, although an investigation of the piriform cortex is hindered by a lack of detailed information about the intrinsic electrical properties of its component neurons. In the present study, we quantify the properties of voltage‐dependent sodium currents and voltage‐ and calcium‐dependent potassium currents in two important classes of excitatory neurons in the main input layer of the piriform cortex. We identify several classes of these currents and show that their properties are similar to those found in better‐studied cortical regions. Our detailed quantitative descriptions of these currents will be valuable to computational neuroscientists who aim to build models that explain how the piriform cortex encodes odours. Abstract The primary olfactory cortex (or piriform cortex, PC) is an anatomically simple palaeocortex that is increasingly used as a model system for investigating cortical sensory processing. However, little information is available on the intrinsic electrical conductances in neurons of the PC, hampering efforts to build realistic computational models of this cortex. In the present study, we used nucleated macropatches and whole‐cell recordings to rigorously quantify the biophysical properties of voltage‐gated sodium (NaV), voltage‐gated potassium (KV) and calcium‐activated potassium (KCa) conductances in two major classes of glutamatergic neurons in layer 2 of the PC, semilunar (SL) cells and superficial pyramidal (SP) cells. We found that SL and SP cells both express a fast‐inactivating NaV current, two types of KV current (A‐type and delayed rectifier‐type) and three types of KCa current (fast‐, medium‐ and slow‐afterhyperpolarization currents). The kinetic and voltage‐dependent properties of the NaV and KV conductances were, with some exceptions, identical in SL and SP cells and similar to those found in neocortical pyramidal neurons. The KCa conductances were also similar across the different types of neurons. Our results are summarized in a series of empirical equations that should prove useful to computational neuroscientists seeking to model the PC. More broadly, our findings indicate that, at the level of single‐cell electrical properties, this palaeocortex is not so different from the neocortex, vindicating efforts to use the PC as a model of cortical sensory processing in general. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5397-5414, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP275824   open full text
• In vivo evidence for reduced ion channel expression in motor axons of patients with amyotrophic lateral sclerosis.
  James Howells, José Manuel Matamala, Susanna B. Park, Nidhi Garg, Steve Vucic, Hugh Bostock, David Burke, Matthew C. Kiernan.
  The Journal of Physiology. November 14, 2018
  --- - |2+ Key points The progressive loss of motor units in amyotrophic lateral sclerosis (ALS) is initially compensated for by the reinnervation of denervated muscle fibres by surviving motor axons. A disruption in protein homeostasis is thought to play a critical role in the pathogenesis of ALS. The changes in surviving motor neurons were studied by comparing the nerve excitability properties of moderately and severely affected single motor axons from patients with ALS with those from single motor axons in control subjects. A mathematical model indicated that approximately 99% of the differences between the ALS and control units could be explained by a non‐selective reduction in the expression of all ion channels. These changes in ALS patients are best explained by a failure in the supply of ion channel and other membrane proteins from the diseased motor neuron. Abstract Amyotrophic lateral sclerosis (ALS) is characterised by a progressive loss of motor units and the reinnervation of denervated muscle fibres by surviving motor axons. This reinnervation preserves muscle function until symptom onset, when some 60–80% of motor units have been lost. We have studied the changes in surviving motor neurons by comparing the nerve excitability properties of 31 single motor axons from patients with ALS with those from 21 single motor axons in control subjects. ALS motor axons were classified as coming from moderately or severely affected muscles according to the compound muscle action potential amplitude of the parent muscle. Compared with control units, thresholds were increased, and there was reduced inward and outward rectification and greater superexcitability following a conditioning impulse. These abnormalities were greater in axons from severely affected muscles, and were correlated with loss of fine motor skills. A mathematical model indicated that 99.1% of the differences between the moderately affected ALS and control units could be explained by a reduction in the expression of all ion channels. For the severely affected units, modelling required, in addition, an increase in the current leak through and under the myelin sheath. This might be expected if the anchoring proteins responsible for the paranodal seal were reduced. We conclude that changes in axonal excitability identified in ALS patients are best explained by a failure in the supply of ion channel and other membrane proteins from the diseased motor neuron, a conclusion consistent with recent animal and in vitro human data. - 'The Journal of Physiology, Volume 596, Issue 22, Page 5379-5396, 15 November 2018. '

  November 14, 2018   doi: 10.1113/JP276624   open full text
• Diverse relaxation rates exist among rat cardiomyocytes isolated from a single myocardial region.
  J. Alexander Clark, Stuart G. Campbell.
  The Journal of Physiology. November 12, 2018
  --- - |2+ Key points Prior studies have shown variation in the functional properties of cardiomyocytes isolated from different regions of the left ventricular myocardium. We found that these region‐dependent variations vanish below a tissue volume of ∼7 mm3 in the adult rat myocardium, revealing a fixed level of intrinsic relaxation rate heterogeneity that is independent of tissue volume. Within these microscopically varying cell populations, fast‐relaxing cells were shown to have elevated phosphorylated troponin I compared to slow‐relaxing cells. Relaxation rate was also correlated with cardiomyocyte length, in that slow‐relaxing cells were longer than fast‐relaxing cells. These results show a new relationship between cardiomyocyte morphology and myofilament relaxation, and suggest that functional diversity among individual myocytes at the microscale may contribute to bulk relaxation of the myocardium. Abstract The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. We designed experiments to quantify the variance in contractile properties among cells within the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free walls of adult Sprague–Dawley rats and then treated with collagenase to isolate individual myocytes. Cardiomyocytes were characterized by measuring sarcomere length changes and calcium transients during electrical pacing. Variance of the time from peak sarcomere shortening to 50% re‐lengthening (RT50) was assessed in each cell population. Isolating cells from progressively shorter strips allowed an estimate of the myocardial volume below which regional variation vanished and only microscale heterogeneity remained (∼7 mm3). The SD of RT50 within this myocardial volume was 28% of the mean. In a series of follow‐up experiments, RT50 was shown to correlate significantly with resting myocyte length, suggesting a connection between cell morphology and intrinsic relaxation behaviour. To explore the mechanistic basis of varying RT50, a novel single‐cell aspirator was employed to collect small batches of cardiomyocytes grouped according to their relaxation rates (fast or slow). Western blot analysis of the two groups revealed significantly elevated troponin I phosphorylation in fast‐relaxing cells. Our observations suggest that cell‐to‐cell heterogeneity of active contractile properties is substantial, with implications for how we understand myocardial relaxation and design drug therapies intended to alter relaxation rate. - 'The Journal of Physiology, EarlyView. '

  November 12, 2018   doi: 10.1113/JP276718   open full text
• GABAB receptors modulate Ca2+ but not G protein‐gated inwardly rectifying K+ channels in cerebrospinal‐fluid contacting neurones of mouse brainstem.
  Nina JURČIĆ, Ghizlane ER‐RAOUI, Coraline AIRAULT, Jérôme TROUSLARD, Nicolas WANAVERBECQ, Riad SEDDIK.
  The Journal of Physiology. November 12, 2018
  --- - |2+ Key points Medullo‐spinal CSF contacting neurones (CSF‐cNs) located around the central canal are conserved in all vertebrates and suggested to be a novel sensory system intrinsic to the CNS. CSF‐cNs receive GABAergic inhibitory synaptic inputs involving ionotropic GABAA receptors but the contribution of metabotropic GABAB receptors (GABAB‐Rs) was not studied yet. Here, we indicate that CSF‐cNs express functional GABAB‐Rs that inhibit postsynaptic calcium channels but fail to activate inhibitory potassium channel of the kir3‐type. We further show that GABAB‐Rs localize presynaptically on GABAergic and glutamatergic synaptic inputs contacting CSF‐cNs where they inhibit the release of GABA and glutamate. Our data are the first to address the function of GABAB‐Rs in CSF‐cNs and show that on the presynaptic side they exert a classical synaptic modulation whereas at the postsynaptic level they have an atypical action by modulating calcium signalling without inducing potassium‐dependent inhibition. Abstract Medullo‐spinal neurones that contact the cerebrospinal fluid (CSF‐cNs) are a population of evolutionary conserved cells located around the central canal. CSF‐cNs activity was shown to be regulated by inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of the G‐protein coupled GABAB receptors was not studied yet. Here, we used a combination of immunofluorescence, electrophysiology and calcium imaging to investigate the expression and function of GABAB‐Rs in CSF‐cNs of the mouse brainstem. We found that CSF‐cNs express GABAB‐Rs, but their selective activation failed to induce G protein‐coupled inwardly‐rectifying potassium (GIRK) currents. Instead, CSF‐cNs express primarily N‐type voltage‐gated calcium (CaV 2.2) channels and GABAB‐Rs recruit Gβγ subunits to inhibit Cav channels activity induced by membrane voltage steps or under physiological conditions by action potentials. Moreover, electrical stimulation evoked in CSF‐cNs GABAergic inhibitory (IPSCs) but also glutamatergic excitatory (EPSCs) synaptic currents, showing that mammalian CSF‐cNs are also under excitatory control by glutamatergic synaptic inputs. We further demonstrate that baclofen reversibly reduced the amplitudes of both IPSCs and EPSCs evoked in CSF‐cNs through a presynaptic mechanism of regulation. In summary, these results are the first to demonstrate the existence of functional postsynaptic GABAB‐Rs in medullar CSF‐cNs as well as presynaptic GABAB auto‐ and heteroreceptors regulating the release of GABA and glutamate. Remarkably, postsynaptic GABAB‐Rs associate with CaV but not GIRK channels indicating that GABAB‐Rs function as a calcium signalling modulator without GIRK‐dependent inhibition in CSF‐cNs. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 12, 2018   doi: 10.1113/JP277172   open full text
• Spinal plasticity with motor imagery practice.
  Sidney Grosprêtre, Florent Lebon, Charalambos Papaxanthis, Alain Martin.
  The Journal of Physiology. November 12, 2018
  --- - |2+ KEY POINT SUMMARY While the effect of Motor Imagery (MI) – the mental simulation of an action – on motor cortical areas has now reached a consensus, less is known about its impact on spinal structures. The current study, using H‐reflex conditioning paradigms, examined the effect of a 20‐min MI practice on several spinal mechanisms of the plantar flexors muscles. We observed modulations of spinal presynaptic circuitry while imagining, even more pronounced following an acute session of MI practice. We suggested that the small cortical output generated during MI may reach specific spinal circuits and that repeating MI may increase the sensitivity of the spinal cord to its effects. The short‐term plasticity induced by MI practice may include spinal network modulation in addition to cortical reorganization. Abstract Kinesthetic Motor imagery (MI) is the mental simulation of a movement with its sensory consequences but without its concomitant execution. While the effect of MI practice on cortical areas is well known, its influence on spinal circuitry remains unclear. Here, we assessed plastic changes in spinal structures following an acute MI practice. Thirteen young healthy participants accomplished two experimental sessions: a 20‐min MI training consisting of four blocks of twenty‐five imagined maximal isometric plantar flexions, and a 20‐min rest (control session). The level of spinal presynaptic inhibition was assessed by conditioning the triceps surae spinal H‐reflex with two methods: i) the stimulation of the common peroneal nerve that induced D1 presynaptic inhibition (HPSI response), and ii) the stimulation of the femoral nerve that induced heteronymous Ia facilitation (HFAC response). We then compared the effects of MI on unconditioned (HTEST) and conditioned (HPSI and HFAC) responses before, immediately after and 10 minutes after the 20‐min session. After resting for 20 minutes, no changes have been observed on the recorded parameters. After MI practice, the amplitude of rest HTEST was unchanged, while HPSI and HFAC significantly increased, showing a reduction of presynaptic inhibition with no impact on afferent‐motoneuronal synapse. The current results revealed the acute effect of MI practice on baseline spinal presynaptic inhibition, increasing the sensitivity of the spinal circuitry to MI. These findings would help understanding the mechanisms of neural plasticity following chronic practice. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 12, 2018   doi: 10.1113/JP276694   open full text
• Spinal control of muscle synergies for adult mammalian locomotion.
  Etienne Desrochers, Jonathan Harnie, Adam Doelman, Marie‐France Hurteau, Alain Frigon.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key points The control of locomotion is thought to be generated by activating groups of muscles that perform similar actions, which are termed muscle synergies. Here, we investigated if muscle synergies are controlled at the level of the spinal cord. We did this by comparing muscle activity in the legs of cats during stepping on a treadmill before and after a complete spinal transection that abolishes commands from the brain. We show that muscle synergies were maintained following spinal transection, validating the concept that muscle synergies for locomotion are primarily controlled by circuits of neurons within the spinal cord. Abstract Locomotion is thought to involve the sequential activation of functional modules or muscle synergies. Here, we tested the hypothesis that muscle synergies for locomotion are organized within the spinal cord. We recorded bursts of muscle activity in the same cats (n = 7) before and after spinal transection during tied‐belt locomotion at three speeds and split‐belt locomotion at three left–right speed differences. We identified seven muscles synergies before (intact state) and after (spinal state) spinal transection. The muscles comprising the different synergies were the same in the intact and spinal states as well as at different speeds or left–right speed differences. However, there were some significant shifts in the onsets and offsets of certain synergies as a function of state, speed and left–right speed differences. The most notable difference between the intact and spinal states was a change in the timing between the knee flexor and hip flexor muscle synergies. In the intact state, the knee flexor synergy preceded the hip flexor synergy, whereas in the spinal state both synergies occurred concurrently. Afferent inputs also appear important for the expression of some muscle synergies, specifically those involving biphasic patterns of muscle activity. We propose that muscle synergies for locomotion are primarily organized within the spinal cord, although their full expression and proper timing requires inputs from supraspinal structures and/or limb afferents. - 'The Journal of Physiology, EarlyView. '

  November 10, 2018   doi: 10.1113/JP277018   open full text
• Evidence against a crucial role of renal medullary perfusion in blood pressure control of hypertensive rats.
  Bożena Bądzyńska, Iwona Baranowska, Olga Gawryś, Janusz Sadowski.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key points The development of new effective methods of treating arterial hypertension is hindered by uncertainty regarding its causes. According to one widespread concept hypertension is caused by abnormal blood circulation in the kidney, specifically by reduction of blood flow through the kidney medulla; however, this causal relationship has never been rigorously verified. We investigated whether in rats with three different forms of experimental hypertension prolonged selective elevation of renal medullary blood flow using local infusion of the vasodilator bradykinin would lower arterial pressure. We found that increasing medullary blood flow by almost 50% did not result in alleviation of hypertension, which argues against a causal role of such changes in the control of arterial pressure and suggests that attempts at improving renal medullary circulation are not likely to be a promising approach to combating hypertension. Abstract The crucial role of renal medullary blood flow (MBF) in the control of arterial pressure (MAP) has been widely accepted but not rigorously verified. We examined the effects of experimental selective MBF elevation on MAP, medullary tissue hypertonicity and renal excretion in hypertensive rats. We used three hypertensive rat models: (1) rats with hypertension induced by chronic angiotensin II infusions (AngII model), (2) rats with hypertension induced by unilateral nephrectomy followed by high salt diet (HS/UNX), and (3) spontaneously hypertensive rats (SHR). In acute experiments, MBF (laser‐Doppler measurement) was selectively increased with an intramedullary infusion of bradykinin (Bk) at 0.27 mg h−1 kg−1 BW over 4 h. MAP, renal artery blood flow (Transonic probe) and renal excretion parameters were measured simultaneously. In chronic studies with AngII and HS/UNX rats, Bk was infused over 2 weeks and MAP (telemetry probe) and renal excretion were repeatedly determined. In acute studies, with AngII, SHR and HS/UNX groups, Bk infusion caused a 47% increase in MBF (P < 0.01–0.001), whereas solvent infusion was without effect. During the experiments MAP decreased slightly and to the same extent with Bk and solvent infusion. Medullary tissue osmolality and [Na+] were lower in Bk‐ than in solvent‐infused AngII rats and in SHR. Two weeks of intramedullary Bk infusion tested in AngII and HS/UNX rats did not alter MAP or renal excretion; though in the latter group a significant MBF increase and medullary hypertonicity decrease was observed. Since no decrease in MAP in hypertensive rats was seen with Bk‐induced major renal medullary hyperperfusion or with a wash‐out of medullary solutes, our data argue against a crucial role of MBF in the pathogenesis of arterial hypertension. - 'The Journal of Physiology, EarlyView. '

  November 10, 2018   doi: 10.1113/JP276342   open full text
• Electrical pulse stimulation induces differential responses in insulin action in myotubes from severely obese individuals.
  Sanghee Park, Kristen D. Turner, Donghai Zheng, Jeffrey J. Brault, Kai Zou, Alec B. Chaves, Thomas S. Nielsen, Charles J. Tanner, Jonas T. Treebak, Joseph A. Houmard.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key Points Exercise/exercise training can enhance insulin sensitivity through adaptations in skeletal muscle, the primary site of insulin‐mediated glucose disposal; however, in humans the range of improvement can vary substantially. The purpose of this study was to determine if obesity influences the magnitude of the exercise response in relation to improving insulin sensitivity in human skeletal muscle. Electrical pulse stimulation (EPS) (24 h) of primary human skeletal muscle myotubes improved insulin action in tissue from both lean and severely obese individuals. However, responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise‐responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. Abstract Exercise/muscle contraction can enhance whole‐body insulin sensitivity; however, in humans the range of improvements can vary substantially. In order, to determine if obesity influences the magnitude of the exercise response, this study compared the effects of electrical pulse stimulation (EPS) ‐induced contractile activity upon primary myotubes derived from lean and severely obese (BMI ≥ 40 kg/m2) women. Prior to muscle contraction, insulin action was compromised in myotubes from the severely obese as evident by reduced insulin‐stimulated glycogen synthesis, glucose oxidation, glucose uptake, insulin signal transduction (IRS1, Akt, TBC1D4), and insulin‐stimulated GLUT4 translocation. EPS (24 h) increased AMP, IMP, AMPK Thr172 phosphorylation, PGC1α content, and insulin action in myotubes of both the lean and severely obese subjects. However, despite normalizing indices of insulin action to levels seen in the lean control (non‐EPS) condition, responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and EPS increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise‐responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 10, 2018   doi: 10.1113/JP276990   open full text
• Presynaptic loss of dynamin‐related protein 1 impairs synaptic vesicle release and recycling at the mouse calyx of Held.
  Mahendra Singh, Henry Denny, Christina Smith, Jorge Granados, Robert Renden.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key points This study characterizes the mechanisms underlying defects in synaptic transmission when dynamin‐related protein 1 (DRP1) is genetically eliminated. Viral‐mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of the synaptic vesicle recycling pool and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing the intracellular calcium buffering capacity with EGTA‐AM, implying close coupling of Ca2+ channels to synaptic vesicles was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not reverse the synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Abstract Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin‐related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of Cre recombinase in ventral cochlear neurons of floxed‐DRP1 mice generated a presynaptic‐specific DRP1 knockout (DRP1‐preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggested SV clusters and mitochondrial content were disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage‐clamp recordings, we found that DRP1‐preKO synapses had larger evoked responses at low frequency stimulation. DRP1‐preKO synapses also had profoundly altered short‐term plasticity, due to defects in SV recycling. Readily releasable pool size, estimated with high‐frequency trains, was dramatically reduced in DRP1‐preKO synapses, suggesting an important role for DRP1 in maintenance of release‐competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1‐preKO synapses. Synaptic transmission defects could be partially rescued with EGTA‐AM, indicating close coupling of Ca2+ channels to SV distance normally found in mature terminals may be compromised by DRP1‐preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation. - 'The Journal of Physiology, EarlyView. '

  November 10, 2018   doi: 10.1113/JP276424   open full text
• Visual response properties of neurons in the superficial layers of the superior colliculus of awake mouse.
  Gioia Franceschi, Samuel G. Solomon.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key points In rodents, including mice, the superior colliculus is the major target of the retina, but its visual response is not well characterized. In the present study, extracellular recordings from single nerve cells in the superficial layers of the superior colliculus were made in awake, head‐restrained mice, and their responses to visual stimuli were measured. It was found that these neurons show brisk, highly sensitive and short latency visual responses, a preference for black over white stimuli, and diverse responses to moving patterns. At least five broad classes can be defined by variation in functional properties among units. The results of the present study demonstrate that eye movements have a measurable impact on visual responses in awake animals and show how they may be mitigated in analyses. Abstract The mouse is an increasingly important animal model of visual function in health and disease. In mice, most retinal signals are routed through the superficial layers of the midbrain superior colliculus, and it is well established that much of the visual behaviour of mice relies on activity in the superior colliculus. The functional organization of visual signals in the mouse superior colliculus is, however, not well established in awake animals. We therefore made extracellular recordings from the superficial layers of the superior colliculus in awake mice, while the animals were viewing visual stimuli including flashed spots and drifting gratings. We find that neurons in the superficial layers of the superior colliculus of awake mouse generally show short latency, brisk responses. Receptive fields are usually ‘ON–OFF’ with a preference for black stimuli, and are weakly non‐linear in response to gratings and other forms of luminance modulation. Population responses to drifting gratings are highly contrast sensitive, with a robust response to spatial frequencies above 0.3 cycles degree−1 and temporal frequencies above 15 Hz. The receptive fields are also often speed‐tuned or direction‐selective. Analysis of the response across multiple stimulus dimensions reveals at least five functionally distinct groups of units. We also find that eye movements affect measurements of receptive field properties in awake animals, and show how these may be mitigated in analyses. Qualitatively similar responses were obtained in urethane‐anaesthetized animals, although receptive fields in awake animals had higher contrast sensitivity, shorter visual latency and a stronger response to high temporal frequencies. - 'The Journal of Physiology, EarlyView. '

  November 10, 2018   doi: 10.1113/JP276964   open full text
• Slow periodic activity in the longitudinal hippocampal slice can self‐propagate non‐synaptically by a mechanism consistent with ephaptic coupling.
  Chia‐Chu Chiang, Rajat S. Shivacharan, Xile Wei, Luis E. Gonzalez‐Reyes, Dominique M. Durand.
  The Journal of Physiology. November 10, 2018
  --- - |2+ Key points Slow periodic activity can propagate with speeds around 0.1 m s−1 and be modulated by weak electric fields. Slow periodic activity in the longitudinal hippocampal slice can propagate without chemical synaptic transmission or gap junctions, but can generate electric fields which in turn activate neighbouring cells. Applying local extracellular electric fields with amplitude in the range of endogenous fields is sufficient to modulate or block the propagation of this activity both in the in silico and in the in vitro models. Results support the hypothesis that endogenous electric fields, previously thought to be too small to trigger neural activity, play a significant role in the self‐propagation of slow periodic activity in the hippocampus. Experiments indicate that a neural network can give rise to sustained self‐propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions. Abstract Slow oscillations are a standard feature observed in the cortex and the hippocampus during slow wave sleep. Slow oscillations are characterized by low‐frequency periodic activity (<1 Hz) and are thought to be related to memory consolidation. These waves are assumed to be a reflection of the underlying neural activity, but it is not known if they can, by themselves, be self‐sustained and propagate. Previous studies have shown that slow periodic activity can be reproduced in the in vitro preparation to mimic in vivo slow oscillations. Slow periodic activity can propagate with speeds around 0.1 m s−1 and be modulated by weak electric fields. In the present study, we show that slow periodic activity in the longitudinal hippocampal slice is a self‐regenerating wave which can propagate with and without chemical or electrical synaptic transmission at the same speeds. We also show that applying local extracellular electric fields can modulate or even block the propagation of this wave in both in silico and in vitro models. Our results support the notion that ephaptic coupling plays a significant role in the propagation of the slow hippocampal periodic activity. Moreover, these results indicate that a neural network can give rise to sustained self‐propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions. - 'The Journal of Physiology, EarlyView. '

  November 10, 2018   doi: 10.1113/JP276904   open full text
• Action potential shortening rescues atrial calcium alternans.
  Giedrius Kanaporis, Zane M. Kalik, Lothar A. Blatter.
  The Journal of Physiology. November 09, 2018
  --- - |2+ Key points Cardiac alternans refers to a beat‐to‐beat alternation in contraction, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and represents a risk factor for cardiac arrhythmia, including atrial fibrillation. We developed strategies to pharmacologically manipulate the AP waveform with the goal to reduce or eliminate the occurrence of CaT and contraction alternans in atrial tissue. With combined patch‐clamp and intracellular Ca2+ measurements we investigated the effect of specific ion channel inhibitors and activators on alternans. In single rabbit atrial myocytes suppression of Ca2+‐activated Cl− channels eliminated AP duration alternans, but prolonged the AP and failed to eliminate CaT alternans. In contrast, activation of K+ currents (IKs and IKr) shortened the AP and eliminated both AP duration and CaT alternans. As demonstrated also at the whole heart level activation of K+ conductances represents a promising strategy to suppress alternans, thus reducing a risk factor for atrial fibrillation. Abstract At the cellular level alternans is observed as beat‐to‐beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). Alternans is a well established risk factor for cardiac arrhythmia, including atrial fibrillation. This study investigates whether pharmacological manipulation of AP morphology is a viable strategy to reduces the risk of arrhythmogenic CaT alternans. Pacing‐induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. Increased AP duration (APD) and beat‐to‐beat alternations in AP morphology lowered the pacing frequency threshold and increased the degree of CaT alternans. Inhibition of Ca2+‐activated Cl− channels reduced beat‐to‐beat AP alternations, but prolonged APD and failed to suppress CaT alternans. In contrast, AP shortening induced by activators of two K+ channels (ML277 for Kv7.1 and NS1643 for Kv11.1) abolished both APD and CaT alternans in field‐stimulated and current‐clamped myocytes. K+ channel activators had no effect on the degree of Ca2+ alternans in voltage‐clamped cells, confirming that suppression of Ca2+ alternans was caused by the changes in AP morphology. Finally, activation of Kv11.1 channel significantly attenuated or even abolished atrial T‐wave alternans in isolated Langendorff perfused hearts. In summary, AP shortening suppressed or completely eliminated both CaT and APD alternans in single atrial myocytes and atrial T‐wave alternans at whole heart level. Therefore, we suggest that AP shortening is a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 09, 2018   doi: 10.1113/JP277188   open full text
• Exendin‐4 Overcomes Cytokine‐Induced Decreases in Gap Junction Coupling via PKA and Epac2 in Mouse and Human Islets.
  Nikki L. Farnsworth, Rachelle Walter, Robert Piscopio, Wolfgang E. Schleicher, Richard K. P. Benninger.
  The Journal of Physiology. November 09, 2018
  --- - |2+ Key Points Summary The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signaling and electrical coupling of the insulin producing β‐cells in the islet We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling In both mouse and human islets Exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signaling by increasing connexin36 gap junction levels on the plasma membrane Our data indicates that PKA regulates β‐cell coupling through a fast mechanism such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover Increases in β‐cell coupling with Exendin‐4 may protect against cytokine mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes Abstract The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β‐cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β‐cells, regulating calcium signaling and insulin secretion dynamics. Cx36 coupling is decreased in pre‐diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro‐inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if Exendin‐4, which increases cAMP, can protect against cytokine‐induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets Exendin‐4 protected against cytokine‐induced decreases in coupling and preserved glucose‐stimulated calcium signaling. Exendin‐4 also protected against protein kinase C delta‐mediated decreases in Cx36 coupling. Exendin‐4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin‐4 regulated Cx36 coupling via both PKA and Epac2 mediated mechanisms in cytokine treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicates that PKA regulates Cx36 coupling through a fast mechanism such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with Exendin‐4 may protect against cytokine mediated β‐cell dysfunction to insulin secretion dynamics during the development of diabetes. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 09, 2018   doi: 10.1113/JP276106   open full text
• Enhanced availability of serotonin increases activation of unfatigued muscle but exacerbates central fatigue during prolonged sustained contractions.
  Justin J. Kavanagh, Amelia J. McFarland, Janet L. Taylor.
  The Journal of Physiology. November 08, 2018
  --- - |2+ Key points Animal preparations have revealed that moderate synaptic release of serotonin (5‐HT) onto motoneurones enhances motor activity via activation of 5‐HT2 receptors, whereas intense release of 5‐HT causes spillover of 5‐HT to extrasynaptic 5‐HT1A receptors on the axon initial segment to reduce motoneurone activity. We explored if increasing extracellular concentrations of endogenously released 5‐HT (via the selective serotonin reuptake inhibitor paroxetine) influences the ability to perform unfatigued and fatigued maximal voluntary contractions in humans. Following the ingestion of paroxetine, voluntary muscle activation and torque generation increased during brief unfatigued maximal contractions. In contrast, the ability to generate maximal torque with increased 5‐HT availability was compromised under fatigued conditions, which was consistent with paroxetine‐induced reductions in motoneurone excitability and voluntary muscle activation. This is the first in vivo human study to provide evidence that 5‐HT released onto the motoneurones could play a role in central fatigue. Abstract Brief stimulation of the raphe–spinal pathway in the turtle spinal cord releases serotonin (5‐HT) onto motoneurones to enhance excitability. However, intense release of 5‐HT via prolonged stimulation results in 5‐HT spillover to the motoneurone axon initial segment to activate inhibitory 5‐HT1A receptors, thus providing a potential spinal mechanism for exercise‐induced central fatigue. We examined how increased extracellular concentrations of 5‐HT affect the ability to perform brief, as well as sustained, maximal voluntary contractions (MVCs) in humans. Paroxetine was used to enhance 5‐HT concentrations by reuptake inhibition, and three studies were performed. Study 1 (n = 14) revealed that 5‐HT reuptake inhibition caused an ∼4% increase in elbow flexion MVC. However, when maximal contractions were sustained, time‐to‐task failure was reduced and self‐perceived fatigue was higher with enhanced availability of 5‐HT. Study 2 (n = 11) used twitch interpolation to reveal that 5‐HT‐based changes in motor performance had a neural basis. Enhanced 5‐HT availability increased voluntary activation for the unfatigued biceps brachii and decreased voluntary activation of the biceps brachii by 2–5% following repeated maximal elbow flexions. The final study (n = 8) investigated whether altered motoneurone excitability may contribute to 5‐HT changes in voluntary activation. F‐waves of the abductor digiti minimi (ADM) were unaffected by paroxetine for unfatigued muscle and marginally affected following a brief 2‐s MVC. However, F‐wave area and persistence were significantly decreased following a prolonged 60‐s MVC of the ADM. Overall, high serotonergic drive provides a spinal mechanism by which higher concentrations of 5‐HT may contribute to central fatigue. - 'The Journal of Physiology, EarlyView. '

  November 08, 2018   doi: 10.1113/JP277148   open full text
• Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis.
  Jiunn‐Tyng Yeh, Ying‐Chun Yu, Tzyh‐Chang Hwang.
  The Journal of Physiology. November 08, 2018
  --- - |2+ Key points Electrophysiological characterization of Q1412X‐CFTR, a C‐terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) that is associated with the severe form of cystic fibrosis (CF), reveals a gating defect not reported previously. Mechanistic investigations of the gating deficit in Q1412X‐CFTR suggest that the reduced open probability in Q1412X‐CFTR is due to a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C‐terminal region of NBD2 reveal the importance of the last two beta‐strands in NBD2 in maintaining proper gating functions. Our studies also show that the application of clinically‐approved drugs (VX‐770 and VX‐809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. Abstract Cystic fibrosis (CF) is caused by loss‐of‐function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation‐activated but ATP‐gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease‐associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C‐terminal truncation mutations such as Q1412X, is defined as decreased membrane expression due to a faster turnover rate. In this study, we characterized the functional properties of Q1412X‐CFTR, a severe‐form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po). Detailed kinetic studies support the idea that the gating defect is due to a dysfunctional ATP‐binding site 2 in the nucleotide binding domains (NBDs). Since the Q1412X mutation results in a deletion of the last two beta‐strands in NBD2 and the whole C‐terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C‐terminus of CFTR while keeping an intact NBD2 (i.e., D1425X and S1455X) assume gating function nearly identical to that of wild‐type channels. However, the deletion of the last beta‐strand in the NBD2 (i.e., N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that the clinically‐approved VX‐809 (Lumacaftor) and VX‐770 (Ivacaftor) significantly enhance the overall function of Q1412X‐CFTR provides the conceptual basis for the treatment of patients carrying this mutation. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 08, 2018   doi: 10.1113/JP277042   open full text
• RAAS inhibitors directly reduce diabetes‐induced renal fibrosis via growth factor inhibition.
  Sandor Koszegi, Agnes Molnar, Lilla Lenart, Judit Hodrea, Dora Bianka Balogh, Tamas Lakat, Edgar Szkibinszkij, Adam Hosszu, Nadja Sparding, Federica Genovese, Laszlo Wagner, Adam Vannay, Attila J Szabo, Andrea Fekete.
  The Journal of Physiology. November 02, 2018
  --- - |2+ Key points Increased activation of the renin‐angiotensin‐aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes‐induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia‐induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis. Abstract In diabetic kidney disease (DKD) increased activation of renin‐angiotensin‐aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin‐induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet‐derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)‐induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha‐smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK‐2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia‐induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non‐antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis. - 'The Journal of Physiology, EarlyView. '

  November 02, 2018   doi: 10.1113/JP277002   open full text
• The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain.
  Jun‐Nan Li, Patrick L. Sheets.
  The Journal of Physiology. November 02, 2018
  --- - |2+ Key points The central nucleus of the amygdala (CeA) encompasses the main output pathways of the amygdala, a temporal lobe structure essential in affective and cognitive dimensions of pain. A major population of neurons in the CeA send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. CeA‐PAG neurons are topographically organized based on their targeted subregion within the PAG. PAG‐projecting neurons in the central medial (CeM) and central lateral (CeL) regions of CeA are intrinsically distinct. CeL‐PAG neurons are a homogeneous population of intrinsically distinct neurons while CeM‐PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM‐PAG subtypes are altered in the complete Freund's adjuvant model of inflammatory pain. Abstract A major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA‐PAG pathway has proved to be a component of descending anti‐nociceptive circuitry, the functional organization of CeA‐PAG neurons remains unclear. We identified CeA‐PAG neurons in C57BL/6 mice of both sexes using intracranial injection of a fluorescent retrograde tracer into the PAG. In acute brain slices, we investigated the topographical and intrinsic characteristics of retrogradely labelled CeA‐PAG neurons using epifluorescence and whole‐cell electrophysiology. We also measured changes to CeA‐PAG neurons in the complete Freund's adjuvant (CFA) model of inflammatory pain. Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL‐PAG neurons consist of a relatively homogeneous population of intrinsically distinct neurons while CeM‐PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM‐PAG subtypes are altered 1 day after induction of the CFA inflammatory pain model. Collectively, our results provide insight into pain‐induced changes to a specific population of CeA neurons that probably play a key role in the integration of noxious input with endogenous analgesia and behavioural coping response. - 'The Journal of Physiology, EarlyView. '

  November 02, 2018   doi: 10.1113/JP276935   open full text
• The dynamics of cortical GABA in human motor learning.
  James Kolasinski, Emily L. Hinson, Amir P. Divanbeighi Zand, Assen Rizov, Uzay E. Emir, Charlotte J. Stagg.
  The Journal of Physiology. November 02, 2018
  --- - |2+ Key points The ability to learn new motor skills is supported by plasticity in the structural and functional organisation of the primary motor cortex in the human brain. Changes inhibitory to signalling by GABA are thought to be crucial in inducing motor cortex plasticity. This study used magnetic resonance spectroscopy (MRS) to quantify the concentration of GABA in human motor cortex during a period of motor learning, as well as during a period of movement and a period at rest. We report evidence for a reduction in the MRS‐measured concentration of GABA specific to learning. Further, the GABA concentration early in the learning task was strongly correlated with the magnitude of subsequent learning: higher GABA concentrations were associated with poorer learning. The results provide initial insight into the neurochemical correlates of cortical plasticity associated with motor learning, specifically relevant in therapeutic efforts to induce cortical plasticity during recovery from stroke. Abstract The ability to learn novel motor skills is a central part of our daily lives and can provide a model for rehabilitation after a stroke. However, there are still fundamental gaps in our understanding of the physiological mechanisms that underpin human motor plasticity. The acquisition of new motor skills is dependent on changes in local circuitry within the primary motor cortex (M1). This reorganisation has been hypothesised to be facilitated by a decrease in local inhibition via modulation of the neurotransmitter GABA, but this link has not been conclusively demonstrated in humans. Here, we used 7 T magnetic resonance spectroscopy to investigate the dynamics of GABA concentrations in human M1 during the learning of an explicit, serial reaction time task. We observed a significant reduction in GABA concentration during motor learning that was not seen in an equivalent motor task lacking a learnable sequence, nor during a passive resting task of the same duration. No change in glutamate was observed in any group. Furthermore, M1 GABA measured early in task performance was strongly correlated with the degree of subsequent learning, such that greater inhibition was associated with poorer subsequent learning. This result suggests that higher levels of cortical inhibition may present a barrier that must be surmounted in order to achieve an increase in M1 excitability, and hence encoding of a new motor skill. These results provide strong support for the mechanistic role of GABAergic inhibition in motor plasticity, raising questions regarding the link between population variability in motor learning and GABA metabolism in the brain. - 'The Journal of Physiology, EarlyView. '

  November 02, 2018   doi: 10.1113/JP276626   open full text
• PKCδ constrains the S‐pathway to phrenic motor facilitation elicited by spinal 5‐HT7 receptors or severe acute intermittent hypoxia​.
  Raphael R. Perim, Daryl P. Fields, Gordon S. Mitchell.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key Points Summary Concurrent 5‐HT2A (Q pathway) and 5‐HT7 (S pathway) serotonin receptor activation cancels phrenic motor facilitation due to mutual cross‐talk inhibition Spinal PKCδ or PKA inhibition restores phrenic motor facilitation with concurrent Q and S pathway activation, demonstrating a key role for these kinases in cross‐talk inhibition Spinal PKCδ inhibition enhances adenosine‐dependent severe AIH‐induced pLTF (S pathway), consistent with relief of cross‐talk inhibition. Abstract Intermittent spinal serotonin receptor activation elicits long‐lasting phrenic motor facilitation (pMF), a form of respiratory motor plasticity. When activated alone, spinal Gq protein‐coupled serotonin 2A receptors (5‐HT2A) initiate pMF by a mechanism that requires ERK‐MAP kinase signaling and new BDNF protein synthesis (Q pathway). Spinal Gs protein‐coupled serotonin 7 (5‐HT7) and adenosine 2A (A2A) receptor activation also elicits pMF, but via distinct mechanisms (S pathway) that require Akt signaling and new TrkB protein synthesis. Although studies have shown inhibitory cross‐talk interactions between these competing pathways, underlying cellular mechanisms are unknown. Hypothesis: a) concurrent 5‐HT2A and 5‐HT7 activation undermines pMF; b) protein kinase A (PKA); and c) NADPH oxidase mediate inhibitory interactions between Q (5‐HT2A) and S (5‐HT7) pathways. Selective 5‐HT2A (DOI‐hydrochloride) and 5HT7 (AS‐19) agonists were administered intrathecally at C4 (3 injections, 5‐min intervals) in anesthetized, vagotomized and ventilated male rats. With either spinal 5‐HT2A or 5‐HT7 activation alone, phrenic amplitude progressively increased (pMF). In contrast, concurrent 5‐HT2A and 5‐HT7 activation failed to elicit pMF. 5‐HT2A‐induced Q pathway was restored by inhibiting PKA activity (Rp‐8‐Br‐cAMPS). NADPH oxidase inhibition did not prevent cross‐talk inhibition. Therefore, we investigated alternative mechanisms to explain Q to S pathway inhibition. Spinal PKC inhibition with Gö6983 or PKCδ peptide inhibitor restored 5‐HT7‐induced S pathway to pMF, revealing PKCδ as the relevant isoform. Spinal PKCδ inhibition enhanced S pathway‐dependent form of pMF elicited by severe AIH. We suggest that powerful constraints between 5‐HT2A and 5‐HT7 or A2A receptor‐induced pMF are mediated by PKCδ and PKA, respectively. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '

  November 01, 2018   doi: 10.1113/JP276731   open full text
• Complex and spatially segregated auditory inputs of the mouse superior colliculus.
  Veronika Bednárová, Benedikt Grothe, Michael H. Myoga.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Although the visual circuits in the superior colliculus (SC) have been thoroughly examined, the auditory circuits lack equivalent scrutiny. SC neurons receiving auditory inputs in mice were characterized and three distinguishable types of neurons were found. The auditory pathways from external nuclei of the inferior colliculus (IC) were characterized, and a novel direct inhibitory connection and an excitation that drives feed‐forward inhibitory circuits within the SC were found. The direct excitatory and inhibitory inputs exhibited distinct arbourization patterns in the SC. These findings suggest functional differences between excitatory and inhibitory sensory information that targets the auditory SC. Abstract The superior colliculus (SC) is a midbrain structure that integrates auditory, somatosensory and visual inputs to drive orientation movements. While much is known about how visual information is processed in the superficial layers of the SC, little is known about the SC circuits in the deep layers that process auditory inputs. We therefore characterized intrinsic neuronal properties in the auditory‐recipient layer of the SC (stratum griseum profundum; SGP) and confirmed three electrophysiologically defined clusters of neurons, consistent with literature from other SC layers. To determine the types of inputs to the SGP, we expressed Channelrhodopsin‐2 in the nucleus of the brachium of the inferior colliculus (nBIC) and external cortex of the inferior colliculus (ECIC) and optically stimulated these pathways while recording from SGP neurons. Probing the connections in this manner, we described a monosynaptic excitation that additionally drives feed‐forward inhibition via circuits intrinsic to the SC. Moreover, we found a profound long‐range monosynaptic inhibition in 100% of recorded SGP neurons, a surprising finding considering that only about 15% of SGP‐projecting neurons in the nBIC/ECIC are inhibitory. Furthermore, we found spatial differences in the cell body locations as well as axon trajectories between the monosynaptic excitatory and inhibitory inputs, suggesting that these inputs may be functionally distinct. Taking this together with recent anatomical evidence suggesting an auditory excitation from the nBIC and a GABAergic multimodal inhibition from the ECIC, we propose that sensory integration in the SGP is more multifaceted than previously thought. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5281-5298, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276370   open full text
• Skeletal muscle ceramides and relationship with insulin sensitivity after 2 weeks of simulated sedentary behaviour and recovery in healthy older adults.
  Paul T. Reidy, Alec I. McKenzie, Ziad Mahmassani, Vincent R. Morrow, Nikol M. Yonemura, Paul N. Hopkins, Robin L. Marcus, Matthew T. Rondina, Yu Kuei Lin, Micah J. Drummond.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Insulin sensitivity (as determined by a hyperinsulinaemic‐euglyceamic clamp) decreased 15% after reduced activity. Despite not fully returning to baseline physical activity levels, insulin sensitivity unexpectedly, rebounded above that recorded before 2 weeks of reduced physical activity by 14% after the recovery period. Changes in insulin sensitivity in response to reduced activity were primarily driven by men but, not women. There were modest changes in ceramides (nuclear/myofibrillar fraction and serum) following reduced activity and recovery but, in the absence of major changes to body composition (i.e. fat mass), ceramides were not related to changes in inactivity‐induced insulin sensitivity in healthy older adults. Abstract Older adults are at risk of physical inactivity as they encounter debilitating life events. It is not known how insulin sensitivity is affected by modest short‐term physical inactivity and recovery in healthy older adults, nor how insulin sensitivity is related to changes in serum and muscle ceramide content. Healthy older adults (aged 64–82 years, five females, seven males) were assessed before (PRE), after 2 weeks of reduced physical activity (RA) and following 2 weeks of recovery (REC). Insulin sensitivity (hyperinsulinaemic‐euglyceamic clamp), lean mass, muscle function, skeletal muscle subfraction, fibre‐specific, and serum ceramide content and indices of skeletal muscle inflammation were assessed. Insulin sensitivity decreased by 15 ± 6% at RA (driven by men) but rebounded above PRE by 14 ± 5% at REC. Mid‐plantar flexor muscle area and leg strength decreased with RA, although only muscle size returned to baseline levels following REC. Body fat did not change and only minimal changes in muscle inflammation were noted across the intervention. Serum and intramuscular ceramides (nuclear/myofibrillar fraction) were modestly increased at RA and REC. However, ceramides were not related to changes in inactivity‐induced insulin sensitivity in healthy older adults. Short‐term inactivity induced insulin resistance in older adults in the absence of significant changes in body composition (i.e. fat mass) are not related to changes in ceramides. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5217-5236, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276798   open full text
• Increased human stretch reflex dynamic sensitivity with height‐induced postural threat.
  Brian C. Horslen, Martin Zaback, J. Timothy Inglis, Jean‐Sébastien Blouin, Mark G. Carpenter.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Threats to standing balance (postural threat) are known to facilitate soleus tendon‐tap reflexes, yet the mechanisms driving reflex changes are unknown. Scaling of ramp‐and‐hold dorsiflexion stretch reflexes to stretch velocity and amplitude were examined as indirect measures of changes to muscle spindle dynamic and static function with height‐induced postural threat. Overall, stretch reflexes were larger with threat. Furthermore, the slope (gain) of the stretch‐velocity vs. short‐latency reflex amplitude relationship was increased with threat. These findings are interpreted as indirect evidence for increased muscle spindle dynamic sensitivity, independent of changes in background muscle activity levels, with a threat to standing balance. We argue that context‐dependent scaling of stretch reflexes forms part of a multisensory tuning process where acquisition and/or processing of balance‐relevant sensory information is continuously primed to facilitate feedback control of standing balance in challenging balance scenarios. Abstract Postural threat increases soleus tendon‐tap (t‐) reflexes. However, it is not known whether t‐reflex changes are a result of central modulation, altered muscle spindle dynamic sensitivity or combined spindle static and dynamic sensitization. Ramp‐and‐hold dorsiflexion stretches of varying velocities and amplitudes were used to examine velocity‐ and amplitude‐dependent scaling of short‐ (SLR) and medium‐latency (MLR) stretch reflexes as an indirect indicator of spindle sensitivity. t‐reflexes were also performed to replicate previous work. In the present study, we examined the effects of postural threat on SLR, MLR and t‐reflex amplitude, as well as SLR‐stretch velocity scaling. Forty young‐healthy adults stood with one foot on a servo‐controlled tilting platform and the other on a stable surface. The platform was positioned on a hydraulic lift. Threat was manipulated by having participants stand in low (height 1.1 m; away from edge) then high (height 3.5 m; at the edge) threat conditions. Soleus stretch reflexes were recorded with surface electromyography and SLRs and MLRs were probed with fixed‐amplitude variable‐velocity stretches. t‐reflexes were evoked with Achilles tendon taps using a linear motor. SLR, MLR and t‐reflexes were 11%, 9.5% and 16.9% larger, respectively, in the high compared to low threat condition. In 22 out of 40 participants, SLR amplitude was correlated to stretch velocity at both threat levels. In these participants, the gain of the SLR–velocity relationship was increased by 36.1% with high postural threat. These findings provide new supportive evidence for increased muscle spindle dynamic sensitivity with postural threat and provide further support for the context‐dependent modulation of human somatosensory pathways. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5251-5265, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276459   open full text
• Neural memory of the genioglossus muscle during sleep is stage‐dependent in healthy subjects and obstructive sleep apnoea patients.
  Luigi Taranto‐Montemurro, Scott A. Sands, Kevin P. Grace, Ali Azarbarzin, Ludovico Messineo, Rebecca Salant, David P. White, D. Andrew Wellman.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points In most patients with obstructive sleep apnoea (OSA), there is a spontaneous resolution of the breathing disorders during slow wave sleep (SWS) for yet unknown reasons related to non‐anatomical factors. Some recently identified forms of neural memory specific of upper airway muscles may play a role in this phenomenon. In the present study, we show for the first time that a form of memory of the genioglossus (tongue) muscle is greatly enhanced during SWS compared to non‐rapid eye movement stage 2 sleep. The present study represents a step forward in understanding the mechanisms responsible for the spontaneous development of stable breathing during SWS in OSA patients and may help the discovery of novel therapeutic strategies for this disease. Abstract Several studies have shown that obstructive sleep apnoea (OSA) improves during slow wave sleep (SWS) for reasons that remain unclear. Recent studies have identified forms of neural memory such as short‐term potentiation or after‐discharge that can occur in response to upper airway obstruction. Neural memory may play a role in the development of stable breathing during SWS by increasing upper airway muscles activity in this sleep stage. We hypothesize that the after‐discharge of the genioglossus muscle following upper airway obstruction is enhanced during SWS compared to non‐rapid eye movement stage 2 (N2). During sleep, we performed five‐breath drops in continuous positive airway pressure (CPAP‐drop) to simulate obstructive events and reflexively activate the genioglossus. Immediately afterwards, CPAP was returned to an optimal level. Once the post‐drop ventilation returned to eupnoea, the genioglossus after‐discharge was measured as the time it took for genioglossus activity to return to baseline levels. In total, 171 CPAP‐drops were analysed from a group of 16 healthy subjects and 19 OSA patients. A mixed‐model analysis showed that after‐discharge duration during SWS was 208% (95% confidence interval = 112% to 387%, P = 0.022) greater than during N2 after adjusting for covariates (ventilatory drive, CPAP levels). There was also a non‐significant trend for a –35% reduction in after‐discharge duration following an arousal vs. no‐arousal from sleep (95% confidence interval = –59.5% to 5%, P = 0.08). Genioglossus after‐discharge is two‐fold greater in SWS vs. N2, which could partly explain the breathing stabilization described in OSA patients during this sleep stage. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5163-5173, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276618   open full text
• Ageing changes in biventricular cardiac function in male and female baboons (Papio spp.).
  Anderson H. Kuo, Cun Li, Hillary F. Huber, Peter W. Nathanielsz, Geoffrey D. Clarke.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Life course changes in cardiovascular function in a non‐human primate have been comprehensively characterized. Age‐related declines in normalized left ventricular stroke volume and cardiac output were found with corresponding decreases in biventricular ejection fractions and filling rates. There were age‐related decreases in male and female baboon normalized left ventricular myocardial mass index, which declined at similar rates. Systolic functional declines in right ventricular function were observed with age, similar to the left ventricle. Sex differences were found in the rates and directions of right ventricular volume changes along with decreased end‐systolic right ventricular sphericity. The results validate the baboon as an appropriate model for translational studies of cardiovascular functional decline with ageing. Abstract Previous studies reported cardiac function declines with ageing. This study determined changes in biventricular cardiac function in a well‐characterized baboon model. Cardiac magnetic resonance imaging measured key biventricular parameters in 47 baboons (22 female, age 4–23 years). ANCOVA assessed sex and age changes with P < 0.05 deemed significant. Stroke volume, cardiac output and other cardiac functional parameters were normalized to body surface area. There were similar, age‐related rates of decrease in male (M) and female (F) normalized left ventricular (LV) myocardial mass index (M: −1.2 g m−2 year−1, F: −0.9 g m−2 year−1). LV ejection fraction declined at −0.96% year−1 (r = −0.43, P = 0.002) and right ventricular (RV) ejection fraction decreased at −1.2% year−1 (r = −0.58, P < 0.001). Normalized LV stroke volume fell at −1.1 ml m−2 year−1 (r = −0.47, P = 0.001), normalized LV ejection rate at −3.8 ml s−1 m−2 year−1 (r = −0.43, P < 0.005) and normalized LV filling rate at −4.1 ml s−1 m−2 year−1 (r = −0.44, P < 0.005). Also, RV wall thickening fraction decreased with age (slope = −1% year−1, P = 0.008). RV ejection rate decreased at −3.6 ml s−1 m−2 year−1 (P = 0.002) and the normalized average RV filling rate dropped at −3.7 ml s−1 m−2 year−1 (P < 0.0001). End‐systolic RV sphericity index also dropped with age (r = −0.33, P = 0.02). Many observed changes parallel previously reported data in human and animal studies. These measured biventricular functional declines in hearts with ageing from the closest experimental primate species to man underscore the utility of the baboon model for investigating mechanisms related to heart ageing. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5083-5098, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276338   open full text
• TRPV1 and BDKRB2 receptor polymorphisms can influence the exercise pressor reflex.
  Karambir Notay, Shannon L. Klingel, Jordan B. Lee, Connor J. Doherty, Jeremy D. Seed, Michal Swiatczak, David M. Mutch, Philip J. Millar.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points The mechanisms responsible for the high inter‐individual variability in blood pressure responses to exercise remain unclear. Common genetic variants of genes related to the vascular transduction of sympathetic outflow have been investigated, but variants influencing skeletal muscle afferent feedback during exercise have not been explored. Single nucleotide polymorphisms in TRPV1 rs222747 and BDKRB2 rs1799722 receptors present in skeletal muscle were associated with differences in the magnitude of the blood pressure response to static handgrip exercise but not mental stress. The combined effects of TRPV1 rs222747 and BDKRB2 rs1799722 on blood pressure and heart rate responses during exercise were additive, and primarily found in men. Genetic differences in skeletal muscle metaboreceptors may be a risk factor for exaggerated blood pressure responses to exercise. Abstract Exercise blood pressure (BP) responses demonstrate high inter‐individual variability, which could relate to differences in metabolically sensitive afferent feedback from the exercising muscle. We hypothesized that single‐nucleotide polymorphisms (SNPs) in genes encoding metaboreceptors present in group III/IV skeletal muscle afferents can influence the exercise pressor response. Two hundred men and women underwent measurements of continuous BP and heart rate at baseline and during 2 min of static handgrip exercise (30% maximal volitional contraction), post‐exercise circulatory occlusion and mental stress (serial subtraction; internal control). Participants were genotyped for SNPs in TRPV1 (rs222747; G/C), ASIC3 (rs2288645; G/A), BDKRB2 (rs1799722; C/T), PTGER2 (rs17197; A/G) and P2RX4 (rs25644; A/G). Exercise systolic BP (19 ± 10 vs. 22 ± 10 mmHg, P = 0.03) was lower in GG versus GC/CC minor allele carriers for TRPV1 rs222747, while exercise diastolic BP (14 ± 7 vs. 17 ± 7 mmHg, P = 0.007) and heart rate (12 ± 8 vs. 15 ± 9 beats min−1, P = 0.03) were lower in CC versus CT/TT minor allele carriers for BDKRB2 rs1799722. Individuals carrying both minor alleles for TRPV1 rs222747 and BDKRB2 rs1799722 had greater systolic (22 ± 11 vs. 17 ± 10 mmHg, P = 0.04) and diastolic (18 ± 7 vs. 14 ± 7 mmHg, P = 0.01) BP responses than those with no minor alleles; these differences were larger in men. No differences in BP or heart rate responses were detected during static handgrip with ASIC3 rs2288645, PTGER2 rs17197 or P2RX4 rs25644. None of the selected SNPs were associated with differences during mental stress. These findings demonstrate that variants in TRPV1 and BDKRB2 receptors can contribute to BP differences during static exercise in an additive manner. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5135-5148, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276526   open full text
• Synaptic entrainment of ectopic action potential generation in hippocampal pyramidal neurons.
  Christian Thome, Fabian C. Roth, Joshua Obermayer, Antonio Yanez, Andreas Draguhn, Alexei V. Egorov.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Ectopic action potentials (EAPs) arise at distal locations in axonal fibres and are often associated with neuronal pathologies such as epilepsy or nerve injury, but they also occur during physiological network conditions. This study investigates whether initiation of such EAPs is modulated by subthreshold synaptic activity. Somatic subthreshold potentials invade the axonal compartment to considerable distances (>350 μm), whereas spread of axonal subthreshold potentials to the soma is inefficient. Ectopic spike generation is entrained by conventional synaptic signalling mechanisms. Excitatory synaptic potentials promote EAPs, whereas inhibitory synaptic potentials block EAPs. The modulation of ectopic excitability depends on propagation of somatic voltage deflections to the axonal EAP initiation site. Synaptic modulation of EAP initiation challenges the view of the distal axon being independent of synaptic activity and may contribute to mechanisms underlying fast network oscillations and pathological network activity. Abstract While most action potentials are generated at the axon initial segment, they can also be triggered at more distal sites along the axon. Such ectopic action potentials (EAPs) occur during several neuronal pathologies such as epilepsy, nerve injuries and inflammation but have also been observed during physiological network activity. EAPs propagate antidromically towards the somato‐dendritic compartment where they modulate synaptic plasticity. Here we investigate the converse signal direction: do somato‐dendritic synaptic potentials affect the generation of ectopic spikes? We measured anti‐ and orthodromic spikes in the soma and axon of mouse hippocampal CA1 pyramidal cells. We found that synaptic potentials propagate reliably through the axon, causing significant voltage transients at distances >350 μm. At these sites, excitatory input efficiently facilitated EAP initiation in distal axons and, conversely, inhibitory input suppressed EAP initiation. Our data reveal a new mechanism by which ectopically generated spikes can be entrained by conventional synaptic signalling during normal and pathological network activity. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5237-5249, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276720   open full text
• Nitric oxide‐dependent attenuation of noradrenaline‐induced vasoconstriction is impaired in the canine model of Duchenne muscular dystrophy.
  Kasun Kodippili, Chady H. Hakim, Hsiao T. Yang, Xiufang Pan, N. Nora Yang, Maurice H. Laughlin, Ronald L. Terjung, Dongsheng Duan.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS‐derived NO‐dependent and NO‐independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO‐independent sympatholysis was not affected in the canine DMD model. Abstract The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane‐associated nNOS to blood flow regulation in dystrophin‐deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO‐independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5199-5216, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP275672   open full text
• Feedforward‐ and motor effort‐dependent increase in prefrontal oxygenation during voluntary one‐armed cranking.
  Kei Ishii, Nan Liang, Ryota Asahara, Makoto Takahashi, Kanji Matsukawa.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Some cortical areas are believed to transmit a descending signal in association with motor intention and/or effort that regulates the cardiovascular system during exercise (termed central command). However, there was no evidence for the specific cortical area responding prior to arbitrary motor execution and in proportion to the motor effort. Using a multichannel near‐infrared spectroscopy system, we found that the oxygenation of the dorsolateral and ventrolateral prefrontal cortices on the right side increases in a feedforward‐ and motor effort‐dependent manner during voluntary one‐armed cranking with the right arm. This finding may suggest a role of the dorsolateral and ventrolateral prefrontal cortices in triggering off central command and may help us to understand impaired regulation of the cardiovascular system in association with lesion of the prefrontal cortex. Abstract Output from higher brain centres (termed central command) regulates the cardiovascular system during exercise in a feedforward‐ and motor effort‐dependent manner. This study aimed to determine a cortical area responding prior to arbitrarily started exercise and in proportion to the effort during exercise. The oxygenation responses in the frontal and frontoparietal areas during one‐armed cranking with the right arm were measured using multichannel near‐infrared spectroscopy, as indexes of regional blood flow responses, in 20 subjects. The intensity of voluntary exercise was 30% and 60% of the maximal voluntary effort (MVE). At the start period of both voluntary cranking tasks, the oxygenation increased (P < 0.05) only in the lateral and dorsal part of the dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex (VLPFC) and sensorimotor cortices. Then, the oxygenation increased gradually in all cortical areas during cranking at 60% MVE, while oxygenation increased only in the frontoparietal area and some of the frontal area during cranking at 30% MVE. The rating of perceived exertion to the cranking tasks correlated (P < 0.05) with the oxygenation responses on the right side of the lateral‐DLPFC (r = 0.46) and VLPFC (r = 0.48) and the frontopolar areas (r = 0.47–0.49). Motor‐driven passive one‐armed cranking decreased the oxygenation in most cortical areas, except the contralateral frontoparietal areas. Accordingly, the lateral‐DLPFC and VLPFC on the right side would respond in a feedforward‐ and motor effort‐dependent manner during voluntary exercise with the right arm. Afferent inputs from mechanosensitive afferents may decrease the cortical oxygenation. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5099-5118, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276956   open full text
• Altered anabolic signalling and reduced stimulation of myofibrillar protein synthesis after feeding and resistance exercise in people with obesity.
  Joseph W. Beals, Sarah K. Skinner, Colleen F. McKenna, Elizabeth G. Poozhikunnel, Samee A. Farooqi, Stephan Vliet, Isabel G. Martinez, Alexander V. Ulanov, Zhong Li, Scott A. Paluska, Nicholas A. Burd.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Lifestyle modifications that include the regular performance of exercise are probably important for counteracting the negative consequences of obesity on postprandial myofibrillar protein synthetic responses to protein dense food ingestion. We show that the interactive effect of resistance exercise and feeding on the stimulation of myofibrillar protein synthesis rates is diminished with obesity compared to normal weight adults. The blunted myofibrillar protein synthetic response with resistance exercise in people with obesity may be underpinned by alterations in muscle anabolic signalling phosphorylation (p70S6K and 4E‐BP1). The results obtained in the present study suggest that further exercise prescription manipulation may be necessary to optimize post‐exercise myofibrillar protein synthesis rates in adults with obesity. Abstract We aimed to determine whether obesity alters muscle anabolic and inflammatory signalling phosphorylation and also muscle protein synthesis within the myofibrillar (MYO) and sarcoplasmic (SARC) protein fractions after resistance exercise. Nine normal weight (NW) (21 ± 1 years, body mass index 22 ± 1 kg m−2) and nine obese (OB) (22 ± 1 years, body mass index 36 ± 2 kg m−2) adults received l‐[ring‐13C6]phenylalanine infusions with blood and muscle sampling at basal and fed‐state of the exercise (EX) and non‐exercise (CON) legs. Participants performed unilateral leg extensions and consumed pork (36 g of protein) immediately after exercise. Basal muscle Toll‐like receptor 4 (TLR4) protein was similar between OB and NW groups (P > 0.05) but increased at 300 min after pork ingestion only in the OB group (P = 0.03). Resistance exercise reduced TLR4 protein in the OB group at 300 min (EX vs. CON leg in OB: P = 0.04). Pork ingestion increased p70S6K phosphorylation at 300 min in CON and EX of the OB and NW groups (P > 0.05), although the response was lower in the EX leg of OB vs. NW at 300 min (P = 0.05). Basal MYO was similar between the NW and OB groups (P > 0.05) and was stimulated by pork ingestion in the EX and CON legs in both groups (Δ from basal NW: CON 0.04 ± 0.01% h−1; EX 0.10 ± 0.02% h−1; OB: CON 0.06 ± 0.01% h−1; EX 0.06 ± 0.01% h−1; P < 0.05). MYO was more strongly stimulated in the EX vs. CON legs in NW (P = 0.02) but not OB (P = 0.26). SARC was feeding sensitive but not further potentiated by resistance exercise in both groups. Our results suggest that obesity may attenuate the effectiveness of resistance exercise to augment fed‐state MYO. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5119-5133, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276210   open full text
• Recovery of respiratory function in mdx mice co‐treated with neutralizing interleukin‐6 receptor antibodies and urocortin‐2.
  David P. Burns, Leonie Canavan, Jane Rowland, Robin O'Flaherty, Molly Brannock, Sarah E. Drummond, Dervla O'Malley, Deirdre Edge, Ken D. O'Halloran.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2‐week intervention in young wild‐type and mdx mice, consisting of either injection of saline or co‐administration of a neutralizing interleukin‐6 receptor antibody (xIL‐6R) and urocortin‐2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL‐6R and Ucn2 co‐treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro‐inflammatory cytokine interleukin‐1β was reduced and the concentration of the anti‐inflammatory cytokine interleukin‐10 was increased in mdx diaphragm following drug co‐treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing. Abstract The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six‐week‐old male mdx (n = 32) and wild‐type (WT; n = 32) mice received either saline (0.9% w/v) or a co‐administration of neutralizing interleukin‐6 receptor antibodies (xIL‐6R; 0.2 mg kg−1) and corticotrophin‐releasing factor receptor 2 agonist (urocortin‐2; 30 μg kg−1) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force‐ and power‐generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co‐treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co‐treatment preserved MyHC type IIx complement in mdx muscle. Drug co‐treatment increased the cross‐sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL‐1β, IL‐6, KC/GRO and TNF‐α were significantly increased in mdx diaphragm compared with WT. Drug co‐treatment significantly decreased IL‐1β and increased IL‐10 in mdx diaphragm. Drug co‐treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5175-5197, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276954   open full text
• Effects of lorazepam and baclofen on short‐ and long‐latency afferent inhibition.
  Claudia V. Turco, Jenin El‐Sayes, Mitchell B. Locke, Robert Chen, Steven Baker, Aimee J. Nelson.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points Short‐latency afferent inhibition (SAI) is modulated by GABAA receptor activity, whereas the pharmacological origin of long‐latency afferent inhibition remains unknown. This is the first study to report that long‐latency afferent inhibition (LAI) is reduced by the GABAA positive allosteric modulator lorazepam, and that both SAI and LAI are not modulated by the GABAB agonist baclofen. These findings advance our understanding of the neural mechanisms underlying afferent inhibition. Abstract The afferent volley evoked by peripheral nerve stimulation has an inhibitory influence on transcranial magnetic stimulation induced motor evoked potentials. This phenomenon, known as afferent inhibition, occurs in two phases: short‐latency afferent inhibition (SAI) and long‐latency afferent inhibition (LAI). SAI exerts its inhibitory influence via cholinergic and GABAergic activity. The neurotransmitter receptors that mediate LAI remain unclear. The present study aimed to determine whether LAI is contributed by GABAA and/or GABAB receptor activity. In a double‐blinded, placebo‐controlled study, 2.5 mg of lorazepam (GABAA agonist), 20 mg of baclofen (GABAB agonist) and placebo were administered to 14 males (mean age 22.7 ± 1.9 years) in three separate sessions. SAI and LAI, evoked by stimulation of the median nerve and recorded from the first dorsal interosseous muscle, were quantified before and at the peak plasma concentration following drug ingestion. Results indicate that lorazepam reduced LAI by ∼40% and, in support of previous work, reduced SAI by ∼19%. However, neither SAI, nor LAI were altered by baclofen. In a follow‐up double‐blinded, placebo‐controlled study, 10 returning participants received placebo or 40 mg of baclofen (double the dosage used in Experiment 1). The results obtained indicate that SAI and LAI were unchanged by baclofen. This is the first study to show that LAI is modulated by GABAA receptor activity, similar to SAI, and that afferent inhibition does not appear to be a GABAB mediated process. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5267-5280, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276710   open full text
• Acute and chronic exercise in patients with heart failure with reduced ejection fraction: evidence of structural and functional plasticity and intact angiogenic signalling in skeletal muscle.
  Fabio Esposito, Odile Mathieu‐Costello, Peter D. Wagner, Russell S. Richardson.
  The Journal of Physiology. November 01, 2018
  --- - |2+ Key points The vascular endothelial growth factor (VEGF) responses to acute submaximal exercise and training effects in patients with heart failure with reduced ejection fraction (HFrEF) were investigated. Six patients and six healthy matched controls performed knee‐extensor exercise (KE) at 50% of maximum work rate before and after (only patients) KE training. Muscle biopsies were taken to assess skeletal muscle structure and the angiogenic response. Before training, during this submaximal KE exercise, patients with HFrEF exhibited higher leg vascular resistance and greater noradrenaline spillover. Skeletal muscle structure and VEGF response were generally not different between groups. Following training, resistance was no longer elevated and noradrenaline spillover was curtailed in the patients. Although, in the trained state, VEGF did not respond to acute exercise, capillarity was augmented. Muscle fibre cross‐sectional area and percentage area of type I fibres increased and mitochondrial volume density exceeded that of controls. Structural/functional plasticity and appropriate angiogenic signalling were observed in skeletal muscle of patients with HFrEF. Abstract This study examined the response to acute submaximal exercise and the effect of training in patients with heart failure with reduced ejection fraction (HFrEF). The acute angiogenic response to submaximal exercise in HFrEF after small muscle mass training is debated. The direct Fick method, with vascular pressures, was performed across the leg during knee‐extensor exercise (KE) at 50% of maximum work rate (WRmax) in patients (n = 6) and controls (n = 6) and then after KE training in patients. Muscle biopsies facilitated the assessment of skeletal muscle structure and vascular endothelial growth factor (VEGF) mRNA levels. Prior to training, HFrEF exhibited significantly higher leg vascular resistance (LVR) (≈15%) and significantly greater noradrenaline spillover (≈385%). Apart from mitochondrial volume density, which was significantly lower (≈22%) in HFrEF, initial skeletal muscle structure, including capillarity, was not different between groups. Resting VEGF mRNA levels, and the increase with exercise, was not different between patients and controls. Following training, LVR was no longer elevated and noradrenaline spillover was curtailed. Skeletal muscle capillarity increased with training, as assessed by capillary‐to‐fibre ratio (≈13%) and number of capillaries around a fibre (NCAF) (≈19%). VEGF mRNA was now not significantly increased by acute exercise. Muscle fibre cross‐sectional area and percentage area of type I fibres both increased significantly with training (≈18% and ≈21%, respectively), while the percentage area of type II fibres fell significantly (≈11%), and mitochondrial volume density now exceeded that of controls. These data reveal structural and functional plasticity and appropriate angiogenic signalling in skeletal muscle of HFrEF patients. - 'The Journal of Physiology, Volume 596, Issue 21, Page 5149-5161, 1 November 2018. '

  November 01, 2018   doi: 10.1113/JP276678   open full text
• SIRT1 overexpression attenuates offspring metabolic and liver disorders as a result of maternal high‐fat feeding.
  Long T. Nguyen, Hui Chen, Amgad Zaky, Carol Pollock, Sonia Saad.
  The Journal of Physiology. October 31, 2018
  --- - |2+ Key points Maternal high‐fat diet (MHF) consumption led to metabolic and liver disorders in male offspring, which are associated with reduced sirtuin (SIRT)1 expression and activity in the offspring liver SIRT1 overexpression in MHF offspring reduced their body weight and adiposity and normalized lipid metabolic markers in epididymal and retroperitoneal adipose tissues SIRT1 overexpression in MHF offspring improved glucose tolerance, as well as systemic and hepatic insulin sensitivity SIRT1 overexpression ameliorated MHF‐induced lipogenesis, oxidative stress and fibrogenesis in the liver of offspring. Abstract Maternal obesity can increase the risk of metabolic disorders in the offspring. However, the underlying mechanism responsible for this is not clearly understood. Previous evidence implied that sirtuin (SIRT)1, a potent regulator of energy metabolism and stress responses, may play an important role. In the present study, we have shown, in C57BL/6 mice, that maternal high‐fat diet (HFD) consumption can induce a pre‐diabetic and non‐alcoholic fatty liver disease phenotype in the offspring, associated with reduced SIRT1 expression in the hypothalamus, white adipose tissues (WAT) and liver. Importantly, the overexpression of SIRT1 in these offspring significantly attenuated the excessive accumulation of epididymal (Epi) white adipose tissue (WAT) and retroperitoneal (Rp)WAT (P < 0.001), glucose intolerance and insulin resistance (both P < 0.05) at weaning age. These changes were associated with the suppression of peroxisome proliferator‐activated receptor gamma (PPAR)γ (P < 0.01), PPARγ‐coactivator 1‐alpha (P < 0.05) and sterol regulatory element‐binding protein‐1c in EpiWAT (P < 0.01), whereas there was increased expression of PPARγ in RpWAT (P < 0.05). In the liver, PPARγ mRNA expression, as well as Akt protein expression and activity, were increased (P < 0.05), whereas fatty acid synthase and carbohydrate response element binding protein were downregulated (P < 0.05), supporting increased insulin sensitivity and reduced lipogenesis in the liver. In addition, hepatic expression of endogenous anti‐oxidants, including glutathione peroxidase 1 and catalase, was increased (P < 0.01 and P < 0.05 respectively), whereas collagen and fibronectin deposition was suppressed (P < 0.01). Collectively, the present study provides direct evidence of the mechanistic significance of SIRT1 in maternal HFD‐induced metabolic dysfunction in offspring and suggests that SIRT1 is a promising target for fetal reprogramming. - 'The Journal of Physiology, EarlyView. '

  October 31, 2018   doi: 10.1113/JP276957   open full text
• Renal reactivity: acid‐base compensation during incremental ascent to high altitude.
  Shaelynn M. Zouboules, Hailey C. Lafave, Ken D. O'Halloran, Tom D. Brutsaert, Heidi E. Nysten, Cassandra E. Nysten, Craig D. Steinback, Mingma T. Sherpa, Trevor A. Day.
  The Journal of Physiology. October 28, 2018
  --- - |2+ Key points Ascent to high altitude imposes an acid‐base challenge in which renal compensation is integral for maintaining pH homeostasis, facilitating acclimatization and helping prevent mountain sicknesses. The time‐course and extent of plasticity of this important renal response during incremental ascent to altitude is unclear. We created a novel index that accurately quantifies renal acid‐base compensation, which may have laboratory, fieldwork and clinical applications. Using this index, we found that renal compensation increased and plateaued after 5 days of incremental altitude exposure, suggesting plasticity in renal acid‐base compensation mechanisms. The time‐course and extent of plasticity in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude. Abstract Ascent to high altitude, and the associated hypoxic ventilatory response, imposes an acid‐base challenge, namely chronic hypocapnia and respiratory alkalosis. The kidneys impart a relative compensatory metabolic acidosis through the elimination of bicarbonate (HCO3−) in urine. The time‐course and extent of plasticity of the renal response during incremental ascent is unclear. We developed an index of renal reactivity (RR), indexing the relative change in arterial bicarbonate concentration ([HCO3−]a) (i.e. renal response) against the relative change in arterial pressure of CO2 () (i.e. renal stimulus) during incremental ascent to altitude (). We aimed to assess whether: (i) RR magnitude was inversely correlated with relative changes in arterial pH (ΔpHa) with ascent and (ii) RR increased over time and altitude exposure (i.e. plasticity). During ascent to 5160 m over 10 days in the Nepal Himalaya, arterial blood was drawn from the radial artery for measurement of blood gas/acid‐base variables in lowlanders at 1045/1400 m and after 1 night of sleep at 3440 m (day 3), 3820 m (day 5), 4240 m (day 7) and 5160 m (day 10) during ascent. At 3820 m and higher, RR significantly increased and plateaued compared to 3440 m (P < 0.04), suggesting plasticity in renal acid‐base compensations. At all altitudes, we observed a strong negative correlation (r ≤ −0.71; P < 0.001) between RR and ΔpHa from baseline. Renal compensation plateaued after 5 days of altitude exposure, despite subsequent exposure to higher altitudes. The time‐course, extent of plasticity and plateau in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude. - 'The Journal of Physiology, EarlyView. '

  October 28, 2018   doi: 10.1113/JP276973   open full text
• A jolt to the field: A self‐generating and self‐propagating ephaptically‐mediated slow spontaneous network activity pattern in the hippocampus.
  Clayton Dickson.
  The Journal of Physiology. October 25, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 25, 2018   doi: 10.1113/JP277233   open full text
• Increased sensitivity of the circadian system to light in delayed sleep–wake phase disorder.
  Lauren A. Watson, Andrew J. K. Phillips, Ihaia T. Hosken, Elise M. McGlashan, Clare Anderson, Leon C. Lack, Steven W. Lockley, Shantha M. W. Rajaratnam, Sean W. Cain.
  The Journal of Physiology. October 25, 2018
  --- - |2+ Key points This is the first study to demonstrate an altered circadian phase shifting response in a circadian rhythm sleep disorder. Patients with delayed sleep–wake phase disorder (DSWPD) demonstrate greater sensitivity of the circadian system to the phase‐delaying effects of light. Increased circadian sensitivity to light is associated with later circadian timing within both control and DSWPD groups. DSWPD patients had a greater sustained pupil response after light exposure. Treatments for DSWPD should consider sensitivity of the circadian system to light as a potential underlying vulnerability, making patients susceptible to relapse. Abstract Patients with delayed sleep–wake phase disorder (DSWPD) exhibit delayed sleep–wake behaviour relative to desired bedtime, often leading to chronic sleep restriction and daytime dysfunction. The majority of DSWPD patients also display delayed circadian timing in the melatonin rhythm. Hypersensitivity of the circadian system to phase‐delaying light is a plausible physiological basis for DSWPD vulnerability. We compared the phase shifting response to a 6.5 h light exposure (∼150 lux) between male patients with diagnosed DSWPD (n = 10; aged 20.8 ± 2.3 years) and male healthy controls (n = 11; aged 22.4 ± 3.3 years). Salivary dim light melatonin onset (DLMO) was measured under controlled conditions in dim light (<3 lux) before and after light exposure. Correcting for the circadian time of the light exposure, DSWPD patients exhibited 31.5% greater phase delay shifts than healthy controls. In both groups, a later initial melatonin phase was associated with a greater magnitude phase shift, indicating that increased circadian sensitivity to light may be a factor that contributes to delayed phase, even in non‐clinical groups. DSWPD patients also had reduced pupil size following the light exposure, and showed a trend towards increased melatonin suppression during light exposure. These findings indicate that, for patients with DSWPD, assessment of light sensitivity may be an important factor that can inform behavioural therapy, including minimization of exposure to phase‐delaying night‐time light. - 'The Journal of Physiology, EarlyView. '

  October 25, 2018   doi: 10.1113/JP275917   open full text
• Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue.
  Stefan Passlick, Ek Raj Thapaliya, Zuxin Chen, Matthew T. Richers, Graham C. R. Ellis‐Davies.
  The Journal of Physiology. October 25, 2018
  --- - |2+ Key points A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one‐ and two‐photon excitation. The caged compound is photolysed with a quantum yield of 0.20. One‐photon uncaging of ABT594 elicited large currents and Ca2+ transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca2+‐permeable nAChRs on axons of MHb neurons. Abstract Photochemical release of neurotransmitters has been instrumental in the study of their underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. We caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one‐ or two‐photon excitation. Brief flashes (0.5–3.0 ms) of 410 nm light evoked large currents and Ca2+ transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR‐mediated Ca2+ signals along the axons of MHb neurons. - The Journal of Physiology, EarlyView.
  

  October 25, 2018   doi: 10.1113/JP276615   open full text
• Guardian of mitochondrial function: an expanded role of Parkin in skeletal muscle.
  J. Botella, N. Saner, C. Granata.
  The Journal of Physiology. October 25, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 25, 2018   doi: 10.1113/JP276841   open full text
• Delivering baking soda to the brain.
  Mark O. Bevensee.
  The Journal of Physiology. October 24, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 24, 2018   doi: 10.1113/JP277119   open full text
• Flowing from sense to action. Are neural integrators necessary?
  Andreas Kardamakis.
  The Journal of Physiology. October 23, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 23, 2018   doi: 10.1113/JP276927   open full text
• Shining light on the paraventricular nucleus: the role of glutamatergic PVN neurons in blood pressure control.
  Bryan K. Becker.
  The Journal of Physiology. October 23, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 23, 2018   doi: 10.1113/JP277043   open full text
• Fat or thin, exercise wins: endurance exercise training reduces inflammatory circulating progenitor cells in lean and obese adults.
  Paul M. Ryan, Ryan T. Sless, Nathaniel E. Hayward.
  The Journal of Physiology. October 22, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 22, 2018   doi: 10.1113/JP277229   open full text
• Don't stop at the top: Plasma volume expansion and pulmonary vasodilation restore left ventricular function at rest but not during exercise at high altitude.
  Elizabeth Karvasarski, Lucas Azevedo, David Granton, Stephen P. Wright.
  The Journal of Physiology. October 18, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 18, 2018   doi: 10.1113/JP277301   open full text
• Investigating cerebral blood flow control to save the newborn brain.
  Vignesh Murali, Cecilia G. Freeman, Ronée E. Harvey, Nicole C. Baig, Jennifer L. Helmond, Noud Helmond.
  The Journal of Physiology. October 18, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 18, 2018   doi: 10.1113/JP277045   open full text
• Sex differences in diaphragmatic fatigue: do young women have an advantage?
  Claire M. DeLucia, Daniel H. Craighead.
  The Journal of Physiology. October 18, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 18, 2018   doi: 10.1113/JP277120   open full text
• NHA2 promotes cyst development in an in vitro model of polycystic kidney disease.
  Hari Prasad, Donna K. Dang, Kalyan C. Kondapalli, Niranjana Natarajan, Valeriu Cebotaru, Rajini Rao.
  The Journal of Physiology. October 17, 2018
  --- - |2+ Key points Significant and selective up‐regulation of the Na+/H+ exchanger NHA2 (SLC9B2) was observed in cysts of patients with autosomal dominant polycystic kidney disease. Using the MDCK cell model of cystogenesis, it was found that NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro. Polycystin‐1 represses NHA2 expression via Ca2+/NFAT signalling whereas the dominant negative membrane‐anchored C‐terminal fragment (PC1‐MAT) increased NHA2 levels. Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression. Taken together, the findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of polycystic kidney disease. Abstract Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin‐1 (PC1) and polycystin‐2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up‐regulation of NHA2, a member of the SLC9B family of Na+/H+ exchangers, that correlated with cyst size and disease severity in ADPKD patients. Using three‐dimensional cultures of MDCK cells to model cystogenesis in vitro, we showed that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store‐dependent and ‐independent pathways, whereas reciprocal activation of Ca2+ influx by the dominant negative membrane‐anchored C‐terminal tail fragment of PC1 elevated NHA2. We showed that NHA2 is a target of Ca2+/NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observed robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up‐regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for polycystic kidney disease and reveal a potential new regulator of transepithelial salt and water transport in the kidney. - 'The Journal of Physiology, EarlyView. '

  October 17, 2018   doi: 10.1113/JP276796   open full text
• Development Of The Cerebral Cortex And The Effect Of The Intrauterine Environment.
  Sebastian Quezada, Margie Castillo‐Melendez, David W Walker, Mary Tolcos.
  The Journal of Physiology. October 16, 2018
  --- - |2+ Key points Normal folding of the cerebral cortex (gyrification) is fundamental for neurodevelopment. Some molecular mechanisms of gyrification during fetal development have been identified, but the impact of maternal health and the intrauterine environment has been largely overlooked. Recent evidence suggests that the intrauterine environment has a significant impact on the normal folding of the fetal cerebral cortex. This article reviews evidence for the effect of the most common intrauterine alterations on the normal development of the cortical folding in the fetus. Abstract The human brain is one of the most complex structures currently under study. Its external shape is highly convoluted, with folds and valleys over the entire surface of the cortex. Disruption of the normal pattern of folding is associated with a number of abnormal neurological outcomes, some serious for the individual. Most of our knowledge of the normal development and folding of the cerebral cortex (gyrification) focuses on the internal, biological (i.e. genetically‐driven) mechanisms of the brain that drive gyrification. However, the impact of an adverse intrauterine and maternal physiological environment on cortical folding during fetal development has been understudied. Accumulating evidence suggests that the state of the intrauterine and maternal environment can have a significant impact on gyrification of the fetal cerebral cortex. This review summarises our current knowledge of how development in a suboptimal intrauterine and maternal environment can affect the normal development of the folded cerebral cortex. Common pregnancy‐related conditions such as intrauterine growth restriction (IUGR), preterm birth, hypoxia, maternal/fetal infections, and multiple fetuses, can have an impact on the normal development and folding of the cerebral cortex This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 16, 2018   doi: 10.1113/JP277151   open full text
• Baroreflex functionality in the eye of diffusion tensor imaging.
  Ching‐Yi Tsai, Yan‐Yuen Poon, Julie Y.H. Chan, Samuel H.H. Chan.
  The Journal of Physiology. October 16, 2018
  --- - |2+ Abstract By application of diffusion tensor imaging (DTI) as a physiological tool to evaluate changes in functional connectivity between key brain stem nuclei in the baroreflex neural circuits of mice and rats, recent work revealed several hitherto unidentified phenomena regarding baroreflex functionality. (1) The presence of robust functional connectivity between nucleus tractus solitarii (NTS) and nucleus ambiguus (NA) or rostral ventrolateral medulla (RVLM) offers a holistic view on the moment‐to‐moment modus operandi of the cardiac vagal baroreflex or baroreflex‐mediated sympathetic vasomotor tone. (2) Under pathophysiological conditions (e.g. neurogenic hypertension), the disruption of functional connectivity between key nuclei in the baroreflex circuits is reversible. However, fatality ensues on progression from pathophysiological to pathological conditions (e.g. hepatic encephalopathy) when the functional connectivity between NTS and NA or RVLM is irreversibly severed. (3) The absence of functional connectivity between the NTS and caudal ventrolateral medulla (CVLM) necessitates partial rewiring of the classical neural circuit that includes CVLM as an inhibitory intermediate between the NTS and RVLM. (4) Sustained functional connectivity between the NTS and NA is responsible for the vital period between brain death and the inevitable cardiac death. (5) Reduced functional connectivity between the NTS and RVLM or NA points to inherent anomalous baroreflex functionality in floxed and Cre‐Lox mice. (6) Disrupted NTS‐NA functional connectivity in Flk‐1 (VEGFR2) deficient mice offers an explanation for the hypertensive side‐effect of anti‐VEGF therapy. These newly identified baroreflex functionalities in the eye of DTI bear clinical and therapeutic implications. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 16, 2018   doi: 10.1113/JP277008   open full text
• Tbx18 sets the pace.
  Wenli Dai, Christopher Weber.
  The Journal of Physiology. October 15, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 15, 2018   doi: 10.1113/JP277180   open full text
• Expression of Concern.
  
  The Journal of Physiology. October 15, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 5063-5064, 15 October 2018.
  

  October 15, 2018   doi: 10.1113/JP277015   open full text
• Issue Information.
  
  The Journal of Physiology. October 15, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4803-4804, 15 October 2018.
  

  October 15, 2018   doi: 10.1113/tjp.12593   open full text
• Role of the C‐terminus of SUR in the differential regulation of β‐cell and cardiac KATP channels by MgADP and metabolism.
  Natascia Vedovato, Olof Rorsman, Konstantin Hennis, Frances M. Ashcroft, Peter Proks.
  The Journal of Physiology. October 14, 2018
  --- - |2+ Key points β‐Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration–inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg‐nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C‐terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg‐nucleotides, which is determined by the final 42 amino acids. Abstract ATP‐sensitive potassium (KATP) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to β‐cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 μm MgADP immediately after patch excision [when the channel open probability (PO) is near maximal]. The results were strikingly different: 100 μm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg‐nucleotide activation. This suggests the C‐terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of β‐cell and cardiac KATP channels. - 'The Journal of Physiology, EarlyView. '

  October 14, 2018   doi: 10.1113/JP276708   open full text
• Oxygen, evolution and redox signalling in the human brain; quantum in the quotidian.
  Damian Miles Bailey.
  The Journal of Physiology. October 13, 2018
  --- - |2+ Abstract Rising atmospheric oxygen (O2) levels provided a selective pressure for the evolution of O2‐dependent micro‐organisms that began with the autotrophic eukaryotes. Since these primordial times, the respiring mammalian cell has become entirely dependent on the constancy of electron flow with molecular O2 serving as the terminal electron acceptor in mitochondrial oxidative phosphorylation. Indeed, the ability to “sense” O2 and maintain homeostasis is considered one of the most important roles of the central nervous system (CNS) and likely represented a major driving force in the evolution of the human brain. Today, modern humans haves evolved with an oversized brain committed to a continually active state and as a consequence, paradoxically vulnerable to failure if the O2 supply is interrupted. However, our pre‐occupation with O2, the elixir of life, obscures the fact that it is a gas with a Janus Face, capable of sustaining life in physiologically controlled amounts yet paradoxically deadly to the CNS when in excess. A closer look at its quantum structure reveals precisely why; the triplet ground state diatomic O2 molecule is paramagnetic and exists in air as a free radical, constrained from reacting aggressively with the brain's organic molecules due to its “spin restriction”, a thermodynamic quirk of evolutionary fate. By further exploring O2’s free radical “quantum quirkiness” including emergent (quantum) physiological phenomena, our understanding of precisely how the human brain senses O2 deprivation (hypoxia) and the elaborate redox‐signalling defence mechanisms that defend O2 homeostasis has the potential to offer unique insights into the pathophysiology and treatment of human brain disease. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 13, 2018   doi: 10.1113/JP276814   open full text
• TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.
  M. Choudhury, N. Black, A. Alghamdi, A. D'Souza, R. Wang, J. Yanni, H. Dobrzynski, P. A. Kingston, H. Zhang, M. R. Boyett, G. M. Morris.
  The Journal of Physiology. October 13, 2018
  --- - |2+ Key points The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or ‘sick sinus syndrome’, can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. ‘Biopacemaking’ utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+/Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. Abstract The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+/Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP‐TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP‐TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18‐induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10−5 ± 2.2 × 10−6 to 2.8 × 10−5 ± 4.3 × 10−6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform‐specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue. - 'The Journal of Physiology, EarlyView. '

  October 13, 2018   doi: 10.1113/JP276508   open full text
• Dynamic structural rearrangements and functional regulation of voltage‐sensing phosphatase.
  Souhei Sakata, Yasushi Okamura.
  The Journal of Physiology. October 12, 2018
  --- - |2+ Abstract The voltage‐sensing phosphatase, VSP, consists of the voltage sensor domain (VSD) and the cytoplasmic catalytic region. The latter contains the phosphatase domain and the C2 domain, showing remarkable similarity to a tumor suppressor enzyme, PTEN. In VSP, membrane depolarization induces the conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome of VSP is enzymatic activity of the cytoplasmic region unlike voltage‐gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change of the cytoplasmic catalytic region to gain insights into operating mechanisms of VSP. This review summarizes a recent study of applying a method of genetic incorporation of a noncanonical amino acid, Anap, to detect dynamic rearrangements of the structure of the catalytic region of sea squirt VSP (Ci‐VSP) under control of membrane voltage. Both the phosphatase domain and the C2 domain move in a similar timing upon membrane depolarization, suggesting that the two regions are coupled to each other. Measurement of FRET between Anap introduced into the C2 domain of Ci‐VSP and dipicrylamine in cell membrane suggested no large motion of the enzyme toward the membrane. Fluorescence change of Anap induced by different membrane potentials indicates the presence of multiple conformations of the active enzyme. A fluorescent unnatural amino acid, Anap, was genetically incorporated into the catalytic domain of Ci‐VSP in Xenopus oocytes. The fluorescence uncovered the conformation change associated with catalytic activity of Ci‐VSP. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 12, 2018   doi: 10.1113/JP274113   open full text
• Why don't mice lacking the mitochondrial Ca2+ uniporter experience an energy crisis?
  Pei Wang, Celia Fernandez‐Sanz, Wang Wang, Shey‐Shing Sheu.
  The Journal of Physiology. October 12, 2018
  --- - |2 Abstract Current dogma holds that the heart balances energy demand and supply effectively and sustainably by sequestering enough Ca2+ into mitochondria during heartbeats to stimulate metabolic enzymes in the tricarboxylic acid (TCA) cycle and electron transport chain (ETC). This process is called excitation‐contraction‐bioenergetics (ECB) coupling. Recent breakthroughs in identifying the mitochondrial Ca2+ uniporter (MCU) and its associated proteins have opened up new windows for interrogating the molecular mechanisms of mitochondrial Ca2+ homeostasis regulation and its role in ECB coupling. Despite remarkable progress made in the past 7 years, it has been surprising, almost disappointing, that germline MCU deficiency in mice with certain genetic background yields viable pups, and knockout of the MCU in adult heart does not cause lethality. Moreover, MCU deficiency results in few adverse phenotypes, normal performance, and preserved bioenergetics in the heart at baseline. In this review, we briefly assess the existing literature on mitochondrial Ca2+ homeostasis regulation and then we consider possible explanations for why MCU‐deficient mice are spared from energy crises under physiological conditions. We propose that MCU and/or mitochondrial Ca2+ may have limited ability to set ECB coupling, that other mitochondrial Ca2+ handling mechanisms may play a role, and that extra‐mitochondrial Ca2+ may regulate ECB coupling. Since the heart needs to regenerate a significant amount of ATP to assure the perpetuation of heartbeats, multiple mechanisms are likely to work in concert to match energy supply with demand. - The Journal of Physiology, EarlyView.
  

  October 12, 2018   doi: 10.1113/JP276636   open full text
• Neurocardiac regulation: From cardiac mechanisms to novel therapeutic approaches.
  E. N. Bardsley, D. J. Paterson.
  The Journal of Physiology. October 11, 2018
  --- - |2+ Cardiac sympathetic over‐activity is a well‐established contributor to the progression of neurogenic hypertension and heart failure, yet the underlying pathophysiology remains unclear. Recent studies have highlighted the importance of acutely regulated cyclic nucleotides and their effectors in the control of intracellular calcium and exocytosis. Emerging evidence now suggests that a significant component of sympathetic over‐activity and enhanced transmission may arise from impaired cyclic nucleotide signalling, resulting from compromised phosphodiesterase activity, as well as alterations in receptor‐coupled G‐protein activation. In this review, we address some of the key cellular and molecular pathways that contribute to sympathetic over‐activity in hypertension and discuss their potential for therapeutic targeting. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 11, 2018   doi: 10.1113/JP276962   open full text
• And the beat goes on.
  Jack R. T. Darby, Janna L. Morrison.
  The Journal of Physiology. October 08, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 08, 2018   doi: 10.1113/JP277026   open full text
• Metaboreceptor polymorphisms: do genes determine your blood pressure response to exercise?
  Jasdeep Kaur, Thales C. Barbosa, Paul J. Fadel.
  The Journal of Physiology. October 08, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 08, 2018   doi: 10.1113/JP276971   open full text
• Timing is everything: maternal circadian rhythms and the developmental origins of health and disease.
  Tamara J. Varcoe.
  The Journal of Physiology. October 08, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 08, 2018   doi: 10.1113/JP276992   open full text
• An ex vivo bladder model with detrusor smooth muscle removed to analyze biologically active mediators released from the suburothelium.
  Leonie Durnin, Benjamin Kwok, Priya Kukadia, Roisin McAvera, Robert D. Corrigan, Sean M. Ward, Ying Zhang, Qi Chen, Sang Don Koh, Kenton M. Sanders, Violeta N. Mutafova‐Yambolieva.
  The Journal of Physiology. October 05, 2018
  --- - |2+ A Key Point List Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure‐volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation, and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop the decentralized (ex vivo) detrusor smooth muscle (DSM)‐denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium‐derived mediators at the luminal and anti‐luminal aspects of the urothelium during filling. Urinary bladders were excised from C57Bl6/J mice and the DSM was removed by fine scissors dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure‐volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate‐ and lipopolysaccharide‐treated denuded bladders were characterized. The preparation wall contains intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacks the DSM and the serosa. Utility of the model for physiology research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g., ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM‐denuded bladder of Cynomolgus monkeys (Macaca fascicularis). The novel model is superior to current models utilized to study properties of the urothelium (e.g., cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enables direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium‐DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 05, 2018   doi: 10.1113/JP276924   open full text
• Sex differences in the regulation of hepatic mitochondrial turnover following physical activity: do males need more quality control than females?
  Catherine Bellissimo, Christopher G.R. Perry.
  The Journal of Physiology. October 04, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  October 04, 2018   doi: 10.1113/JP276896   open full text
• What short‐term potentiation is and why it may be relevant to obstructive sleep apnoea.
  Magdy Younes.
  The Journal of Physiology. October 04, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 04, 2018   doi: 10.1113/JP276848   open full text
• When gain is greater than loss: effects of physical activity on insulin sensitivity after short‐term inactivity in older subjects.
  Jaume Padilla, Nathan C. Winn, Lauren K. Walsh.
  The Journal of Physiology. October 04, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 04, 2018   doi: 10.1113/JP277110   open full text
• Parkin: one of the guardians of mitochondrial function and skeletal muscle contractility.
  Gabriel S. Arini, Ancély F. dos Santos.
  The Journal of Physiology. October 01, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  October 01, 2018   doi: 10.1113/JP276872   open full text
• Issue Information.
  
  The Journal of Physiology. October 01, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4549-4550, 1 October 2018.
  

  October 01, 2018   doi: 10.1113/tjp.12592   open full text
• Haptic exploration attenuates and alters somatosensory cortical oscillations.
  Max J. Kurz, Alex I. Wiesman, Nathan M. Coolidge, Tony W. Wilson.
  The Journal of Physiology. September 21, 2018
  --- - |2+ Key points Several behavioural studies have shown the sensory perceptions are reduced during movement; yet the neurophysiological reason for this is not clear. Participants underwent stimulation of the median nerve when either sitting quietly (i.e. passive stimulation condition) or performing haptic exploration of a ball with the left hand. Magnetoencephalographic brain imaging and advanced beamforming methods were used to identify the differences in somatosensory cortical responses. We show that the neural populations active during the passive stimulation condition were strongly gated during the haptic exploration task. These results imply that the reduced haptic perceptions might be governed by gating of certain somatosensory neural populations. Abstract Several behavioural studies have shown that children have reduced sensory perceptions during movement; however, the neurophysiological nexus for these altered perceptions remains unknown. We used magnetoencephalographic brain imaging and advanced beamforming methods to address this knowledge gap. In our experiment, a cohort of children (aged 10–18 years) underwent stimulation of the median nerve when either sitting quietly (i.e. passive stimulation condition) or performing haptic exploration of a ball with the left hand. Our results revealed two novel observations. First, there was a relationship between the child's age and the strength of the beta (18–26 Hz) response seen within the somatosensory cortices during the passive stimulation condition. This suggests that there may be an age‐dependent change in the processing of peripheral feedback by the somatosensory cortices. Second, all of the cortical regions that were active during the passive stimulation condition were almost completely gated during the haptic task. Instead, the haptic task involved neural oscillations within Brodmann area 2, which is known to convey less spatially precise tactile information but is involved in the processing of more complex somatosensations across the respective digits. These results imply that the reduced somatosensory perceptions seen during movements in healthy children may be related to the gating of certain neural generators, as well as activation of haptic‐specific neural generators within the somatosensory cortices. The utilization of such haptic‐specific circuits during development may lead to the enhanced somatosensory processing during haptic exploration seen in healthy adults. - The Journal of Physiology, Volume 596, Issue 20, Page 5051-5061, 15 October 2018.
  

  September 21, 2018   doi: 10.1113/JP276263   open full text
• Turnaround in the history of carotid chemoreflex contribution for cardiorespiratory control in COPD: what are the upcoming chapters?
  Diogo Machado Oliveira, Indyanara Cristina Ribeiro, Tamires Silva Cesar, Tiago Obeid Freitas, Liliane Cunha Aranda.
  The Journal of Physiology. September 20, 2018
  --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
  

  September 20, 2018   doi: 10.1113/JP276986   open full text
• The effect of maternal metabolic status on offspring health: a role for skeletal muscle?
  Jasmine Mikovic, Séverine Lamon.
  The Journal of Physiology. September 20, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  September 20, 2018   doi: 10.1113/JP276929   open full text
• Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.
  Tyler Basting, Jiaxi Xu, Snigdha Mukerjee, Joel Epling, Robert Fuchs, Srinivas Sriramula, Eric Lazartigues.
  The Journal of Physiology. September 20, 2018
  --- - |2+ Key points Recurrent periods of over‐excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over‐activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin‐2 leads to an immediate frequency‐dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)‐salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. Abstract Neuro‐cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over‐excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin‐2 using an adeno‐associated virus (CamKII‐ChR2‐eYFP‐AAV2) in wildtype mice (n = 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency‐dependent (2, 10 and 20 Hz) increase in BP from baseline by ∼9 mmHg at 20 Hz stimulation (P < 0.001). Additionally, in vGlut2‐cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre‐dependent caspase (AAV2‐flex‐taCASP3‐TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA‐salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P < 0.05) resulted in an attenuated rise in BP following DOCA‐salt treatment (P < 0.05 at 7 day time point, n = 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA‐salt treatment in the lesioned animals (P < 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP. - The Journal of Physiology, EarlyView.
  

  September 20, 2018   doi: 10.1113/JP276229   open full text
• A simple decision to move in response to touch reveals basic sensory memory and mechanisms for variable response times.
  Stella Koutsikou, Robert Merrison‐Hort, Edgar Buhl, Andrea Ferrario, Wen‐Chang Li, Roman Borisyuk, Stephen R. Soffe, Alan Roberts.
  The Journal of Physiology. September 19, 2018
  --- - |2+ Key points Short‐term working memory and decision‐making are usually studied in the cerebral cortex; in many models of simple decision making, sensory signals build slowly and noisily to threshold to initiate a motor response after long, variable delays. When touched, hatchling frog tadpoles decide whether to swim; we define the long and variable delays to swimming and use whole‐cell recordings to uncover the neurons and processes responsible. Firing in sensory and sensory pathway neurons is short latency, and too brief and invariant to explain these delays, while recordings from hindbrain reticulospinal neurons controlling swimming reveal a prolonged and variable build‐up of synaptic excitation which can reach firing threshold and initiate swimming. We propose this excitation provides a sensory memory of the stimulus and may be generated by small reverberatory hindbrain networks. Our results uncover fundamental network mechanisms that allow animals to remember brief sensory stimuli and delay simple motor decisions. Abstract Many motor responses to sensory input, like locomotion or eye movements, are much slower than reflexes. Can simpler animals provide fundamental answers about the cellular mechanisms for motor decisions? Can we observe the ‘accumulation’ of excitation to threshold proposed to underlie decision making elsewhere? We explore how somatosensory touch stimulation leads to the decision to swim in hatchling Xenopus tadpoles. Delays measured to swimming in behaving and immobilised tadpoles are long and variable. Activity in their extensively studied sensory and sensory pathway neurons is too short‐lived to explain these response delays. Instead, whole‐cell recordings from the hindbrain reticulospinal neurons that drive swimming show that these receive prolonged, variable synaptic excitation lasting for nearly a second following a brief stimulus. They fire and initiate swimming when this excitation reaches threshold. Analysis of the summation of excitation requires us to propose extended firing in currently undefined presynaptic hindbrain neurons. Simple models show that a small excitatory recurrent‐network inserted in the sensory pathway can mimic this process. We suggest that such a network may generate slow, variable summation of excitation to threshold. This excitation provides a simple memory of the sensory stimulus. It allows temporal and spatial integration of sensory inputs and explains the long, variable delays to swimming. The process resembles the ‘accumulation’ of excitation proposed for cortical circuits in mammals. We conclude that fundamental elements of sensory memory and decision making are present in the brainstem at a surprisingly early stage in development. - The Journal of Physiology, EarlyView.
  

  September 19, 2018   doi: 10.1113/JP276356   open full text
• Somatic modulation of ectopic action potential initiation in distal axons.
  Aurélie Fékété, Dominique Debanne.
  The Journal of Physiology. September 19, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  September 19, 2018   doi: 10.1113/JP277012   open full text
• VEGF‐A165b to the rescue: vascular integrity and pain sensitization.
  Madhavi Jere, Ryan M. Cassidy.
  The Journal of Physiology. September 19, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  September 19, 2018   doi: 10.1113/JP276902   open full text
• Inhibition of GluN2A NMDA receptors ameliorates synaptic plasticity deficits in the Fmr1−/y mouse model.
  Camilla J. Lundbye, Anna Karina H. Toft, Tue G. Banke.
  The Journal of Physiology. September 19, 2018
  --- - |2+ Key points Fragile X syndrome (FXS) is a genetic condition that is the most common form of inherited intellectual impairment and causes a range of neurodevelopmental complications including learning disabilities and intellectual disability and shares many characteristics with autism spectrum disorder (ASD). In the FXS mouse model, Fmr1−/y, impaired synaptic plasticity was restored by pharmacologically inhibiting GluN2A‐containing NMDA receptors but not GluN2B‐containing receptors. Similar results were obtained by crossing Fmr1−/y with GluN2A knock‐out (Grin2A−/−) mice. These results suggest that dampening the elevated levels of GluN2A‐containing NMDA receptors in Fmr1−/y mice has the potential to restore hyperexcitability of the neural circuitry to (a more) normal‐like level of brain activity. Abstract NMDA receptors (NMDARs) play important roles in synaptic plasticity at central excitatory synapses, and dysregulation of their function may lead to severe disorders such Fragile X syndrome (FXS). FXS is caused by transcriptional silencing of the FMR1 gene followed by lack of the encoding protein. Here we examined the effects of pharmacological and genetic manipulation of hippocampal NMDAR functions in long‐term potentiation (LTP) and depression (LTD). We found impaired NMDAR‐dependent LTP in the Fmr1‐deficient mice, which could be fully restored when GluN2A‐containing NMDARs was pharmacological inhibited. Interestingly, similar LTP effects were observed when the GluN2A gene (Grin2a) was deleted in Fmr1−/y mice (Fmr1−/y/Grin2a−/− double knockout). In addition, GluN2A inhibition improved elevated mGluR5‐dependent LTD to normal level in the Fmr1−/y mouse. These findings suggest that GluN2A is a promising target in FXS research that could help us better understand the disorder. - The Journal of Physiology, Volume 596, Issue 20, Page 5017-5031, 15 October 2018.
  

  September 19, 2018   doi: 10.1113/JP276304   open full text
• Gestational chronodisruption leads to persistent changes in the rat fetal and adult adrenal clock and function.
  E. R. Salazar, H. G. Richter, C. Spichiger, N. Mendez, D. Halabi, K. Vergara, I. P. Alonso, F. A. Corvalán, C. Azpeleta, M. Seron‐Ferre, C. Torres‐Farfan.
  The Journal of Physiology. September 18, 2018
  --- - |2+ Key points Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Here we explored the developmental impact of gestational chronodisruption (chronic photoperiod shift, CPS) on adult and fetal adrenal biorhythms and function. We found that gestational chronodisruption altered fetal and adult adrenal function, at the molecular, morphological and physiological levels. The differences between control and CPS offspring suggest desynchronization of the adrenal circadian clock and steroidogenic pathway, leading to abnormal stress responses and metabolic adaptation, potentially increasing the risk of developing chronic diseases. Abstract Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long‐term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down‐regulated and 437 up‐regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48–20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long‐term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases. - The Journal of Physiology, EarlyView.
  

  September 18, 2018   doi: 10.1113/JP276083   open full text
• P2X4 receptor re‐sensitization depends on a protonation/deprotonation cycle mediated by receptor internalization and recycling.
  Giorgio Fois, Karl J. Föhr, Carolin Kling, Michael Fauler, Oliver H. Wittekindt, Paul Dietl, Manfred Frick.
  The Journal of Physiology. September 17, 2018
  --- - |2+ Key points Re‐sensitization of P2X4 receptors depends on a protonation/de‐protonation cycle Protonation and de‐protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartments Protonation and de‐protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptors Re‐sensitization is blocked in the presence of the receptor agonist ATP Abstract P2X4 receptors are members of the P2X receptor family of cation‐permeable, ligand‐gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re‐sensitization is required to return the receptor into its resting state. Desensitization and re‐sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re‐sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re‐sensitization. Re‐sensitization depends on a protonation/de‐protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re‐sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP. - The Journal of Physiology, Volume 596, Issue 20, Page 4893-4907, 15 October 2018.
  

  September 17, 2018   doi: 10.1113/JP275448   open full text
• Astrocyte‐mediated primary afferent depolarization: a new twist to a complicated tale?
  Arlette Kolta.
  The Journal of Physiology. September 15, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4809-4810, 15 October 2018.
  

  September 15, 2018   doi: 10.1113/JP276949   open full text
• Differential targeting and signalling of voltage‐gated T‐type Cav3.2 and L‐type Cav1.2 channels to ryanodine receptors in mesenteric arteries.
  Gang Fan, Mario Kaßmann, Ahmed M. Hashad, Donald G. Welsh, Maik Gollasch.
  The Journal of Physiology. September 15, 2018
  --- - |2+ Key points In arterial smooth muscle, Ca2+ sparks are elementary Ca2+‐release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+‐sensitive K+ (BKCa) channels. This study elucidated the contribution of T‐type Cav3.2 channels in caveolae and their functional interaction with L‐type Cav1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L‐type Cav1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T‐type Cav3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T‐type Cav3.2 channels and RyRs to ignite Ca2+ sparks. Abstract Recent data suggest that T‐type Cav3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav3.2 channels and their functional interaction with Cav1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen‐inducible smooth muscle‐specific Cav1.2−/− (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+, Cd2+ and methyl‐β‐cyclodextrin were used to inhibit Cav3.2 channels, Cav1.2 channels and caveolae, respectively. Ni2+ (50 μmol L−1) and methyl‐β‐cyclodextrin (10 mmol L−1) decreased Ca2+ spark frequency by ∼20–30% in mesenteric VSMCs in a non‐additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 μmol L−1) suppressed Ca2+ sparks in mesenteric arteries by ∼70–80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl‐β‐cyclodextrin. Our data demonstrate that Ca2+ influx through CaV1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries. - The Journal of Physiology, Volume 596, Issue 20, Page 4863-4877, 15 October 2018.
  

  September 15, 2018   doi: 10.1113/JP276923   open full text
• Spatial receptive field shift by preceding cross‐modal stimulation in the cat superior colliculus.
  Jinghong Xu, Tingting Bi, Jing Wu, Fanzhu Meng, Kun Wang, Jiawei Hu, Xiao Han, Jiping Zhang, Xiaoming Zhou, Les Keniston, Liping Yu.
  The Journal of Physiology. September 15, 2018
  --- - |2+ Key points It has been known for some time that sensory information of one type can bias the spatial perception of another modality. However, there is a lack of evidence of this occurring in individual neurons. In the present study, we found that the spatial receptive field of superior colliculus multisensory neurons could be dynamically shifted by a preceding stimulus in a different modality. The extent to which the receptive field shifted was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. This result provides a neural mechanism that could underlie the process of cross‐modal spatial calibration. Abstract Psychophysical studies have shown that the different senses can be spatially entrained by each other. This can be observed in certain phenomena, such as ventriloquism, in which a visual stimulus can attract the perceived location of a spatially discordant sound. However, the neural mechanism underlying this cross‐modal spatial recalibration has remained unclear, as has whether it takes place dynamically. We explored these issues in multisensory neurons of the cat superior colliculus (SC), a midbrain structure that involves both cross‐modal and sensorimotor integration. Sequential cross‐modal stimulation showed that the preceding stimulus can shift the receptive field (RF) of the lagging response. This cross‐modal spatial calibration took place in both auditory and visual RFs, although auditory RFs shifted slightly more. By contrast, if a preceding stimulus was from the same modality, it failed to induce a similarly substantial RF shift. The extent of the RF shift was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. A narrow time gap and high stimulus salience were able to induce larger RF shifts. In addition, when both visual and auditory stimuli were presented simultaneously, a substantial RF shift toward the location‐fixed stimulus was also induced. These results, taken together, reveal an online cross‐modal process and reflect the details of the organization of SC inter‐sensory spatial calibration. - The Journal of Physiology, Volume 596, Issue 20, Page 5033-5050, 15 October 2018.
  

  September 15, 2018   doi: 10.1113/JP275427   open full text
• Specialized mechanoreceptor systems in rodent glabrous skin.
  Jan Walcher, Julia Ojeda‐Alonso, Julia Haseleu, Maria K. Oosthuizen, Ashlee H. Rowe, Nigel C. Bennett, Gary R. Lewin.
  The Journal of Physiology. September 15, 2018
  --- - |2+ Key points An ex vivo preparation was developed to record from single sensory fibres innervating the glabrous skin of the mouse forepaw. The density of mechanoreceptor innervation of the forepaw glabrous skin was found to be three times higher than that of hindpaw glabrous skin. Rapidly adapting mechanoreceptors that innervate Meissner's corpuscles were severalfold more responsive to slowly moving stimuli in the forepaw compared to those innervating hindpaw skin. We found a distinct group of small hairs in the centre of the mouse hindpaw glabrous skin that were exclusively innervated by directionally sensitive D‐hair receptors. The directional sensitivity, but not the end‐organ anatomy, were the opposite to D‐hair receptors in the hairy skin. Glabrous skin hairs in the hindpaw are not ubiquitous in rodents, but occur in African and North American species that diverged more than 65 million years ago. Abstract Rodents use their forepaws to actively interact with their tactile environment. Studies on the physiology and anatomy of glabrous skin that makes up the majority of the forepaw are almost non‐existent in the mouse. Here we developed a preparation to record from single sensory fibres of the forepaw and compared anatomical and physiological receptor properties to those of the hindpaw glabrous and hairy skin. We found that the mouse forepaw skin is equipped with a very high density of mechanoreceptors; >3 times more than hindpaw glabrous skin. In addition, rapidly adapting mechanoreceptors that innervate Meissner's corpuscles of the forepaw were severalfold more sensitive to slowly moving mechanical stimuli compared to their counterparts in the hindpaw glabrous skin. All other mechanoreceptor types as well as myelinated nociceptors had physiological properties that were invariant regardless of which skin area they occupied. We discovered a novel D‐hair receptor innervating a small group of hairs in the middle of the hindpaw glabrous skin in mice. These glabrous skin D‐hair receptors were direction sensitive albeit with an orientation sensitivity opposite to that described for hairy skin D‐hair receptors. Glabrous skin hairs do not occur in all rodents, but are present in North American and African rodent species that diverged more than 65 million years ago. The function of these specialized hairs is unknown, but they are nevertheless evolutionarily very ancient. Our study reveals novel physiological specializations of mechanoreceptors in the glabrous skin that likely evolved to facilitate tactile exploration. - The Journal of Physiology, Volume 596, Issue 20, Page 4995-5016, 15 October 2018.
  

  September 15, 2018   doi: 10.1113/JP276608   open full text
• Pulmonary vascular dysfunction in metabolic syndrome.
  Conor Willson, Makiko Watanabe, Atsumi Tsuji‐Hosokawa, Ayako Makino.
  The Journal of Physiology. September 13, 2018
  --- - |2+ Abstract Metabolic syndrome is a critically important precursor to the onset of many diseases, such as cardiovascular disease, and cardiovascular disease is the leading cause of death worldwide. The primary risk factors of metabolic syndrome include hyperglycaemia, abdominal obesity, dyslipidaemia, and high blood pressure. It has been well documented that metabolic syndrome alters vascular endothelial and smooth muscle cell functions in the heart, brain, kidney and peripheral vessels. However, there is less information available regarding how metabolic syndrome can affect pulmonary vascular function and ultimately increase an individual's risk of developing various pulmonary vascular diseases, such as pulmonary hypertension. Here, we review in detail how metabolic syndrome affects pulmonary vascular function. - The Journal of Physiology, EarlyView.
  

  September 13, 2018   doi: 10.1113/JP275856   open full text
• ‘Compliance’ to exercise: how much is really needed for a healthy heart (and mind)?
  Theodore M. DeConne, Joseph M. Stock, Kamila U. Migdal.
  The Journal of Physiology. September 13, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4817-4818, 15 October 2018.
  

  September 13, 2018   doi: 10.1113/JP277013   open full text
• Effect of coil orientation on motor‐evoked potentials in humans with tetraplegia.
  Hang Jin Jo, Vincenzo Di Lazzaro, Monica A. Perez.
  The Journal of Physiology. September 13, 2018
  --- - |2+ Key points Although corticospinal function changes following spinal cord injury (SCI), the extent to which we can activate the corticospinal tract after injury remains poorly understood. To address this question, we used transcranial magnetic stimulation over the hand representation of the primary motor cortex to elicit motor‐evoked potentials (MEPs) using posterior–anterior and anterior–posterior induced currents in the brain and compared them with responses evoked using lateral–medial currents in participants with and without cervical incomplete SCI during small levels of index finger abduction. We found prolonged MEP latencies in all coil orientations in SCI compared to control subjects. However, the latencies of MEPs elicited by posterior–anterior and anterior–posterior compared to lateral–medial stimulation were shorter in SCI compared to controls, particularly for MEPs elicited by anterior–posterior currents. Our findings demonstrate for the first time that corticospinal responses elicited by different directions of the induced current in the brain are differentially affected after SCI. Abstract The corticospinal tract undergoes reorganization following spinal cord injury (SCI). However, the extent to which we can activate corticospinal neurons using non‐invasive stimulation after injury remains poorly understood. To address this question, we used transcranial magnetic stimulation over the hand representation of the primary motor cortex to elicit motor‐evoked potentials (MEPs) using posterior–anterior (PA) and anterior–posterior (AP) induced currents in the brain and compared them with the responses evoked by direct activation of corticospinal axons using lateral–medial (LM) currents. Testing was completed during small levels of index finger abduction in humans with and without (controls) cervical incomplete SCI. We found prolonged MEP latencies in individuals with SCI in all coil orientations compared to controls. However, latencies of MEPs elicited by PA and AP stimulation relative to those elicited by LM stimulation were shorter in SCI compared to control subjects. Notably, the largest difference between SCI and control subjects was present in MEPs elicited by AP currents. Using a novel controllable pulse parameter transcranial magnetic stimulation, we also found that MEPs elicited by AP currents with 30 μs compared to 60 and 120 μs pulse width had increased latency in controls but not in SCI subjects. Our findings demonstrate that differences between corticospinal responses elicited by AP and PA induced currents were not preserved in humans with tetraplegia and suggest that neural structures activated by AP currents change largely after the injury. - The Journal of Physiology, Volume 596, Issue 20, Page 4909-4921, 15 October 2018.
  

  September 13, 2018   doi: 10.1113/JP275798   open full text
• Passive heat therapy protects against endothelial cell hypoxia‐reoxygenation via effects of elevations in temperature and circulating factors.
  Vienna E. Brunt, Karen Wiedenfeld‐Needham, Lindan N. Comrada, Christopher T. Minson.
  The Journal of Physiology. September 12, 2018
  --- - |2+ Key Points Accumulating evidence indicates that passive heat therapy (chronic use of hot tubs or saunas) has widespread physiological benefits, including enhanced resistance against novel stressors (‘stress resistance’). Using a cell culture model to isolate the key stimuli that are likely to underlie physiological adaptation with heat therapy, we showed that both mild elevations in temperature (to 39°C) and exposure to serum from human subjects who have undergone 8 weeks of heat therapy (i.e. altered circulating factors) independently prevented oxidative and inflammatory stress associated with hypoxia‐reoxygenation in cultured endothelial cells. Our results elucidate some of the mechanisms (i.e. direct effects of temperature vs. circulating factors) by which heat therapy seems to improve resistance against oxidative and inflammatory stress. Heat therapy may be a promising intervention for reducing cellular damage following ischaemic events, which has broad implications for patients with cardiovascular diseases and conditions characterized by ‘chronic’ ischaemia (e.g. peripheral artery disease, metabolic diseases, obesity). Abstract Repeated exposure to passive heat stress (‘heat therapy’) has widespread physiological benefits, including cellular protection against novel stressors. Increased heat shock protein (HSP) expression and upregulation of circulating factors may impart this protection. We tested the isolated abilities of mild heat pretreatment and serum from human subjects (n = 10) who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia‐reoxygenation (H/R), a model of ischaemic cardiovascular events. Cultured human umbilical vein endothelial cells were incubated for 24 h at 37°C (control), 39°C (heat pretreatment) or 37°C with 10% serum collected before and after 8 weeks of passive heat therapy (four to five times per week to increase rectal temperature to ≥ 38.5°C for 60 min). Cells were then collected before and after incubation at 1% O2 for 16 h (hypoxia; 37°C), followed by 20% O2 for 4 h (reoxygenation; 37°C) and assessed for markers of cell stress. In control cells, H/R increased nuclear NF‐κB p65 protein (i.e. activation) by 106 ± 38%, increased IL‐6 release by 37 ± 8% and increased superoxide production by 272 ± 45%. Both heat pretreatment and exposure to heat therapy serum prevented H/R‐induced NF‐κB activation and attenuated superoxide production; by contrast, only exposure to serum attenuated IL‐6 release. H/R also decreased cytoplasmic haemeoxygenase‐1 (HO‐1) protein (known to suppress NF‐κB), in control cells (−25 ± 8%), whereas HO‐1 protein increased following H/R in cells pretreated with heat or serum‐exposed, providing a possible mechanism of protection against H/R. These data indicate heat therapy is capable of imparting resistance against inflammatory and oxidative stress via direct heat and humoral factors. - The Journal of Physiology, Volume 596, Issue 20, Page 4831-4845, 15 October 2018.
  

  September 12, 2018   doi: 10.1113/JP276559   open full text
• Acute hydrocortisone administration reduces cardiovagal baroreflex sensitivity and heart rate variability in young men.
  Ahmed M. Adlan, Jet J. C. S. Veldhuijzen van Zanten, Gregory Y. H. Lip, Julian F. R. Paton, George D. Kitas, James P. Fisher.
  The Journal of Physiology. September 12, 2018
  --- - |2+ Key points A surge in cortisol during acute physiological and pathophysiological stress may precipitate ventricular arrhythmia and myocardial infarction. Reduced cardiovagal baroreflex sensitivity and heart rate variability are observed during acute stress and are associated with an increased risk of acute cardiac events. In the present study, healthy young men received either a single iv bolus of saline (placebo) or hydrocortisone, 1 week apart, in accordance with a randomized, placebo‐controlled, cross‐over study design. Hydrocortisone acutely increased heart rate and blood pressure and reduced cardiovagal baroreflex sensitivity and heart rate variability in young men. These findings suggest that, by reducing cardiovagal baroreflex sensitivity and heart rate variability, acute surges in cortisol facilitate a pro‐arrhythmic milieu and provide an important mechanistic link between stress and acute cardiac events Abstract Surges in cortisol concentration during acute stress may increase cardiovascular risk. To better understand the interactions between cortisol and the autonomic nervous system, we determined the acute effects of hydrocortisone administration on cardiovagal baroreflex sensitivity (BRS), heart rate variability (HRV) and cardiovascular reactivity. In a randomized, placebo‐controlled, single‐blinded cross‐over study, 10 healthy males received either a single iv bolus of saline (placebo) or 200 mg of hydrocortisone, 1 week apart. Heart rate (HR), blood pressure (BP) and limb blood flow were monitored 3 h later, at rest and during the sequential infusion of sodium nitroprusside and phenylephrine (modified Oxford Technique), a cold pressor test and a mental arithmetic stress task. HRV was assessed using the square root of the mean of the sum of the squares of differences between successive R‐R intervals (rMSSD). Hydrocortisone markedly increased serum cortisol 3 h following infusion and also compared to placebo. In addition, hydrocortisone elevated resting HR (+7 ± 4 beats min−1; P < 0.001) and systolic BP (+5 ± 5 mmHg; P = 0.008); lowered cardiovagal BRS [geometric mean (95% confidence interval) 15.6 (11.1–22.1) ms/mmHg vs. 26.2 (17.4––39.5) ms/mmHg, P = 0.011] and HRV (rMSSD 59 ± 29 ms vs. 84 ± 38 ms, P = 0.004) and increased leg vasoconstrictor responses to cold pressor test (Δ leg vascular conductance −45 ± 20% vs. −23 ± 26%; P = 0.023). In young men, an acute cortisol surge is accompanied by increases in HR and BP, as well as reductions in cardiovagal BRS and HRV, potentially providing a pro‐arrhythmic milieu that may precipitate ventricular arrhythmia or myocardial infarction and increase cardiovascular risk. - The Journal of Physiology, Volume 596, Issue 20, Page 4847-4861, 15 October 2018.
  

  September 12, 2018   doi: 10.1113/JP276644   open full text
• Rac1 supports muscle glucose uptake independently of Akt.
  Daniel M. Marko, Hesham Shamshoum.
  The Journal of Physiology. September 09, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4815-4816, 15 October 2018.
  

  September 09, 2018   doi: 10.1113/JP276851   open full text
• To clot or not to clot? That is a free radical question.
  Daniel R. Crabtree, David Muggeridge, Stephen J. Leslie, Ian L. Megson, James N. Cobley.
  The Journal of Physiology. September 09, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4805-4806, 15 October 2018.
  

  September 09, 2018   doi: 10.1113/JP276786   open full text
• Nutritional programming by maternal obesity: insights into the development of non‐alcoholic fatty liver disease.
  Judy Ghalayini, Shin‐Hann Lee.
  The Journal of Physiology. September 09, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4813-4814, 15 October 2018.
  

  September 09, 2018   doi: 10.1113/JP276965   open full text
• Optimal dissection of a model circuit.
  Takeshi Sakaba.
  The Journal of Physiology. September 09, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4807-4808, 15 October 2018.
  

  September 09, 2018   doi: 10.1113/JP276895   open full text
• Chronic aerobic exercise reduces the aortic age of an elderly cohort.
  Mina Amin Iskandar, Tharmegan Tharmaratnam, Zunair Ahmad.
  The Journal of Physiology. September 09, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 20, Page 4811-4812, 15 October 2018.
  

  September 09, 2018   doi: 10.1113/JP276961   open full text
• The application of stable‐isotope tracers to study human musculoskeletal protein turnover: a tale of bag filling and bag enlargement.
  D. Joe Millward, Ken Smith.
  The Journal of Physiology. September 07, 2018
  --- - |2+ Abstract The nutritional regulation of protein and amino acid balance in human skeletal muscle carried out by the authors with Mike Rennie is reviewed in the context of a simple physiological model for the regulation of the maintenance and growth of skeletal muscle, the “Bag Theory”. Beginning in London in the late 1970s the work has involved the use of stable isotopes to probe muscle protein synthesis and breakdown with two basic experimental models, primed‐dose continuous tracer infusions combined with muscle biopsies and arterio‐venous (A‐V) studies across a limb, most often the leg, allowing both protein synthesis and breakdown as well as net balance to be measured. In this way, over a 30 year period, the way in which amino acids and insulin mediate the anabolic effect of a meal has been elaborated in great detail confirming the original concepts of bag filling within the muscle endomysial “bag”, which is limited by the “bag” size unless bag enlargement occurs requiring new collagen synthesis. Finally we briefly review some new developments involving 2H2O labelling of muscle proteins. - The Journal of Physiology, EarlyView.
  

  September 07, 2018   doi: 10.1113/JP275430   open full text
• A systems perspective on placental amino acid transport.
  Jane K. Cleal, Emma M. Lofthouse, Bram G. Sengers, Rohan M. Lewis.
  The Journal of Physiology. September 07, 2018
  --- - |2+ Abstract Placental amino acid transfer is a complex process that is essential for fetal development. Impaired amino acid transfer causes fetal growth restriction, which may have lifelong health consequences. Transepithelial transfer of amino acids across the placental syncytiotrophoblast requires accumulative, exchange and facilitated transporters on the apical and basal membranes to work in concert. However, transporters alone do not determine amino acid transfer and factors that affect substrate availability, such as blood flow and metabolism, may also become rate‐limiting for transfer. In order to determine the rate‐limiting processes, it is necessary to take a systems approach which recognises the interdependence of these processes. New technologies have the potential to deliver targeted interventions to the placenta and help poorly growing fetuses. While many factors are necessary for amino acid transfer, novel therapies need to target the rate‐limiting factors if they are going to be effective. This review will outline the factors which determine amino acid transfer and describe how they become interdependent. It will also highlight the role of computational modelling as a tool to understand this process. - The Journal of Physiology, EarlyView.
  

  September 07, 2018   doi: 10.1113/JP274883   open full text
• ERG3 potassium channel‐mediated suppression of neuronal intrinsic excitability and prevention of seizure generation in mice.
  Kuo Xiao, Zhiming Sun, Xueqin Jin, Weining Ma, Yan Song, Shirong Lai, Qian Chen, Minghua Fan, Jingliang Zhang, Weihua Yue, Zhuo Huang.
  The Journal of Physiology. September 07, 2018
  --- - |2+ Key points ERG3 channels have a high expression level in the central nervous system. Knockdown of ERG3 channels enhances neuronal intrinsic excitability (caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials) in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. The expression of ERG3 protein is reduced in human and mouse hippocampal epileptogenic foci. Knockdown of ERG3 channels in hippocampus enhanced seizure susceptibility, while mice treated with the ERG channel activator NS‐1643 were less prone to epileptogenesis. The results provide strong evidence that ERG3 channels have a crucial role in the regulation of neuronal intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells and are critically involved in the onset and development of epilepsy. Abstract The input–output relationship of neuronal networks depends heavily on the intrinsic properties of their neuronal elements. Profound changes in intrinsic properties have been observed in various physiological and pathological processes, such as learning, memory and epilepsy. However, the cellular and molecular mechanisms underlying acquired changes in intrinsic excitability are still not fully understood. Here, we demonstrate that ERG3 channels are critically involved in the regulation of intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Knock‐down of ERG3 channels significantly increases neuronal intrinsic excitability, which is mainly caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials. Interestingly, the expression level of ERG3 protein is significantly reduced in human and mouse brain tissues with temporal lobe epilepsy. Moreover, ERG3 channel knockdown in hippocampus significantly enhanced seizure susceptibility, while mice treated with the ERG channel activator NS‐1643 were less prone to epileptogenesis. Taken together, our results suggest ERG3 channels play an important role in determining the excitability of hippocampal neurons and dysregulation of these channels may be involved in the generation of epilepsy. ERG3 channels may thus be a novel therapeutic target for the prevention of epilepsy. - The Journal of Physiology, Volume 596, Issue 19, Page 4729-4752, 1 October 2018.
  

  September 07, 2018   doi: 10.1113/JP275970   open full text
• A mother's gift: consequences of unhealthy diet for offspring metabolism.
  Elisa Karen Silva Ramos, Paola Visnardi Fassina, Michelle Andrade Lemos.
  The Journal of Physiology. September 07, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4575-4577, 1 October 2018.
  

  September 07, 2018   doi: 10.1113/JP276769   open full text
• The importance of exercise intensity, volume and metabolic signalling events in the induction of mitochondrial biogenesis.
  Heather L. Petrick, Kaitlyn M. J. H. Dennis, Paula M. Miotto.
  The Journal of Physiology. September 07, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4571-4572, 1 October 2018.
  

  September 07, 2018   doi: 10.1113/JP276802   open full text
• Oestrogen and a Goldilocks zone for post‐damage muscle inflammation and repair?
  Peter M. Tiidus.
  The Journal of Physiology. September 07, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4563-4564, 1 October 2018.
  

  September 07, 2018   doi: 10.1113/JP276870   open full text
• Finding the metabolic stress ‘sweet spot’: implications for sprint interval training‐induced muscle remodelling.
  Lauren E. Skelly, Jenna B. Gillen.
  The Journal of Physiology. September 06, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4573-4574, 1 October 2018.
  

  September 06, 2018   doi: 10.1113/JP276912   open full text
• Sex differences in diaphragmatic fatigue and the metaboreflex following inspiratory pressure‐threshold loading.
  Christina D. Bruce, Alexandra F. Yacyshyn, Luca Ruggiero.
  The Journal of Physiology. September 05, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4579-4580, 1 October 2018.
  

  September 05, 2018   doi: 10.1113/JP276978   open full text
• Deep continuous theta burst stimulation of the operculo‐insular cortex selectively affects Aδ‐fibre heat pain.
  Cédric Lenoir, Maxime Algoet, André Mouraux.
  The Journal of Physiology. September 04, 2018
  --- - |2+ Key points Deep continuous theta burst stimulation (cTBS) of the right operculo‐insular cortex delivered with a double cone coil selectively impairs the ability to perceive thermonociceptive input conveyed by Aδ‐fibre thermonociceptors without concomitantly affecting the ability to perceive innocuous warm, cold or vibrotactile sensations. Unlike deep cTBS, superficial cTBS of the right operculum delivered with a figure‐of‐eight coil does not affect the ability to perceive thermonociceptive input conveyed by Aδ‐fibre thermonociceptors. The effect of deep operculo‐insular cTBS on the perception of Aδ‐fibre input was present at both the contralateral and the ipsilateral hand. The magnitude of the increase in Aδ‐heat detection threshold induced by the deep cTBS was significantly correlated with the intensity of the cTBS pulses. Deep cTBS delivered over the operculo‐insular cortex is associated with a risk of transcranial magnetic stimulation‐induced seizure. Abstract Previous studies have suggested a pivotal role of the insular cortex in nociception and pain perception. Using a double‐cone coil designed for deep transcranial magnetic stimulation, our objective was to assess (1) whether continuous theta burst stimulation (cTBS) of the operculo‐insular cortex affects differentially the perception of different types of thermal and mechanical somatosensory inputs, (2) whether the induced after‐effects are lateralized relative to the stimulated hemisphere, and (3) whether the after‐effects are due to neuromodulation of the insula or neuromodulation of the more superficial opercular cortex. Seventeen participants took part in two experiments. In Experiment 1, thresholds and perceived intensity of Aδ‐ and C‐fibre heat pain elicited by laser stimulation, non‐painful cool sensations elicited by contact cold stimulation and mechanical vibrotactile sensations were assessed at the left hand before, immediately after and 20 min after deep cTBS delivered over the right operculo‐insular cortex. In Experiment 2, Aδ‐fibre heat pain and vibrotactile sensations elicited by stimulating the contralateral and ipsilateral hands were evaluated before and after deep cTBS or superficial cTBS delivered using a flat figure‐of‐eight coil. Only the threshold to detect Aδ‐fibre heat pain was significantly increased 20 min after deep cTBS. This effect was present at both hands. No effect was observed after superficial cTBS. Neuromodulation of the operculo‐insular cortex using deep cTBS induces a bilateral reduction of the ability to perceive Aδ‐fibre heat pain, without concomitantly affecting the ability to perceive innocuous warm, cold or vibrotactile sensations. - The Journal of Physiology, Volume 596, Issue 19, Page 4767-4787, 1 October 2018.
  

  September 04, 2018   doi: 10.1113/JP276359   open full text
• Evolving systems biology approaches to understanding non‐coding RNAs in pulmonary hypertension.
  Lloyd D. Harvey, Stephen Y. Chan.
  The Journal of Physiology. September 03, 2018
  --- - |2 Abstract Our appreciation of the roles of non‐coding RNAs, in particular microRNAs, in the manifestation of pulmonary hypertension (PH) has advanced considerably over the past decade. Comprised of small nucleotide sequences, microRNAs have demonstrated critical and broad regulatory roles in the pathogenesis of PH via the direct binding to messenger RNA transcripts for degradation or inhibition of translation, thereby exerting a profound influence on cellular activity. Yet, as inherently pleiotropic molecules, microRNAs have been difficult to study using traditional, reductionist approaches alone. With the advent of high‐throughput –omics technologies and more advanced computational modelling, the study of microRNAs and their multi‐faceted and complex functions in human disease serves as a fertile platform for the application of systems biology methodologies in combination with traditional experimental techniques. Here, we offer our viewpoint of past successes of systems biology in elucidating the otherwise hidden actions of microRNAs in PH, as well as areas for future development to integrate these strategies into the discovery of RNA pathobiology in this disease. We contend that such successful applications of systems biology in elucidating the functional architecture of microRNA regulation will further reveal the molecular mechanisms of disease, while simultaneously revealing potential diagnostic and therapeutic strategies in disease amelioration. - The Journal of Physiology, EarlyView.
  

  September 03, 2018   doi: 10.1113/JP275855   open full text
• Fatigue‐related group III/IV muscle afferent feedback facilitates intracortical inhibition during locomotor exercise.
  Simranjit K. Sidhu, Joshua C. Weavil, Taylor S. Thurston, Dorothea Rosenberger, Jacob E. Jessop, Eivind Wang, Russell S. Richardson, Chris J. McNeil, Markus Amann.
  The Journal of Physiology. September 03, 2018
  --- - |2+ Key Points This study investigated the influence of group III/IV muscle afferents on corticospinal excitability during cycling exercise and focused on GABAB neuron‐mediated inhibition as a potential underlying mechanism. The study provides novel evidence to demonstrate that group III/IV muscle afferent feedback facilitates inhibitory intracortical neurons during whole body exercise. Firing of these interneurons probably contributes to the development of central fatigue during physical activity. Abstract We investigated the influence of group III/IV muscle afferents in determining corticospinal excitability during cycling exercise and focused on GABAB neuron‐mediated inhibition as a potential underlying mechanism. Both under control conditions (CTRL) and with lumbar intrathecal fentanyl (FENT) impairing feedback from group III/IV leg muscle afferents, subjects (n = 11) cycled at a comparable vastus‐lateralis EMG signal (∼0.26 mV) before (PRE; 100 W) and immediately after (POST; 90 ± 2 W) fatiguing constant‐load cycling exercise (80% Wpeak; 221 ± 10 W; ∼8 min). During, PRE and POST cycling, single and paired‐pulse (100 ms interstimulus interval) transcranial magnetic stimulations (TMS) were applied to elicit unconditioned and conditioned motor‐evoked potentials (MEPs), respectively. To distinguish between cortical and spinal contributions to the MEPs, cervicomedullary stimulations (CMS) were used to elicit unconditioned (CMS only) and conditioned (TMS+CMS, 100 ms interval) cervicomedullary motor‐evoked potentials (CMEPs). While unconditioned MEPs were unchanged from PRE to POST in CTRL, unconditioned CMEPs increased significantly, resulting in a decrease in unconditioned MEP/CMEP (P < 0.05). This paralleled a reduction in conditioned MEP (P < 0.05) and no change in conditioned CMEP. During FENT, unconditioned and conditioned MEPs and CMEPs were similar and comparable during PRE and POST (P > 0.2). These findings reveal that feedback from group III/IV muscle afferents innervating locomotor muscle decreases the excitability of the motor cortex during fatiguing cycling exercise. This impairment is, at least in part, determined by the facilitating effect of these sensory neurons on inhibitory GABAB intracortical interneurons. - The Journal of Physiology, Volume 596, Issue 19, Page 4789-4801, 1 October 2018.
  

  September 03, 2018   doi: 10.1113/JP276460   open full text
• Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain.
  Shinichiro Kume, Takushi Shimomura, Michihiro Tateyama, Yoshihiro Kubo.
  The Journal of Physiology. September 03, 2018
  --- - |2+ Key points In the human ether‐a‐go‐go related gene (hERG) channel, both the ether‐a‐go‐go (EAG) domain in the N‐terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C‐terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C‐linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C‐linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage‐dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C‐terminal domains is a result of the lack of interaction between the EAG and CNBH domains. Abstract The human ether‐a‐go‐go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N‐terminal cytoplasmic ether‐a‐go‐go (EAG) domain and the C‐terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C‐linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter‐domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C‐linker domain, which is reported to be critical for the function of CN‐regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter‐domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage‐dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain. - The Journal of Physiology, Volume 596, Issue 19, Page 4629-4650, 1 October 2018.
  

  September 03, 2018   doi: 10.1113/JP276208   open full text
• Ultrastructural basis of strong unitary inhibition in a binaural neuron.
  Enida Gjoni, Clémentine Aguet, Daniela A. Sahlender, Graham Knott, Ralf Schneggenburger.
  The Journal of Physiology. September 02, 2018
  --- - |2+ Key points Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1–11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. Abstract Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr2+ method, we find a constant quantal size but a variable quantal content between ‘weak’ and ‘strong’ unitary IPSCs. A 3‐D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7 μm), which contain several active zones (range 1–11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi‐active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons. - The Journal of Physiology, Volume 596, Issue 20, Page 4969-4982, 15 October 2018.
  

  September 02, 2018   doi: 10.1113/JP276015   open full text
• Phase waves and trigger waves: emergent properties of oscillating and excitable networks in the gut.
  Sean P. Parsons, Jan D. Huizinga.
  The Journal of Physiology. August 31, 2018
  --- - |2 Abstract The gut is enmeshed by a number of cellular networks, but there is only a limited understanding of how these networks generate the complex patterns of activity that drive gut contractile functions. Here we review two fundamental types of cell behaviour, excitable and oscillating, and the patterns that networks of such cells generate, trigger waves and phase waves, respectively. We use both the language of biophysics and the theory of nonlinear dynamics to define these behaviours and understand how they generate patterns. Based on this we look for evidence of trigger and phase waves in the gut, including some of our recent work on the small intestine. - The Journal of Physiology, Volume 596, Issue 20, Page 4819-4829, 15 October 2018.
  

  August 31, 2018   doi: 10.1113/JP273425   open full text
• Keto diets: good, bad or ugly?
  Mark Evans.
  The Journal of Physiology. August 31, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4561-4561, 1 October 2018.
  

  August 31, 2018   doi: 10.1113/JP276703   open full text
• Blocking slow exocytosis with slow Ca2+ buffers slows recovery from depression.
  Skyler Jackman, Henrique Gersdorff.
  The Journal of Physiology. August 31, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4555-4557, 1 October 2018.
  

  August 31, 2018   doi: 10.1113/JP276673   open full text
• Redox‐regulation of haemostasis in hypoxic exercising humans: a randomised double‐blind placebo‐controlled antioxidant study.
  Lewis Fall, Julien V. Brugniaux, Danielle Davis, Christopher J. Marley, Bruce Davies, Karl J. New, Jane McEneny, Ian S. Young, Damian M. Bailey.
  The Journal of Physiology. August 29, 2018
  --- - |2+ Key points In vitro evidence has identified that coagulation is activated by increased oxidative stress, though the link and underlying mechanism in humans have yet to be established. We conducted the first randomised controlled trial in healthy participants to examine if oral antioxidant prophylaxis alters the haemostatic responses to hypoxia and exercise given their synergistic capacity to promote free radical formation. Systemic free radical formation was shown to increase during hypoxia and was further compounded by exercise, responses that were attenuated by antioxidant prophylaxis. In contrast, antioxidant prophylaxis increased thrombin generation at rest in normoxia, and this was normalised only in the face of prevailing oxidation. Collectively, these findings suggest that human free radical formation is an adaptive phenomenon that serves to maintain vascular haemostasis. Abstract In vitro evidence suggests that blood coagulation is activated by increased oxidative stress although the link and underlying mechanism in humans have yet to be established. We conducted the first randomised controlled trial to examine if oral antioxidant prophylaxis alters the haemostatic responses to hypoxia and exercise. Healthy males were randomly assigned double‐blind to either an antioxidant (n = 20) or placebo group (n = 16). The antioxidant group ingested two capsules/day that each contained 500 mg of l‐ascorbic acid and 450 international units (IU) of dl‐α‐tocopherol acetate for 8 weeks. The placebo group ingested capsules of identical external appearance, taste and smell (cellulose). Both groups were subsequently exposed to acute hypoxia and maximal physical exercise with venous blood sampled pre‐supplementation (normoxia), post‐supplementation at rest (normoxia and hypoxia) and following maximal exercise (hypoxia). Systemic free radical formation (electron paramagnetic resonance spectroscopic detection of the ascorbate radical (A•−)) increased during hypoxia (15,152 ± 1193 AU vs. 14,076 ± 810 AU at rest, P < 0.05) and was further compounded by exercise (16,569 ± 1616 AU vs. rest, P < 0.05), responses that were attenuated by antioxidant prophylaxis. In contrast, antioxidant prophylaxis increased thrombin generation as measured by thrombin–antithrombin complex, at rest in normoxia (28.7 ± 6.4 vs. 4.3 ± 0.2 μg mL−1 pre‐intervention, P < 0.05) and was restored but only in the face of prevailing oxidation. Collectively, these findings are the first to suggest that human free radical formation likely reflects an adaptive response that serves to maintain vascular haemostasis. - The Journal of Physiology, Volume 596, Issue 20, Page 4879-4891, 15 October 2018.
  

  August 29, 2018   doi: 10.1113/JP276414   open full text
• Maternal obesity has sex‐dependent effects on insulin, glucose and lipid metabolism and the liver transcriptome in young adult rat offspring.
  Consuelo Lomas‐Soria, Luis A. Reyes‐Castro, Guadalupe L. Rodríguez‐González, Carlos A. Ibáñez, Claudia J. Bautista, Laura A. Cox, Peter W. Nathanielsz, Elena Zambrano.
  The Journal of Physiology. August 29, 2018
  --- - |2+ Key points Maternal high‐fat diet consumption predisposes to metabolic dysfunction in male and female offspring at young adulthood. Maternal obesity programs non‐alcoholic fatty liver disease (NAFLD) in a sex‐dependent manner. We demonstrate sex‐dependent liver transcriptome profiles in rat offspring of obese mothers. In this study, we focused on pathways related to insulin, glucose and lipid signalling. These results improve understanding of the mechanisms by which a maternal high‐fat diet affects the offspring. Abstract Maternal obesity (MO) predisposes offspring (F1) to obesity, insulin resistance (IR) and non‐alcoholic fatty liver disease (NAFLD). MO's effects on the F1 liver transcriptome are poorly understood. We used RNA‐seq to determine the liver transcriptome of male and female F1 of MO and control‐fed mothers. We hypothesized that MO‐F1 are predisposed to sex‐dependent adult liver dysfunction. Female Wistar rat mothers ate a control (C) or obesogenic (MO) diet from the time they were weaned through breeding at postnatal day (PND) 120, delivery and lactation. After weaning, all male and female F1 ate a control diet. At PND 110, F1 serum, liver and fat were collected to analyse metabolites, histology and liver differentially expressed genes. Male and female MO‐F1 showed increased adiposity index, triglycerides, insulin and homeostatic model assessment vs. C‐F1 with similar body weight and glucose serum concentrations. MO‐F1 males presented greater physiological and histological NAFLD characteristics than MO‐F1 females. RNA‐seq revealed 1365 genes significantly changed in male MO‐F1 liver and only 70 genes in female MO‐F1 compared with controls. GO and KEGG analysis identified differentially expressed genes related to metabolic processes. Male MO‐F1 liver showed the following altered pathways: insulin signalling (22 genes), phospholipase D signalling (14 genes), NAFLD (13 genes) and glycolysis/gluconeogenesis (7 genes). In contrast, few genes were altered in these pathways in MO‐F1 females. In summary, MO programs sex‐dependent F1 changes in insulin, glucose and lipid signalling pathways, leading to liver dysfunction and insulin resistance. - The Journal of Physiology, Volume 596, Issue 19, Page 4611-4628, 1 October 2018.
  

  August 29, 2018   doi: 10.1113/JP276372   open full text
• The muscle anabolic effect of protein ingestion during a hyperinsulinaemic euglycaemic clamp in middle‐aged women is not caused by leucine alone.
  Stephan Vliet, Gordon I. Smith, Lane Porter, Raja Ramaswamy, Dominic N. Reeds, Adewole L. Okunade, Jun Yoshino, Samuel Klein, Bettina Mittendorfer.
  The Journal of Physiology. August 29, 2018
  --- - |2+ Key points It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic‐euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased muscle p‐eIF2αSer51 and increased muscle protein synthesis. Abstract It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We tested this hypothesis by measuring muscle p‐mTORSer2448, p‐p70S6KThr389 and p‐eIF2αSer51, as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45 g protein kg−1 fat‐free mass (containing 0.0513 g leucine kg−1 fat‐free mass) or a control drink (n = 14) or 0.0513 g leucine kg−1 fat‐free mass or a control drink (n = 14) during a hyperinsulinaemic‐euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p‐mTORSer2448 and p‐p70S6KThr389 by >50% (all P < 0.05) but had no effect on p‐eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased (by ∼35%) p‐eIF2αSer51 and increased (by ∼100%) MPS (all P < 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation. - The Journal of Physiology, Volume 596, Issue 19, Page 4681-4692, 1 October 2018.
  

  August 29, 2018   doi: 10.1113/JP276504   open full text
• Legacy of excess: consequences of maternal obesity for the adult offspring.
  Alison J. Forhead.
  The Journal of Physiology. August 29, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4559-4560, 1 October 2018.
  

  August 29, 2018   doi: 10.1113/JP276762   open full text
• Age‐dependent effects on sympathetic responsiveness in cardiac action potential conduction and calcium handling.
  Xavier Alexander Lee, Neal Ingraham Callaghan.
  The Journal of Physiology. August 29, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4569-4570, 1 October 2018.
  

  August 29, 2018   doi: 10.1113/JP276950   open full text
• Spinal dorsal horn astrocytes release GABA in response to synaptic activation.
  Rasmus Kordt Christensen, Rodolfo Delgado‐Lezama, Raúl E. Russo, Barbara Lykke Lind, Emanuel Loeza Alcocer, Martin Fredensborg Rath, Gabriela Fabbiani, Nicole Schmitt, Martin Lauritzen, Anders Victor Petersen, Eva Meier Carlsen, Jean‐François Perrier.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Key points GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing. Abstract Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red‐eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABAA receptors. By positioning the sensor recorded in the whole‐cell patch‐clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca2+, we concluded that they originated from non‐neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca2+ increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca2+ in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs. - The Journal of Physiology, Volume 596, Issue 20, Page 4983-4994, 15 October 2018.
  

  August 28, 2018   doi: 10.1113/JP276562   open full text
• Hepatic mitochondrial adaptations to physical activity: impact of sexual dimorphism, PGC1α and BNIP3‐mediated mitophagy.
  Alex Schulze, Colin S. McCoin, Chiemela Onyekere, Julie Allen, Paige Geiger, Gerald W. Dorn, E. Matthew Morris, John P. Thyfault.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Key points Hepatic mitochondrial adaptations to physical activity may be regulated by mitochondrial biogenesis (PGC1α) and mitophagy (BNIP3). Additionally, these adaptations may be sex‐dependent. Chronic increase in physical activity lowers basal mitochondrial respiratory capacity in mice. Female mice have higher hepatic electron transport system protein content, elevated respiratory capacity, lowered mitophagic flux, and emit less mitochondrial H2O2 independent of physical activity. Males require chronic daily physical activity to attain a similar mitochondrial phenotype compared to females. In contrast, females have limited hepatic adaptations to chronic physical activity. Livers deficient in PGC1α and BNIP3 display similar mitochondrial adaptations to physical activity to those found in wild‐type mice. Abstract Hepatic mitochondrial adaptations to physical activity may be regulated by biogenesis‐ and mitophagy‐associated pathways in a sex‐dependent manner. Here, we tested if mice with targeted deficiencies in liver‐specific peroxisome proliferator‐activated receptor γ coactivator 1α (PGC1α; LPGC1α+/−) and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3)‐mediated mitophagy (BNIP3−/−) would have reduced physical activity‐induced adaptations in respiratory capacity, H2O2 emission and mitophagy compared to wild‐type (WT) controls and if these effects were impacted by sex. Male and female WT, LPGC1α+/− and BNIP3−/− C57BL6/J mice were divided into groups that remained sedentary or had access to daily physical activity via voluntary wheel running (VWR) (n = 6–10/group) for 4 weeks. Mice had ad libitum access to low‐fat diet and water. VWR reduced basal mitochondrial respiration, increased mitochondrial coupling and altered ubiquitin‐mediated mitophagy in a sex‐specific manner in WT mice. Female mice of all genotypes displayed higher electron transport system content, displayed increased ADP‐stimulated respiration, produced less mitochondrially derived reactive oxygen species, exhibited reduced mitophagic flux, and were less responsive to VWR compared to males. Males responded more robustly to VWR‐induced changes in hepatic mitochondrial function resulting in a match to adaptations found in females. Deficiencies in PGC1α and BNIP3 alone did not largely alter mitochondrial adaptations to VWR. However, VWR restored sex‐dependent abnormalities in mitophagic flux in LPGC1α+/−. Finally, BNIP3−/− mice had elevated mitochondrial content and increased mitochondrial respiration putatively through repressed mitophagic flux. In conclusion, hepatic mitochondrial adaptations to physical activity are more dependent on sex than PGC1α and BNIP3. - The Journal of Physiology, EarlyView.
  

  August 28, 2018   doi: 10.1113/JP276539   open full text
• Short‐chain fatty acids: microbial metabolites that alleviate stress‐induced brain–gut axis alterations.
  Marcel de Wouw, Marcus Boehme, Joshua M. Lyte, Niamh Wiley, Conall Strain, Orla O'Sullivan, Gerard Clarke, Catherine Stanton, Timothy G. Dinan, John F. Cryan.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Key points Chronic (psychosocial) stress changes gut microbiota composition, as well as inducing behavioural and physiological deficits. The microbial metabolites short‐chain fatty acids (SCFAs) have been implicated in gastrointestinal functional, (neuro)immune regulation and host metabolism, but their role in stress‐induced behavioural and physiological alterations is poorly understood. Administration of SCFAs to mice undergoing psychosocial stress alleviates enduring alterations in anhedonia and heightened stress‐responsiveness, as well as stress‐induced increases in intestinal permeability. In contrast, chronic stress‐induced alterations in body weight gain, faecal SCFAs and the gene expression of the SCFA receptors FFAR2 and FFAR3 remained unaffected by SCFA supplementation. These results present novel insights into mechanisms underpinning the influence of the gut microbiota on brain homeostasis, behaviour and host metabolism, informing the development of microbiota‐targeted therapies for stress‐related disorders. Abstract There is a growing recognition of the involvement of the gastrointestinal microbiota in the regulation of physiology and behaviour. Microbiota‐derived metabolites play a central role in the communication between microbes and their host, with short‐chain fatty acids (SCFAs) being perhaps the most studied. SCFAs are primarily derived from fermentation of dietary fibres and play a pivotal role in host gut, metabolic and immune function. All these factors have previously been demonstrated to be adversely affected by stress. Therefore, we sought to assess whether SCFA supplementation could counteract the enduring effects of chronic psychosocial stress. C57BL/6J male mice received oral supplementation of a mixture of the three principle SCFAs (acetate, propionate and butyrate). One week later, mice underwent 3 weeks of repeated psychosocial stress, followed by a comprehensive behavioural analysis. Finally, plasma corticosterone, faecal SCFAs and caecal microbiota composition were assessed. SCFA treatment alleviated psychosocial stress‐induced alterations in reward‐seeking behaviour, and increased responsiveness to an acute stressor and in vivo intestinal permeability. In addition, SCFAs exhibited behavioural test‐specific antidepressant and anxiolytic effects, which were not present when mice had also undergone psychosocial stress. Stress‐induced increases in body weight gain, faecal SCFAs and the colonic gene expression of the SCFA receptors free fatty acid receptors 2 and 3 remained unaffected by SCFA supplementation. Moreover, there were no collateral effects on caecal microbiota composition. Taken together, these data show that SCFA supplementation alleviates selective and enduring alterations induced by repeated psychosocial stress and these data may inform future research into microbiota‐targeted therapies for stress‐related disorders. - The Journal of Physiology, Volume 596, Issue 20, Page 4923-4944, 15 October 2018.
  

  August 28, 2018   doi: 10.1113/JP276431   open full text
• Sensors and signals: the role of reactive oxygen species in hypoxic pulmonary vasoconstriction.
  Kimberly A. Smith, Paul T. Schumacker.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Abstract When lung cells experience hypoxia, the functional response, termed hypoxic pulmonary vasoconstriction, activates a multitude of pathways with the goal of optimizing gas exchange. While previously controversial, overwhelming evidence now suggests that increased reactive oxygen species – produced at complex III of the mitochondrial electron transport chain and released into the intermembrane space – is the cellular oxygen signal responsible for triggering hypoxic pulmonary vasoconstriction. The increased reactive oxygen species (ROS) activate many downstream targets that ultimately lead to increased intracellular ionized calcium concentration and contraction of pulmonary arterial smooth muscle cells. While the specific targets of ROS signals are not completely understood, it is clear that this signalling pathway is critical for development and for normal lung function in newborns and adults. - The Journal of Physiology, EarlyView.
  

  August 28, 2018   doi: 10.1113/JP275852   open full text
• Specific synaptic input strengths determine the computational properties of excitation–inhibition integration in a sound localization circuit.
  Enida Gjoni, Friedemann Zenke, Brice Bouhours, Ralf Schneggenburger.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Key points During the computation of sound localization, neurons of the lateral superior olive (LSO) integrate synaptic excitation arising from the ipsilateral ear with inhibition from the contralateral ear. We characterized the functional connectivity of the inhibitory and excitatory inputs onto LSO neurons in terms of unitary synaptic strength and convergence. Unitary IPSCs can generate large conductances, although their strength varies over a 10‐fold range in a given recording. By contrast, excitatory inputs are relatively weak. The conductance associated with IPSPs needs to be at least 2‐fold stronger than the excitatory one to guarantee effective inhibition of action potential (AP) firing. Computational modelling showed that strong unitary inhibition ensures an appropriate slope and midpoint of the tuning curve of LSO neurons. Conversely, weak but numerous excitatory inputs filter out spontaneous AP firing from upstream auditory neurons. Abstract The lateral superior olive (LSO) is a binaural nucleus in the auditory brainstem in which excitation from the ipsilateral ear is integrated with inhibition from the contralateral ear. It is unknown whether the strength of the unitary inhibitory and excitatory inputs is adapted to allow for optimal tuning curves of LSO neuron action potential (AP) firing. Using electrical and optogenetic stimulation of afferent synapses, we found that the strength of unitary inhibitory inputs to a given LSO neuron can vary over a ∼10‐fold range, follows a roughly log‐normal distribution, and, on average, causes a large conductance (9 nS). Conversely, unitary excitatory inputs, stimulated optogenetically under the bushy‐cell specific promoter Math5, were numerous, and each caused a small conductance change (0.7 nS). Approximately five to seven bushy cell inputs had to be active simultaneously to bring an LSO neuron to fire. In double stimulation experiments, the effective inhibition window caused by IPSPs was short (1−3 ms) and its length depended on the inhibitory conductance; an ∼2‐fold stronger inhibition than excitation was needed to suppress AP firing. Computational modelling suggests that few, but strong, unitary IPSPs create a tuning curve of LSO neuron firing with an appropriate slope and midpoint. Furthermore, weak but numerous excitatory inputs reduce the spontaneous AP firing that LSO neurons would otherwise inherit from their upstream auditory neurons. Thus, the specific connectivity and strength of unitary excitatory and inhibitory inputs to LSO neurons is optimized for the computations performed by these binaural neurons. - The Journal of Physiology, Volume 596, Issue 20, Page 4945-4967, 15 October 2018.
  

  August 28, 2018   doi: 10.1113/JP276012   open full text
• Insulin transport across the blood–brain barrier can occur independently of the insulin receptor.
  Elizabeth M. Rhea, Christian Rask‐Madsen, William A. Banks.
  The Journal of Physiology. August 28, 2018
  --- - |2+ Key points Insulin enters the brain from the blood via a saturable transport system. It is unclear how insulin is transported across the blood–brain barrier (BBB). Using two models of the signalling‐related insulin receptor loss or inhibition, we show insulin transport can occur in vivo without the signalling‐related insulin receptor. Insulin in the brain has multiple roles including acting as a metabolic regulator and improving memory. Understanding how insulin is transported across the BBB will aid in developing therapeutics to further increase CNS concentrations. Abstract A saturable system transports insulin from blood across the blood–brain barrier (BBB) and into the central nervous system. Whether or not the classic or signalling‐related insulin receptor plays a role in mediating this transport in vivo is controversial. Here, we employed kinetics methods that distinguish between transport across the brain endothelial cell and reversible luminal surface receptor binding. Using a previously established line of mice with endothelial‐specific loss of the signalling‐related insulin receptor (EndoIRKO) or inhibiting the insulin receptor with the selective antagonist S961, we show insulin transport across the BBB is maintained. Rates of insulin transport were similar in all groups and transport was still saturable. Unlike transport, binding of insulin to the brain endothelial cell was decreased with the loss or inhibition of the signalling‐related insulin receptor. These findings demonstrate that the signalling‐related insulin receptor is not required for insulin transport across the BBB. - The Journal of Physiology, Volume 596, Issue 19, Page 4753-4765, 1 October 2018.
  

  August 28, 2018   doi: 10.1113/JP276149   open full text
• Jack‐of‐many‐trades: discovering new roles for troponin C.
  Kerry S. McDonald.
  The Journal of Physiology. August 28, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4553-4554, 1 October 2018.
  

  August 28, 2018   doi: 10.1113/JP276790   open full text
• Effects of living at moderate altitude on pulmonary vascular function and exercise capacity in mice with sickle cell anaemia.
  Scott K. Ferguson, Katherine Redinius, Ayla Yalamanoglu, Julie W. Harral, Jin Hyen Baek, David Pak, Zoe Loomis, Daniel Hassell, Paul Eigenberger, Eva Nozik‐Grayck, Rachelle Nuss, Kathryn Hassell, Kurt R. Stenmark, Paul W. Buehler, David C. Irwin.
  The Journal of Physiology. August 26, 2018
  --- - |2+ Key points Sickle cell disease (SCD) results in cardiopulmonary dysfunction, which may be exacerbated by prolonged exposure to environmental hypoxia. It is currently unknown whether exposure to mild and moderate altitude exacerbates SCD associated cardiopulmonary and systemic complications. Three months of exposure to mild (1609 m) and moderate (2438 m) altitude increased rates of haemolysis and right ventricular systolic pressures in mice with SCD compared to healthy wild‐type cohorts and SCD mice at sea level. The haemodynamic changes in SCD mice that had lived at mild and moderate altitude were accompanied by changes in the balance between pulmonary vascular endothelial nitric oxide synthase and endothelin receptor expression and impaired exercise tolerance. These data demonstrate that chronic altitude exposure exacerbates the complications associated with SCD and provides pertinent information for the clinical counselling of SCD patients. Abstract Exposure to high altitude worsens symptoms and crises in patients with sickle cell disease (SCD). However, it remains unclear whether prolonged exposure to low barometric pressures exacerbates SCD aetiologies or impairs quality of life. We tested the hypothesis that, relative to wild‐type (WT) mice, Berkley sickle cell mice (BERK‐SS) residing at sea level, mild (1609 m) and moderate (2438 m) altitude would have a higher rate of haemolysis, impaired cardiac function and reduced exercise tolerance, and that the level of altitude would worsen these decrements. Following 3 months of altitude exposure, right ventricular systolic pressure was measured (solid‐state transducer). In addition, the adaptive balance between pulmonary vascular endothelial nitric oxide synthase and endothelin was assessed in lung tissue to determine differences in pulmonary vascular adaptation and the speed/duration relationship (critical speed) was used to evaluate treadmill exercise tolerance. At all altitudes, BERK‐SS mice had a significantly lower percentage haemocrit and higher total bilirubin and free haemoglobin concentration (P < 0.05 for all). right ventricular systolic pressures in BERK‐SS were higher than WT at moderate altitude and also compared to BERK‐SS at sea level (P < 0.05, for both). Critical speed was significantly lower in BERK‐SS at mild and moderate altitude (P < 0.05). BERK‐SS demonstrated exacerbated SCD complications and reduced exercise capacity associated with an increase in altitude. These results suggest that exposure to mild and moderate altitude enhances the progression of SCD in BERK‐SS mice compared to healthy WT cohorts and BERK‐SS mice at sea level and provides crucial information for the clinical counselling of SCD patients. - The Journal of Physiology, EarlyView.
  

  August 26, 2018   doi: 10.1113/JP275810   open full text
• Renewed excitement for paraventricular neurons and sympathetic nerve activity.
  Susan M. Barman.
  The Journal of Physiology. August 25, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4551-4552, 1 October 2018.
  

  August 25, 2018   doi: 10.1113/JP276813   open full text
• Emerging views of how changes in blood pressure influence cerebral blood flow.
  Hannah G. Caldwell.
  The Journal of Physiology. August 25, 2018
  --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4565-4567, 1 October 2018.
  

  August 25, 2018   doi: 10.1113/JP276800   open full text
• Long‐term pulmonary vascular consequences of perinatal insults.
  Kara Goss.
  The Journal of Physiology. August 24, 2018
  --- - |2+ Abstract Development of the pulmonary circulation is a critical component of fetal lung development, and continues throughout infancy and childhood, marking an extended window of susceptibility to vascular maldevelopment and maladaptation. Perinatal vascular insults may result in abnormal vascular structure or function, including decreased angiogenic signaling and vascular endowment, impaired vasoreactivity through increased pulmonary artery endothelial dysfunction and remodeling, or enhanced genetic susceptibility to pulmonary vascular disease through epigenetic modifications or germline mutations. Although some infants develop early onset pulmonary hypertension, due to the unique adaptive capabilities of the immature host many do not have clinically evident early pulmonary vascular dysfunction. These individuals remain at increased risk for development of late‐onset pulmonary hypertension, and may be particularly susceptible to secondary insults. This review will address the role of perinatal vascular insults in the development of late pulmonary vascular dysfunction with an effort to highlight areas of critical research need. - The Journal of Physiology, EarlyView.
  

  August 24, 2018   doi: 10.1113/JP275859   open full text
• A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated.
  Gengyun Le, Susan A. Novotny, Tara L. Mader, Sarah M. Greising, Sunny S. K. Chan, Michael Kyba, Dawn A. Lowe, Gordon L. Warren.
  The Journal of Physiology. August 24, 2018
  --- - |2+ Key points The female hormone oestrogen may protect muscle from injury by reducing inflammation but this is debatable. In this study, the inflammatory response of injured muscle from oestrogen‐replete mice was comprehensively compared to that from oestrogen‐deficient mice. We show that oestrogen markedly promotes movement of neutrophils, an inflammatory white blood cell type, into muscle over the first few days after injury but has only a minor effect on the movement of macrophages, another inflammatory cell type. Despite the enhancement of inflammation by oestrogen in injured muscle, we found strength in oestrogen‐replete mice to recover faster and to a greater extent than it does in oestrogen‐deficient mice. Our study and others indicate that lower doses of oestrogen, such as that used in our study, may affect muscle inflammation and injury differently from higher doses. Abstract Oestrogen has been shown to protect against skeletal muscle injury and a reduced inflammatory response has been suggested as a possible protective mechanism. There are, however, dissenting reports. Our objective was to conduct an unbiased, comprehensive study of the effect of oestradiol on the inflammatory response following muscle injury. Female C57BL6/J mice were ovariectomized and supplemented with and without oestradiol. Tibialis anterior muscles were freeze injured and studied primarily at 1–4 days post‐injury. Oestradiol supplementation increased injured muscle gene expression of neutrophil chemoattractants (Cxcl1 and Cxcl5) and to a lesser extent that of monocyte/macrophage chemoattractants (Ccl2 and Spp1). Oestradiol markedly increased gene expression of the neutrophil cell surface marker (Ly6g) but had less consistent effects on the monocyte/macrophage cell surface markers (Cd68, Cd163 and Cd206). These results were confirmed at the protein level by immunoblot with oestradiol increasing LY6G/C content and having no significant effect on CD163 content. These findings were confirmed with fluorescence‐activated cell sorting counts of neutrophils and macrophages in injured muscles; oestradiol increased the proportion of CD45+ cells that were neutrophils (LY6G+) but not the proportion that were macrophages (CD68+ or CD206+). Physiological impact of the oestradiol‐enhanced neutrophil response was assessed by strength measurements. There was no significant difference in strength between oestradiol‐supplemented and ‐unsupplemented mice until 2 weeks post‐injury; strength was 13–24% greater in supplemented mice at 2–6 weeks post‐injury. In conclusion, a moderate level of oestradiol supplementation enhances neutrophil infiltration in injured muscle and this is associated with a beneficial effect on strength recovery. - The Journal of Physiology, Volume 596, Issue 19, Page 4665-4680, 1 October 2018.
  

  August 24, 2018   doi: 10.1113/JP276432   open full text
• Angiogenesis in the lung.
  Lindsey Eldridge, Elizabeth M. Wagner.
  The Journal of Physiology. August 18, 2018
  --- - |2+ Abstract Both systemic (tracheal and bronchial) and pulmonary circulations perfuse the lung. However, documentation of angiogenesis of either is complicated by the presence of the other. Well‐documented angiogenesis of the systemic circulations have been identified in asthma, cystic fibrosis, chronic thromboembolism and primary carcinomas. Angiogenesis of the vasa vasorum, which are branches of bronchial arteries, is seen in the walls of large pulmonary vessels after a period of chronic hypoxia. Documentation of increased pulmonary capillaries has been shown in models of chronic hypoxia, after pneumonectomy and in some carcinomas. Although endothelial cell proliferation may occur as part of the repair process in several pulmonary diseases, it is separate from the unique establishment of new functional perfusing networks defined as angiogenesis. Identification of the mechanisms driving the expansion of new vascular beds in the adult needs further investigation. Yet the growth factors and molecular mechanisms of lung angiogenesis remain difficult to separate from underlying disease sequelae. - The Journal of Physiology, EarlyView.
  

  August 18, 2018   doi: 10.1113/JP275860   open full text
• The impact of immobilisation and inflammation on the regulation of muscle mass and insulin resistance: different routes to similar end‐points.
  Hannah Crossland, Sarah Skirrow, Zudin A. Puthucheary, Dumitru Constantin‐Teodosiu, Paul L. Greenhaff.
  The Journal of Physiology. August 18, 2018
  --- - |2 Abstract Loss of muscle mass and insulin sensitivity are common phenotypic traits of immobilisation and increased inflammatory burden. The suppression of muscle protein synthesis is the primary driver of muscle mass loss in human immobilisation, and includes blunting of post‐prandial increases in muscle protein synthesis. However, the mechanistic drivers of this suppression are unresolved. Immobilisation also induces limb insulin resistance in humans, which appears to be attributable to the reduction in muscle contraction per se. Again mechanistic insight is missing such that we do not know how muscle senses its “inactivity status” or whether the proposed drivers of muscle insulin resistance are simply arising as a consequence of immobilisation. A heightened inflammatory state is associated with major and rapid changes in muscle protein turnover and mass, and dampened insulin‐stimulated glucose disposal and oxidation in both rodents and humans. A limited amount of research has attempted to elucidate molecular regulators of muscle mass loss and insulin resistance during increased inflammatory burden, but rarely concurrently. Nevertheless, there is evidence that Akt (protein kinase B) signalling and FOXO transcription factors form part of a common signalling pathway in this scenario, such that molecular cross‐talk between atrophy and insulin signalling during heightened inflammation is believed to be possible. To conclude, whilst muscle mass loss and insulin resistance are common end‐points of immobilisation and increased inflammatory burden, a lack of understanding of the mechanisms responsible for these traits exists such that a substantial gap in understanding of the pathophysiology in humans endures. - The Journal of Physiology, EarlyView.
  

  August 18, 2018   doi: 10.1113/JP275444   open full text
• Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.
  Satoshi Koba, Eri Hanai, Nao Kumada, Naoya Kataoka, Kazuhiro Nakamura, Tatsuo Watanabe.
  The Journal of Physiology. August 18, 2018
  --- - |2+ Key points Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN‐RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN‐RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. Abstract The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN‐RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN‐RVLM neurons. Male rats received microinjections into the PVN with an adeno‐associated virus (AAV) vector that encoded a hybrid of channelrhodopsin‐2/1 with the reporter tdTomato (ChIEF‐tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2‐GFPretro). Under anaesthesia with urethane and α‐chloralose, photostimulation (473 nm wavelength) of PVN‐RVLM neurons, achieved by laser illumination of either RVLM of ChIEF‐tdTomato rats (n = 8) or PVN of ChR2‐GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF‐tdTomato rats were closely associated with tdTomato‐labelled, PVN‐derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF‐tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN‐RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons. - The Journal of Physiology, Volume 596, Issue 19, Page 4581-4595, 1 October 2018.
  

  August 18, 2018   doi: 10.1113/JP276223   open full text
• Novel mechanisms regulating endothelial barrier function in the pulmonary microcirculation.
  Szandor Simmons, Lasti Erfinanda, Christoph Bartz, Wolfgang M. Kuebler.
  The Journal of Physiology. August 14, 2018
  --- - |2 Abstract The pulmonary epithelial and vascular endothelial cell layers provide two sequential physical and immunological barriers that together form a semi‐permeable interface and prevent alveolar and interstitial oedema formation. In this review, we focus specifically on the continuous endothelium of the pulmonary microvascular bed that warrants strict control of the exchange of gases, fluid, solutes and circulating cells between the plasma and the interstitial space. The present review provides an overview of emerging molecular mechanisms that permit constant transcellular exchange between the vascular and interstitial compartment, and cause, prevent or reverse lung endothelial barrier failure under experimental conditions, yet with a clinical perspective. Based on recent findings and at times seemingly conflicting results we discuss emerging paradigms of permeability regulation by altered ion transport as well as shifts in the homeostasis of sphingolipids, angiopoietins and prostaglandins. - The Journal of Physiology, EarlyView.
  

  August 14, 2018   doi: 10.1113/JP276245   open full text
• The choroid plexus sodium‐bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.
  Henriette L. Christensen, Dagne Barbuskaite, Aleksandra Rojek, Hans Malte, Inga B. Christensen, Annette C. Füchtbauer, Ernst‐Martin Füchtbauer, Tobias Wang, Jeppe Praetorius, Helle H. Damkier.
  The Journal of Physiology. August 12, 2018
  --- - |2+ Key points Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2. The low pH, however, is harmful and bases such as HCO3− are imported across the brain barriers in order to normalize brain pH. We show that the HCO3− transporter NBCe2 in the choroid plexus of the blood‐cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2. This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid‐base disturbances, such as chronic lung disease. Abstract The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE‐mediated CSF pH regulation by extrusion of acid‐base equivalents was tested by determining the contribution of the electrogenic Na+‐HCO3− cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE‐targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia‐induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild‐type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating‐induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia‐induced acidosis. - The Journal of Physiology, Volume 596, Issue 19, Page 4709-4728, 1 October 2018.
  

  August 12, 2018   doi: 10.1113/JP275489   open full text
• Short‐term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high‐fat diet.
  Gerald Grandl, Leon Straub, Carla Rudigier, Myrtha Arnold, Stephan Wueest, Daniel Konrad, Christian Wolfrum.
  The Journal of Physiology. August 08, 2018
  --- - |2+ Key points A ketogenic diet is known to lead to weight loss and is considered metabolically healthy; however there are conflicting reports on its effect on hepatic insulin sensitivity. KD fed animals appear metabolically healthy in the fasted state after 3 days of dietary challenge, whereas obesogenic high‐fat diet (HFD) fed animals show elevated insulin levels. A glucose challenge reveals that both KD and HFD fed animals are glucose intolerant. Glucose intolerance correlates with increased lipid oxidation and lower respiratory exchange ratio (RER); however, all animals respond to glucose injection with an increase in RER. Hyperinsulinaemic–euglycaemic clamps with double tracer show that the effect of KD is a result of hepatic insulin resistance and increased glucose output but not impaired glucose clearance or tissue glucose uptake in other tissues. Abstract Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high‐fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high‐fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short‐term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP‐derived glucose. - The Journal of Physiology, Volume 596, Issue 19, Page 4597-4609, 1 October 2018.
  

  August 08, 2018   doi: 10.1113/JP275173   open full text
• Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.
  Cheng‐Jun Hu, Hui Zhang, Aya Laux, Soni S. Pullamsetti, Kurt R. Stenmark.
  The Journal of Physiology. August 07, 2018
  --- - |2+ Abstract Chronic pulmonary hypertension (PH) is characterized by the accumulation of persistently activated cell types in the pulmonary vessel exhibiting aberrant expression of genes involved in apoptosis resistance, proliferation, inflammation and extracellular matrix (ECM) remodelling. Current therapies for PH, focusing on vasodilatation, do not normalize these activated phenotypes. Furthermore, current approaches to define additional therapeutic targets have focused on determining the initiating signals and their downstream effectors that are important in PH onset and development. Although these approaches have produced a large number of compelling PH treatment targets, many promising human drugs have failed in PH clinical trials. Herein, we propose that one contributing factor to these failures is that processes important in PH development may not be good treatment targets in the established phase of chronic PH. We hypothesize that this is due to alterations of chromatin structure in PH cells, resulting in functional differences between the same factor or pathway in normal or early PH cells versus cells in chronic PH. We propose that the high expression of genes involved in the persistently activated phenotype of PH vascular cells is perpetuated by an open chromatin structure and multiple transcription factors (TFs) via the recruitment of high levels of epigenetic regulators including the histone acetylases P300/CBP, histone acetylation readers including BRDs, the Mediator complex and the positive transcription elongation factor (Abstract figure). Thus, determining how gene expression is controlled by examining chromatin structure, TFs and epigenetic regulators associated with aberrantly expressed genes in pulmonary vascular cells in chronic PH, may uncover new PH therapeutic targets. - The Journal of Physiology, EarlyView.
  

  August 07, 2018   doi: 10.1113/JP275857   open full text
• Apparent calcium dependence of vesicle recruitment.
  Andreas Ritzau‐Jost, Lukasz Jablonski, Julio Viotti, Noa Lipstein, Jens Eilers, Stefan Hallermann.
  The Journal of Physiology. August 07, 2018
  --- - |2+ Key points Synaptic transmission relies on the recruitment of neurotransmitter‐filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium‐dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium‐dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium‐independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast‐recruited vesicles but does not change the vesicle recruitment rates themselves. Abstract During sustained synaptic transmission, recruitment of new transmitter‐filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity‐dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin–Munc13 pathway, which has been proposed to mediate Ca2+‐dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+‐independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin–Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+‐dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well‐established two‐pool models predict an apparent Ca2+‐dependence of vesicle recruitment. Thus, previous conclusions of Ca2+‐dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution. - The Journal of Physiology, Volume 596, Issue 19, Page 4693-4707, 1 October 2018.
  

  August 07, 2018   doi: 10.1113/JP275911   open full text
• Sickle cell vasculopathy: vascular phenotype on fire!
  Gregory J. Kato.
  The Journal of Physiology. August 03, 2018
  --- - - The Journal of Physiology, EarlyView.
  

  August 03, 2018   doi: 10.1113/JP276705   open full text
• The impact of exercise and nutrition on the regulation of skeletal muscle mass.
  Chris McGlory, Stephan Vliet, Tanner Stokes, Bettina Mittendorfer, Stuart M. Phillips.
  The Journal of Physiology. August 02, 2018
  --- - |2 Abstract The maintenance of skeletal muscle mass and strength throughout life is a key determinant of human health and well‐being. There is a gradual loss of both skeletal muscle mass and strength with ageing (a process termed sarcopenia) that increases the risk of functional dependence, morbidity and mortality. Understanding the factors that regulate the size of human muscle mass, particularly during the later years of life, has therefore become an area of intense scientific inquiry. The amount of muscle mass is determined by coordinated changes in muscle protein synthesis (MPS) and muscle protein breakdown (MPB). In this review, we assess both classical and contemporary work that has examined how resistance exercise and nutrition impact on MPS and MPB. Special consideration is given to the role of different sources of dietary protein (food vs. supplements) and non‐protein nutrients such as omega‐3 fatty acids in regulating MPS. We also critically evaluate recent studies that have employed novel ‘omic’ technologies such as dynamic protein profiling to probe for changes in rates of MPS and MPB at the individual protein level following exercise. Finally, we provide suggestions for future research that we hope will yield important information for the development of exercise and nutritional strategies to counteract muscle loss in a variety of clinical settings. - The Journal of Physiology, EarlyView.
  

  August 02, 2018   doi: 10.1113/JP275443   open full text
• Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.
  Svetlana Tikunova, Natalya Belevych, Kelly Doan, Peter J. Reiser.
  The Journal of Physiology. August 02, 2018
  --- - |2+ Key points The Ca2+‐desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (‐log [Ca]) relationship in slow‐twitch mouse fibres. The D73N mutation led to a rightward shift in the force–stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross‐sectional area of slow‐twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. Abstract The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow‐twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force–stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross‐sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation. - The Journal of Physiology, Volume 596, Issue 19, Page 4651-4663, 1 October 2018.
  

  August 02, 2018   doi: 10.1113/JP276296   open full text
• Mechanoadaptation: articular cartilage through thick and thin.
  Tonia L. Vincent, Angus K. T. Wann.
  The Journal of Physiology. July 29, 2018
  --- - |2 Abstract The articular cartilage is exquisitely sensitive to mechanical load. Its structure is largely defined by the mechanical environment and destruction in osteoarthritis is the pathophysiological consequence of abnormal mechanics. It is often overlooked that disuse of joints causes profound loss of volume in the articular cartilage, a clinical observation first described in polio patients and stroke victims. Through the 1980s, the results of studies exploiting experimental joint immobilisation supported this. Importantly, this substantial body of work was also the first to describe metabolic changes that resulted in decreased synthesis of matrix molecules, especially sulfated proteoglycans. The molecular mechanisms that underlie disuse atrophy are poorly understood despite the identification of multiple mechanosensing mechanisms in cartilage. Moreover, there has been a tendency to equate cartilage loss with osteoarthritic degeneration. Here, we review the historic literature and clarify the structural, metabolic and clinical features that clearly distinguish cartilage loss due to disuse atrophy and those due to osteoarthritis. We speculate on the molecular sensing pathways in cartilage that may be responsible for cartilage mechanoadaptation. - The Journal of Physiology, EarlyView.
  

  July 29, 2018   doi: 10.1113/JP275451   open full text
• Inspiratory pre‐motor potentials during quiet breathing in ageing and chronic obstructive pulmonary disease.
  David A. T. Nguyen, Claire L. Boswell‐Ruys, Rachel A. McBain, Danny J. Eckert, Simon C. Gandevia, Jane E. Butler, Anna L. Hudson.
  The Journal of Physiology. July 29, 2018
  --- - |2+ Key points A cortical contribution to breathing, as indicated by a Bereitschaftspotential (BP) in averaged electroencephalographic signals, occurs in healthy individuals when external inspiratory loads are applied. Chronic obstructive pulmonary disease (COPD) is a condition where changes in the lung, chest wall and respiratory muscles produce an internal inspiratory load. These changes also occur in normal ageing, although to a lesser extent. In the present study, we determined whether BPs are present during quiet breathing and breathing with an external inspiratory load in COPD compared to age‐matched and young healthy controls. We demonstrated that increased age, rather than COPD, is associated with a cortical contribution to quiet breathing. A cortical contribution to inspiratory loading is associated with more severe dyspnoea (i.e. the sensation of breathlessness). We propose that cortical mechanisms may be engaged to defend ventilation in ageing with dyspnoea as a consequence. Abstract A cortical contribution to breathing is determined by the presence of a Bereitschaftspotential, a low amplitude negativity in the averaged electroencephalographic (EEG) signal, which begins ∼1 s before inspiration. It occurs in healthy individuals when external inspiratory loads to breathing are applied. In chronic obstructive pulmonary disease (COPD), changes in the lung, chest wall and respiratory muscles produce an internal inspiratory load. We hypothesized that there would be a cortical contribution to quiet breathing in COPD and that a cortical contribution to breathing with an inspiratory load would be linked to dyspnoea, a major symptom of COPD. EEG activity was analysed in 14 participants with COPD (aged 57–84 years), 16 healthy age‐matched (57–87 years) and 15 young (18–26 years) controls during quiet breathing and inspiratory loading. The presence of Bereitschaftspotentials, from ensemble averages of EEG epochs at Cz and FCz, were assessed by blinded assessors. Dyspnoea was rated using the Borg scale. The incidence of a cortical contribution to quiet breathing was significantly greater in participants with COPD (6/14) compared to the young (0/15) (P = 0.004) but not the age‐matched controls (6/16) (P = 0.765). A cortical contribution to inspiratory loading was associated with higher Borg ratings (P = 0.007), with no effect of group (P = 0.242). The data show that increased age, rather than COPD, is associated with a cortical contribution to quiet breathing. A cortical contribution to inspiratory loading is associated with more severe dyspnoea. We propose that cortical mechanisms may be engaged to defend ventilation with dyspnoea as a consequence. - The Journal of Physiology, EarlyView.
  

  July 29, 2018   doi: 10.1113/JP275764   open full text
• Maternal exercise in rats upregulates the placental insulin‐like growth factor system with diet‐ and sex‐specific responses: minimal effects in mothers born growth restricted.
  Yeukai T. M. Mangwiro, James S. M. Cuffe, Jessica F. Briffa, Dayana Mahizir, Kristina Anevska, Andrew J. Jefferies, Sogand Hosseini, Tania Romano, Karen M. Moritz, Mary E. Wlodek.
  The Journal of Physiology. July 26, 2018
  --- - |2+ Key points The placental insulin‐like growth factor (IGF) system is critical for normal fetoplacental growth, which is dysregulated following several pregnancy perturbations including uteroplacental insufficiency and maternal obesity. We report that the IGF system was altered in placentae of mothers born growth restricted compared to normal birth weight mothers, with maternal diet‐ and fetal sex‐specific responses. Additionally, we report increased body weight and plasma IGF1 concentrations in fetuses from chow‐fed normal birth weight mothers that exercised prior to and continued during pregnancy compared to sedentary mothers. Exercise initiated during pregnancy, on the other hand, resulted in placental morphological alterations and increased IGF1 and IGF1R protein expression, which may in part be modulated by reduced Let 7f‐1 miRNA abundance. Growth restriction of mothers before birth and exercise differentially regulate the placental IGF system with diet‐ and sex‐specific responses, probably as a means to improve fetoplacental growth and development, and hence neonatal survival. This increased neonatal survival may prevent adult disease onset. Abstract The insulin‐like growth factor (IGF) system regulates fetoplacental growth and plays a role in disease programming. Dysregulation of the IGF system is implicated in several pregnancy perturbations associated with altered fetal growth, including intrauterine growth restriction and maternal obesity. Limited human studies have demonstrated that maternal exercise enhances fetoplacental growth and decreases cord IGF ligands, which may restore the placental IGF system in complicated pregnancies. This study investigated the impact maternal exercise has on the placental IGF system in placentae from mothers born growth restricted and if these outcomes are dependent on maternal diet or fetal sex. Uteroplacental insufficiency (Restricted) or sham (Control) surgery was induced on embryonic day (E) 18 in Wistar–Kyoto rats. F1 offspring were fed a chow or high‐fat diet from weaning, and at 16 weeks were randomly allocated an exercise protocol: Sedentary, Exercised prior to and during pregnancy (Exercise), or Exercised during pregnancy only (PregEx). Females were mated (20 weeks) with placentae associated with F2 fetuses collected at E20. The placental IGF system mRNA abundance and placental morphology was altered in mothers born growth restricted. Exercise increased fetal weight and Control plasma IGF1 concentrations, and decreased female placental weight. PregEx did not influence fetoplacental growth but increased placental IGF1 and IGF1R (potentially modulated by reduced Let 7f‐1 miRNA) and decreased placental IGF2 protein. Importantly, these placental IGF system changes occurred with sex‐specific responses. These data highlight that exercise differently influences fetoplacental growth and the placental IGF system depending on maternal exercise initiation, which may prevent the transgenerational transmission of deficits and dysfunction. - The Journal of Physiology, EarlyView.
  

  July 26, 2018   doi: 10.1113/JP275758   open full text
• Antenatal prevention of cerebral palsy and childhood disability: is the impossible possible?
  Stacey J. Ellery, Meredith Kelleher, Peta Grigsby, Irina Burd, Jan B. Derks, Jon Hirst, Suzanne L. Miller, Larry S. Sherman, Mary Tolcos, David W. Walker.
  The Journal of Physiology. July 22, 2018
  --- - |2+ Abstract This review covers our current knowledge of the causes of perinatal brain injury leading to cerebral palsy‐like outcomes, and argues that much of this brain damage is preventable. We review the experimental evidence that there are treatments that can be safely administered to women in late pregnancy that decrease the likelihood and extent of perinatal brain damage that occurs because of acute and severe hypoxia that arises during some births, and the additional impact of chronic fetal hypoxia, infection, inflammation, growth restriction and preterm birth. We discuss the types of interventions required to ameliorate or even prevent apoptotic and necrotic cell death, and the vulnerability of all the major cell types in the brain (neurons, astrocytes, oligodendrocytes, microglia, cerebral vasculature) to hypoxia/ischaemia, and whether a pan‐protective treatment given to the mother before birth is a realistic prospect. - The Journal of Physiology, EarlyView.
  

  July 22, 2018   doi: 10.1113/JP275595   open full text
• The impact of loading, unloading, ageing and injury on the human tendon.
  S. Peter Magnusson, Michael Kjaer.
  The Journal of Physiology. July 20, 2018
  --- - |2+ Abstract A tendon transfers force from the contracting muscle to the skeletal system to produce movement and is therefore a crucial component of the entire muscle‐tendon complex and its function. However, tendon research has for some time focused on mechanical properties without any major appreciation of potential cellular and molecular changes. At the same time, methodological developments have permitted determination of the mechanical properties of human tendons in vivo, which was previously not possible. Here we review the current understanding of how tendons respond to loading, unloading, ageing and injury from cellular, molecular and mechanical points of view. A mechanistic understanding of tendon tissue adaptation will be vital for development of adequate guidelines in physical training and rehabilitation, as well as for optimal injury treatment. - The Journal of Physiology, EarlyView.
  

  July 20, 2018   doi: 10.1113/JP275450   open full text
• Omecamtiv Mercabil and Blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.
  Thomas Kampourakis, Xuemeng Zhang, Yin‐Biao Sun, Malcolm Irving.
  The Journal of Physiology. October 20, 2017
  Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules‐ Omecamtiv Mecarbil (OM) and Blebbistatin (BS) ‐ that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. This article is protected by copyright. All rights reserved

  October 20, 2017   doi: 10.1113/JP275050   open full text
• Heteromeric α/β glycine receptors regulate excitability in parvalbumin‐expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.
  M. A. Gradwell, K. A. Boyle, R. J. Callister, D. I. Hughes, B. A. Graham.
  The Journal of Physiology. October 19, 2017
  Key points Spinal parvalbumin‐expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn. This study assessed the inhibitory regulation of the parvalbumin‐expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability. Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin‐expressing interneuron excitability. Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/β subunit‐containing receptors underlie both synaptic and tonic glycinergic currents. Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin‐expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. Abstract The dorsal horn (DH) of the spinal cord is an important site for modality‐specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin‐expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch‐clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine‐mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/β glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.

  October 19, 2017   doi: 10.1113/JP274926   open full text
• Molecular mechanism for muscarinic M1 receptor‐mediated endocytosis of TWIK‐related acid‐sensitive K+ 1 channels in rat adrenal medullary cells.
  Hidetada Matsuoka, Masumi Inoue.
  The Journal of Physiology. October 19, 2017
  Key points The muscarinic acetylcholine receptor (mAChR)‐mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)‐related acid‐sensitive K+ (TASK)1 channels. In this study we focused on the molecular mechanism of mAChR‐mediated inhibition of TASK1 channels. Exposure to muscarine resulted in a clathrin‐dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R). This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non‐receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1. The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin‐dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. Abstract Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK‐related acid‐sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR‐mediated inhibition. TASK1‐like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine‐induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose–response curves for muscarinic agonist‐induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine‐induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1‐like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1R stimulation results in internalization of TASK1 channels through the PLC–PKC–Src pathway with the consequent phosphorylation of tyrosine and that this M1R‐mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.

  October 19, 2017   doi: 10.1113/JP275039   open full text
• Endomorphins potentiate ASIC currents and enhance the lactic acid‐mediated increase in arterial blood pressure—effects amplified in hindlimb ischemia.
  Mohamed Farrag, Julie K. Drobish, Henry L. Puhl, Joyce S. Kim, Paul B. Herold, Marc P. Kaufman, Victor Ruiz‐Velasco.
  The Journal of Physiology. October 16, 2017
  Chronic muscle ischemia leads to accumulation of lactic acid and other inflammatory mediators with a subsequent drop in interstitial pH. Acid‐sensing ion channels (ASICs), expressed in thin muscle afferents, sense the decrease in pH and evoke a pressor reflex known to increase mean arterial pressure. The naturally occurring endomorphins are also released by primary afferents under ischemic conditions. We examined whether high affinity mu opioid receptor (MOR) agonists, endomorphin‐1 (E‐1) and ‐2 (E‐2), modulate ASIC currents and the lactic acid‐mediated pressor reflex. In rat dorsal root ganglion (DRG) neurons, exposure to E‐2 in acidic solutions significantly potentiated ASIC currents when compared to acidic solutions alone. The potentiation was significantly greater in DRG neurons isolated from rats whose femoral arteries were ligated for 72 hr. Sustained ASIC current potentiation was also observed in neurons pretreated with pertussis toxin, an uncoupler of G proteins and MOR. The endomorphin‐mediated potentiation was a result of a leftward shift of the activation curve to more basic pH values and a slight shift of the inactivation curve to more acidic pH values. Intra‐arterial co‐administration of lactic acid and E‐2 led to a significantly greater pressor reflex than lactic acid alone in the presence of naloxone. Finally, E‐2 effects were inhibited by pretreatment with the ASIC3 blocker (APETx2) and enhanced by pretreatment with the ASIC1a blocker psalmotoxin‐1. These findings have uncovered a novel role of endomorphins by which the opioids can enhance the lactic acid‐mediated reflex increase in arterial pressure that is MOR stimulation‐independent and APETx2‐sensitive. This article is protected by copyright. All rights reserved

  October 16, 2017   doi: 10.1113/JP275058   open full text
• Altered NMDA receptor‐evoked intracellular Ca2+ dynamics in magnocellular neurosecretory neurons of hypertensive rats.
  Meng Zhang, Javier E. Stern.
  The Journal of Physiology. October 15, 2017
  A growing body of evidence supports an elevated NMDA receptor‐mediated glutamate excitatory function in the SON and PVN of hypertensive rats that contributes to neurohumoral activation in this disease. Still, the precise mechanisms underlying altered NMDAR signalling in hypertension remains to be elucidated. In this study, we performed simultaneous electrophysiology and fast confocal Ca2+ imaging to determine whether an altered NMDAR‐mediated changes in intracellular Ca2+ levels (NMDAR‐ΔCa2+) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in renovascular hypertensive (RVH) rats. We found that despite evoking a similar excitatory inward current, activation of NMDARs resulted in a larger and prolonged ΔCa2+ in MNCs from RVH rats. Changes in NMDAR‐ΔCa2+ dynamics were observed both in somatic and dendritic compartments. Inhibition of the ER SERCA pump activity with thapsigargin prolonged NMDAR‐ΔCa2+ responses in MNCs of sham rats, but this effect was occluded in RVH rats, thus equalizing the magnitude and time course of the NMDA‐ΔCa2 responses between the two experimental groups. Taken together, our results support (1) an exacerbated NMDAR‐ΔCa2+ response in somatodendritic compartments of MNCs of RVH rats, and (2) that a blunted ER Ca2+ buffering capacity contributes to the altered NMDAR‐ΔCa2+ dynamics in this condition. Thus, an altered spatiotemporal dynamics of NMDAR‐ΔCa2+ response stands as an underlying mechanisms contributing to neurohumoral activation in neurogenic hypertension. This article is protected by copyright. All rights reserved

  October 15, 2017   doi: 10.1113/JP275169   open full text
• Asymmetry between ON and OFF α ganglion cells of mouse retina: integration of signal and noise from synaptic inputs.
  Michael A. Freed.
  The Journal of Physiology. October 15, 2017
  Key points Bipolar and amacrine cells presynaptic to the ON sustained α cell of mouse retina provide currents with a higher signal‐to‐noise power ratio (SNR) than those presynaptic to the OFF sustained α cell. Yet the ON cell loses proportionately more SNR from synaptic inputs to spike output than the OFF cell does. The higher SNR of ON bipolar cells at the beginning of the ON pathway compensates for losses incurred by the ON ganglion cell, and improves the processing of positive contrasts. Abstract ON and OFF pathways in the retina include functional pairs of neurons that, at first glance, appear to have symmetrically similar responses to brightening and darkening, respectively. Upon careful examination, however, functional pairs exhibit asymmetries in receptive field size and response kinetics. Until now, descriptions of how light‐adapted retinal circuitry maintains a preponderance of signal over the noise have not distinguished between ON and OFF pathways. Here I present evidence of marked asymmetries between members of a functional pair of sustained α ganglion cells in the mouse retina. The ON cell exhibited a proportionately greater loss of signal‐to‐noise power ratio (SNR) from its presynaptic arrays to its postsynaptic currents. Thus the ON cell combines signal and noise from its presynaptic arrays of bipolar and amacrine cells less efficiently than the OFF cell does. Yet the inefficiency of the ON cell is compensated by its presynaptic arrays providing a higher SNR than the arrays presynaptic to the OFF cell, apparently to improve visual processing of positive contrasts. Dynamic clamp experiments were performed that introduced synaptic conductances into ON and OFF cells. When the amacrine‐modulated conductance was removed, the ON cell's spike train exhibited an increase in SNR. The OFF cell, however, showed the opposite effect of removing amacrine input, which was a decrease in SNR. Thus ON and OFF cells have different modes of synaptic integration with direct effects on the SNR of the spike output.

  October 15, 2017   doi: 10.1113/JP274736   open full text
• Laminar‐specific encoding of texture elements in rat barrel cortex.
  Benjamin J. Allitt, Dasuni S. Alwis, Ramesh Rajan.
  The Journal of Physiology. October 15, 2017
  Key points For rats texture discrimination is signalled by the large face whiskers by stick‐slip events. Neural encoding of repetitive stick‐slip events will be influenced by intrinsic properties of adaptation. We show that texture coding in the barrel cortex is laminar specific and follows a power function. Our results also show layer 2 codes for novel feature elements via robust firing rates and temporal fidelity. We conclude that texture coding relies on a subtle neural ensemble to provide important object information. Abstract Texture discrimination by rats is exquisitely guided by fine‐grain mechanical stick‐slip motions of the face whiskers as they encounter, stick to and slip past successive texture‐defining surface features such as bumps and grooves. Neural encoding of successive stick‐slip texture events will be shaped by adaptation, common to all sensory systems, whereby receptor and neural responses to a stimulus are affected by responses to preceding stimuli, allowing resetting to signal novel information. Additionally, when a whisker is actively moved to contact and brush over surfaces, that motion itself generates neural responses that could cause adaptation of responses to subsequent stick‐slip events. Nothing is known about encoding in the rat whisker system of stick‐slip events defining textures of different grain or the influence of adaptation from whisker protraction or successive texture‐defining stick‐slip events. Here we recorded responses from halothane‐anaesthetized rats in response to texture‐defining stimuli applied to passive whiskers. We demonstrate that: across the columnar network of the whisker‐recipient barrel cortex, adaptation in response to repetitive stick‐slip events is strongest in uppermost layers and equally lower thereafter; neither whisker protraction speed nor stick‐slip frequency impede encoding of stick‐slip events at rates up to 34.08 Hz; and layer 2 normalizes responses to whisker protraction to resist effects on texture signalling. Thus, within laminar‐specific response patterns, barrel cortex reliably encodes texture‐defining elements even to high frequencies.

  October 15, 2017   doi: 10.1113/JP274865   open full text
• N1366S mutation of human skeletal muscle sodium channel causes paramyotonia congenita.
  Qing Ke, Jia Ye, Siyang Tang, Jin Wang, Benyan Luo, Fang Ji, Xu Zhang, Ye Yu, Xiaoyang Cheng, Yuezhou Li.
  The Journal of Physiology. October 15, 2017
  Key points Paramyotonia congenita is a hereditary channelopathy caused by missense mutations in the SCN4A gene, which encodes the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. Affected individuals suffered from myotonia and paralysis of muscles, which were aggravated by exposure to cold. We report a three‐generation Chinese family with patients presenting paramyotonia congenita and identify a novel N1366S mutation of NaV1.4. Whole‐cell electrophysiological recordings of the N1366S channel reveal a gain‐of‐function change of gating in response to cold. Modelling and molecular dynamic simulation data suggest that an arginine‐to‐serine substitution at position 1366 increases the distance from N1366 to R1454 and disrupts the hydrogen bond formed between them at low temperature. We demonstrate that N1366S is a disease‐causing mutation and that the temperature‐sensitive alteration of N1366S channel activity may be responsible for the pronounced paramyotonia congenita symptoms of these patients. Abstract Paramyotonia congenita is an autosomal dominant skeletal muscle channelopathy caused by missense mutations in SCN4A, the gene encoding the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. We report a three‐generation family in which six members present clinical symptoms of paramyotonia congenita characterized by a marked worsening of myotonia by cold and by the presence of clear episodes of paralysis. We identified a novel mutation in SCN4A (Asn1366Ser, N1366S) in all patients in the family but not in healthy relatives or in 500 normal control subjects. Functional analysis of the channel protein expressed in HEK293 cells by whole‐cell patch clamp recording revealed that the N1366S mutation led to significant alterations in the gating process of the NaV1.4 channel. The N1366S mutant displayed a cold‐induced hyperpolarizing shift in the voltage dependence of activation and a depolarizing shift in fast inactivation, as well as a reduced rate of fast inactivation and accelerated recovery from fast inactivation. In addition, homology modelling and molecular dynamic simulation of N1366S and wild‐type NaV1.4 channels indicated that the arginine‐to‐serine substitution disrupted the hydrogen bond formed between N1366 and R1454. Together, our results suggest that N1366S is a gain‐of‐function mutation of NaV1.4 at low temperature and the mutation may be responsible for the clinical symptoms of paramyotonia congenita in the affected family and constitute a basis for studies into its pathogenesis.

  October 15, 2017   doi: 10.1113/JP274877   open full text
• Dietary sodium induces a redistribution of the tubular metabolic workload.
  Khalil Udwan, Ahmed Abed, Isabelle Roth, Eva Dizin, Marc Maillard, Carla Bettoni, Johannes Loffing, Carsten A. Wagner, Aurélie Edwards, Eric Feraille.
  The Journal of Physiology. October 15, 2017
  Key points Body Na+ content is tightly controlled by regulated urinary Na+ excretion. The intrarenal mechanisms mediating adaptation to variations in dietary Na+ intake are incompletely characterized. We confirmed and expanded observations in mice that variations in dietary Na+ intake do not alter the glomerular filtration rate but alter the total and cell‐surface expression of major Na+ transporters all along the kidney tubule. Low dietary Na+ intake increased Na+ reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments. High dietary Na+ intake decreased Na+ reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency. The abundance of apical transporters and Na+ delivery are the main determinants of Na+ reabsorption along the kidney tubule. Tubular O2 consumption and the efficiency of sodium reabsorption are dependent on sodium diet. Abstract Na+ excretion by the kidney varies according to dietary Na+ intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na+ reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na+, a normal sodium diet (NSD) containing 0.18% Na+ and a moderately high sodium diet (HSD) containing 1.25% Na+. As expected, LSD did not alter measured GFR and increased the abundance of total and cell‐surface NHE3, NKCC2, NCC, α‐ENaC and cleaved γ‐ENaC compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na+ delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O2 consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) was related to the changes in tubular Na+ reabsorption. Our data show that fractional Na+ reabsorption is distributed differently according to dietary Na+ intake and induces changes in tubular O2 consumption and sodium transport efficiency.

  October 15, 2017   doi: 10.1113/JP274927   open full text
• Task‐dependent output of human parasternal intercostal motor units across spinal levels.
  Anna L. Hudson, Simon C. Gandevia, Jane E. Butler.
  The Journal of Physiology. October 13, 2017
  Key points During breathing, there is differential activity in the human parasternal intercostal muscles and the activity is tightly coupled to the known mechanical advantages for inspiration of the same regions of muscles. It is not known whether differential activity is preserved for the non‐respiratory task of ipsilateral trunk rotation. In the present study, we compared single motor units during resting breathing and axial rotation of the trunk during apnoea. We not only confirmed non‐uniform recruitment of motor units across parasternal intercostal muscles in breathing, but also demonstrated that the same motor units show an altered pattern of recruitment in the non‐respiratory task of trunk rotation. The output of parasternal intercostal motoneurones is modulated differently across spinal levels depending on the task and these results help us understand the mechanisms that may govern task‐dependent differences in motoneurone output. Abstract During inspiration, there is differential activity in the human parasternal intercostal muscles across interspaces. We investigated whether the earlier recruitment of motor units in the rostral interspaces compared to more caudal spaces during inspiration is preserved for the non‐respiratory task of ipsilateral trunk rotation. Single motor unit activity (SMU) was recorded from the first, second and fourth parasternal interspaces on the right side in five participants in two tasks: resting breathing and ‘isometric’ axial rotation of the trunk during apnoea. Recruitment of the same SMUs was compared between tasks (n = 123). During resting breathing, differential activity was indicated by earlier recruitment of SMUs in the first and second interspaces compared to the fourth space in inspiration (P < 0.01). By contrast, during trunk rotation, the same motor units showed an altered pattern of recruitment because SMUs in the first interspace were recruited later and at a higher rotation torque than those in the second and fourth interspaces (P < 0.05). Tested for a subset of SMUs, the reliability of the breathing and rotation tasks, as well as the SMU recruitment measures, was good–excellent [intraclass correlation (2,1): 0.69–0.91]. Thus, the output of parasternal intercostal motoneurones is modulated differently across spinal levels depending on the task. Given that the differential inspiratory output of parasternal intercostal muscles is linked to their relative mechanical effectiveness for inspiration and also that this output is altered in trunk rotation, we speculate that a mechanism matching neural drive to muscle mechanics underlies the task‐dependent differences in output of axial motoneurone pools.

  October 13, 2017   doi: 10.1113/JP274866   open full text
• Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport, and osmotic enhancement of delivery.
  Michelle E. Pizzo, Daniel J. Wolak, Niyanta N. Kumar, Eric Brunette, Christina L. Brunnquell, Melanie‐Jane Hannocks, N. Joan Abbott, M. Elizabeth Meyerand, Lydia Sorokin, Danica B. Stanimirovic, Robert G. Thorne.
  The Journal of Physiology. October 12, 2017
  The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance as well as several ongoing intrathecal clinical trials. Here, we clarify how immunoglobulin G (IgG) and smaller single‐domain antibodies (sdAb) distribute throughout the whole brain in a size‐dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo 3D magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along perivascular spaces of all vessel types, with sdAb accessing 4–7 times greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size‐dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points for substances in the CSF to enter the perivascular space. Osmolyte co‐infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by approximately 50%. Our results reveal potential distribution mechanisms for endogenous IgG, one of the most abundant proteins in the CSF, as well as provide new insights needed to understand and improve drug delivery of macromolecules to the central nervous system via the intrathecal route. This article is protected by copyright. All rights reserved

  October 12, 2017   doi: 10.1113/JP275105   open full text
• An investigation of fetal behavioural states during maternal sleep in healthy late gestation pregnancy: an observational study.
  Peter R. Stone, Wendy Burgess, Jordan McIntyre, Alistair J. Gunn, Christopher A. Lear, Laura Bennet, Edwin A Mitchell, John M. D. Thompson,.
  The Journal of Physiology. October 11, 2017
  Background Fetal behavioural states (FBS) are measures of fetal wellbeing. Maternal position affects FBS with supine being associated with an increased likelihood of fetal quiescence consistent with the human fetus adapting to a lower oxygen consuming state. A number of studies now confirm the association of sleep position with risk of late intrauterine death. We designed this study to observe the effects of maternal sleep positions overnight in healthy late gestation pregnancy. Method Twenty nine healthy women had continuous fetal ECG recordings overnight. Two blinded observers, assigned fetal states in 5 minute blocks. Measures of fetal heart rate variability (FHRV) were calculated from ECG beat to beat data. Maternal position was determined from infrared video recording. Results Compared to state 2F (active sleep), 4F (active awake‐high activity) occurred almost exclusively when the mother was in a left or right lateral position. State 1F (quiet sleep) was more common when mother was supine (OR 1.30, 95%CI, 1.11‐1.52) and less common on maternal right side with the left being referent position (OR 0.81, 95%CI, 0.70‐0.93). State 4F was more common between 2100 and 0100 than between 0100 and 0700 (OR 2 2.83, 95%CI, 2.32‐3.47). In each fetal state, maternal position had significant effects on fetal heart rate (FHR) and measures of FHRV. Conclusion In healthy late gestation pregnancy, maternal sleep position affects FBS and heart rate variability. These effects are likely fetal adaptations to positions which may produce a mild hypoxic stress. This article is protected by copyright. All rights reserved

  October 11, 2017   doi: 10.1113/JP275084   open full text
• Physiological tremor increases when skeletal muscle is shortened: implications for fusimotor control.
  Kian Jalaleddini, Akira Nagamori, Christopher M. Laine, Mahsa A. Golkar, Robert E. Kearney, Francisco J. Valero‐Cuevas.
  The Journal of Physiology. October 11, 2017
  The involuntary force fluctuations associated with physiological (as distinct from pathological) tremor are an unavoidable component of human motor control. While the origins of physiological tremor are known to depend on muscle afferentation, it is possible that the mechanical properties of muscle‐tendon systems also affect its generation, amplification and maintenance. In this paper, we investigated the dependence of physiological tremor on muscle length in healthy individuals. We measured physiological tremor during tonic, isometric plantarflexion torque at 30% of maximum at three ankle angles. The amplitude of physiological tremor increased as calf muscles shortened in contrast to the stretch reflex whose amplitude decreases as muscle shortens. We used a published closed‐loop simulation model of afferented muscle to explore the mechanisms responsible for this behaviour. We demonstrate that changing muscle lengths does not suffice to explain our experimental findings. Rather, the model consistently required the modulation of γ‐static fusimotor drive to produce increases in physiological tremor with muscle shortening—while successfully replicating the concomitant reduction in stretch reflex amplitude. This need to control γ‐static fusimotor drive explicitly as a function of muscle length has important implications. First, it permits the amplitudes of physiological tremor and stretch reflex to be decoupled. Second, it postulates neuromechanical interactions that require length‐dependent γ drive modulation to be independent from α drive to the parent muscle. Lastly, it suggests that physiological tremor can be used as a simple, non‐invasive measure of the afferent mechanisms underlying healthy motor function, and their disruption in neurological conditions. This article is protected by copyright. All rights reserved

  October 11, 2017   doi: 10.1113/JP274899   open full text
• KLF2 mediates enhanced chemoreflex sensitivity, disordered breathing and autonomic dysregulation in heart failure.
  Noah J. Marcus, Rodrigo Del Rio, Yanfeng Ding, Harold D. Schultz.
  The Journal of Physiology. October 11, 2017
  Key points Enhanced carotid body chemoreflex activity contributes to development of disordered breathing patterns, autonomic dysregulation and increases in incidence of arrhythmia in animal models of reduced ejection fraction heart failure. Chronic reductions in carotid artery blood flow are associated with increased carotid body chemoreceptor activity. Krüppel‐like Factor 2 (KLF2) is a shear stress‐sensitive transcription factor that regulates the expression of enzymes which have previously been shown to play a role in increased chemoreflex sensitivity. We investigated the impact of restoring carotid body KLF2 expression on chemoreflex control of ventilation, sympathetic nerve activity, cardiac sympatho‐vagal balance and arrhythmia incidence in an animal model of heart failure. The results indicate that restoring carotid body KLF2 in chronic heart failure reduces sympathetic nerve activity and arrhythmia incidence, and improves cardiac sympatho‐vagal balance and breathing stability. Therapeutic approaches that increase KLF2 in the carotid bodies may be efficacious in the treatment of respiratory and autonomic dysfunction in heart failure. Abstract Oscillatory breathing and increased sympathetic nerve activity (SNA) are associated with increased arrhythmia incidence and contribute to mortality in chronic heart failure (CHF). Increased carotid body chemoreflex (CBC) sensitivity plays a role in this process and can be precipitated by chronic blood flow reduction. We hypothesized that downregulation of a shear stress‐sensitive transcription factor, Krüppel‐like Factor 2 (KLF2), mediates increased CBC sensitivity in CHF and contributes to associated autonomic, respiratory and cardiac sequelae. Ventilation (Ve), renal SNA (RSNA) and ECG were measured at rest and during CBC activation in sham and CHF rabbits. Oscillatory breathing was quantified as the apnoea–hypopnoea index (AHI) and respiratory rate variability index (RRVI). AHI (control 6 ± 1/h, CHF 25 ± 1/h), RRVI (control 9 ± 3/h, CHF 29 ± 3/h), RSNA (control 22 ± 2% max, CHF 43 ± 5% max) and arrhythmia incidence (control 50 ± 10/h, CHF 300 ± 100/h) were increased in CHF at rest (FIO2 21%), as were CBC responses (Ve, RSNA) to 10% FIO2 (all P < 0.05 vs. control). In vivo adenoviral transfection of KLF2 to the carotid bodies in CHF rabbits restored KLF2 expression, and reduced AHI (7 ± 2/h), RSNA (18 ± 2% max) and arrhythmia incidence (46 ± 13/h) as well as CBC responses to hypoxia (all P < 0.05 vs. CHF empty virus). Conversely, lentiviral KLF2 siRNA in the carotid body decreased KLF2 expression, increased chemoreflex sensitivity, and increased AHI (6 ± 2/h vs. 14 ± 3/h), RRVI (5 ± 3/h vs. 20 ± 3/h) and RSNA (24 ± 4% max vs. 34 ± 5% max) relative to scrambled‐siRNA rabbits. In conclusion, down‐regulation of KLF2 in the carotid body increases CBC sensitivity, oscillatory breathing, RSNA and arrhythmia incidence during CHF.

  October 11, 2017   doi: 10.1113/JP273805   open full text
• Plasma membrane Ca2+ ATPase 1 is required for maintaining atrial Ca2+ homeostasis and electrophysiological stability in the mouse.
  Yanwen Wang, Claire Wilson, Elizabeth J. Cartwright, Ming Lei.
  The Journal of Physiology. October 10, 2017
  To determine the role of PMCA1 in maintaining Ca2+ homeostasis and electrical stability in the atrium under physiological and stress conditions, mice with a cardiomyocyte‐specific deletion of PMCA1 (PMCA1cko) and their control littermates (PMCA1loxP/loxP) were studied at the organ and cellular levels.   At the organ level, the PMCA1cko hearts became more susceptible to atrial arrhythmias under rapid programmed electrical stimulation (PES) compared with the PMCA1loxP/loxP hearts, and such arrhythmic events became more severe under Ca2+ overload conditions. At the cellular level, the occurrence of irregular‐type APs of PMCA1cko atrial myocytes increased significantly under Ca2+ overload conditions and/or at higher frequency of stimulation. The decay of Na+‐Ca2+ exchanger (NCX) current that followed a stimulation protocol was significantly prolonged in PMCA1cko atrial myocytes under basal conditions, with Ca2+ overload leading to even greater prolongation. In conclusion, PMCA1 is required for maintaining Ca2+ homeostasis and electrical stability in the atrium. This is particularly critical during fast removal of Ca2+ from the cytosol which is required under stress conditions. This article is protected by copyright. All rights reserved

  October 10, 2017   doi: 10.1113/JP274110   open full text
• Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus.
  Ellen Paula Santos da Conceição, Christopher J. Madden, Shaun F. Morrison.
  The Journal of Physiology. October 10, 2017
  We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling brown adipose tissue (BAT) thermogenesis. Nanoinjections of the muscarinic acetylcholine receptor (mAChR) receptor agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold‐evoked BAT sympathetic nerve activity (SNA, nadirs: −72%, and −95%), BAT temperature (TBAT, −0.5°C and −0.6°C), expired CO2 (Exp. CO2, −0.3% and −0.5%), and heart rate (HR, −22 bpm and −41 bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), TBAT (+1.8°C), Exp. CO2 (+0.7%), core temperature (TCORE, +0.5°C), and HR (+54 bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically‐active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa. This article is protected by copyright. All rights reserved

  October 10, 2017   doi: 10.1113/JP275299   open full text
• Calcium and electrical dynamics in lymphatic endothelium.
  Erik J. Behringer, Joshua P. Scallan, Mohammad Jafarnejad, Jorge A. Castorena‐Gonzalez, Scott D. Zawieja, James E. Moore, Michael J. Davis, Steven S. Segal.
  The Journal of Physiology. October 09, 2017
  Subsequent to a rise in intracellular Ca2+ ([Ca2+]i), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼−70 mV. Acetylcholine (ACh) increased [Ca2+]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained “plateau”). In striking contrast to the endothelium‐derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+‐activated K+ channels (KCa) as confirmed with PCR, persisted in the absence of extracellular Ca2+, was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4−/− mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion. This article is protected by copyright. All rights reserved

  October 09, 2017   doi: 10.1113/JP274842   open full text
• Leukoencephalopathy‐causing CLCN2 mutations are associated with impaired Cl− channel function and trafficking.
  Héctor Gaitán‐Peñas, Pirjo M Apaja, Tanit Arnedo, Aida Castellanos, Xabier Elorza‐Vidal, David Soto, Xavier Gasull, Gergely L Lukacs, Raúl Estévez.
  The Journal of Physiology. October 09, 2017
  Key points Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC‐2. GlialCAM, a regulatory subunit of ClC‐2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC‐2 mutant by affecting ClC‐2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC‐2 at the plasma membrane by GlialCAM depends on its localisation at cell–cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC‐2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC‐2 form a protein complex in glial cells. Abstract Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC‐2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V‐ClC‐2 functional expression. Co‐expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC‐2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC‐2 localisation to cell–cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC‐2 at the PM. Wild‐type, but not mutant, ClC‐2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy‐causing CLCN2 mutations reduce the functional expression of ClC‐2, which is partly counteracted by GlialCAM/MLC1‐mediated increase in the gating and stability of the channel.

  October 09, 2017   doi: 10.1113/JP275087   open full text
• Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.
  David P. Burns, Arijit Roy, Eric F. Lucking, Fiona B. McDonald, Sam Gray, Richard J. Wilson, Deirdre Edge, Ken D. O'Halloran.
  The Journal of Physiology. October 09, 2017
  Key points Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO2 production is equivalent in wild‐type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild‐type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. Abstract Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8‐week‐old male wild‐type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force‐generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO2 production was not different between wild‐type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild‐type and mdx mice. Diaphragm force was severely depressed in mdx mice, with evidence of fibre remodelling and damage. Diaphragm EMG responses to chemoactivation were enhanced in mdx mice. We conclude that there is evidence of chronic hypoventilation in young mdx mice. Diaphragm dysfunction confers mechanical deficiency in mdx resulting in impaired capacity to generate normal tidal volume at rest and decreased absolute ventilation during chemoactivation. Enhanced mdx diaphragm EMG responsiveness suggests compensatory neuroplasticity facilitating respiratory motor output, which may extend to accessory muscles of breathing. Our results may have relevance to emerging treatments for human DMD aiming to preserve ventilatory capacity.

  October 09, 2017   doi: 10.1113/JP274792   open full text
• Empowering human cardiac progenitor cells by P2Y14 nucleotide receptor overexpression.
  Farid G. Khalafalla, Waqas Kayani, Arwa Kassab, Kelli Ilves, Megan M. Monsanto, Roberto Alvarez, Monica Chavarria, Benjamin Norman, Walter P. Dembitsky, Mark A. Sussman.
  The Journal of Physiology. October 05, 2017
  Autologous cardiac progenitor cell (hCPC) therapy is a promising alternative approach to current inefficient therapies for heart failure (HF). However, ex vivo expansion and pharmacological/genetic modification of hCPCs are necessary interventions to rejuvenate aged/diseased cells and improve their regenerative capacities. This study was designed to assess the potential of improving hCPC functional capacity by targeting P2Y14 purinergic receptor (P2Y14R), which has been previously reported to induce regenerative and anti‐senescence responses in a variety of experimental models. c‐Kit+ hCPCs were isolated from cardiac biopsies of multiple HF patients undergoing left ventricular assist device (LVAD) implantation surgery. Significant correlations existed between expression of P2Y14R in hCPCs and clinical parameters of HF patients. P2Y14R was downregulated in hCPCs derived from patients with relatively lower ejection fraction and patients diagnosed with diabetes. hCPC lines with lower P2Y14R expression did not respond to P2Y14R agonist UDP‐glucose (UDP‐Glu) while hCPCs with higher P2Y14R expression showed enhanced proliferation in response to UDP‐Glu stimulation. Mechanistically, UDP‐Glu stimulation enhanced activation of canonical growth signalling pathways ERK1/2 and AKT. Restoring P2Y14R expression levels in functionally compromised hCPCs via lentiviral‐mediated overexpression improved proliferation, migration and survival under stress stimuli. Additionally, P2Y14R overexpression reversed senescence‐associated morphology and reduced levels of molecular markers of senescence p16INK4a, p53, p21 and mitochondrial reactive oxygen species (ROS). Findings from this study unveil novel biological roles of the UDP‐sugar receptor P2Y14 in hCPCs and suggest purinergic signalling modulation as a promising strategy to improve phenotypic properties of functionally impaired hCPCs. This article is protected by copyright. All rights reserved

  October 05, 2017   doi: 10.1113/JP274980   open full text
• Rapid versus slow ascending vasodilatation: intercellular conduction versus flow‐mediated signalling with tetanic versus rhythmic muscle contractions.
  Shenghua Y. Sinkler, Steven S. Segal.
  The Journal of Physiology. October 05, 2017
  In response to exercise, vasodilatation initiated within the microcirculation of skeletal muscle ascends the resistance network into upstream feed arteries (FAs) located external to the tissue. Ascending vasodilatation (AVD) is essential to reducing FA resistance that otherwise restricts blood flow into the microcirculation. We tested the hypothesis that signalling events underlying AVD vary with the intensity and duration of muscle contraction. In the gluteus maximus muscle of anaesthetized male C57BL/6 mice (age, 3‐4 months), brief tetanic contraction (100 Hz, 500 ms) evoked rapid onset vasodilatation (ROV) in FAs that peaked within 4 s. In contrast, during rhythmic twitch contractions (4 Hz), slow onset vasodilatation (SOV) of FAs began after ∼10 s and plateaued within 30 s. Selectively damaging the endothelium with light‐dye treatment midway between a FA and its primary arteriole eliminated ROV in the FA along with conducted vasodilatation of the FA initiated on the arteriole using ACh microiontophoresis. Superfusion of SKCa and IKCa channel blockers UCL 1684 + TRAM 34 attenuated ROV, implicating endothelial hyperpolarization as the underlying signal. Nevertheless, SOV of FAs during rhythmic contractions persisted until superfusion of NO synthase with L‐NAME. Thus, ROV of FAs reflects hyperpolarization of downstream arterioles that conducts along the endothelium into proximal FAs. In contrast, SOV of FAs reflects local production of NO by the endothelium in response to luminal shear stress, which increases secondary to arteriolar dilatation downstream. Thus, AVD ensures increased oxygen delivery to active muscle fibres by reducing upstream resistance via complementary signalling pathways that reflect the intensity and duration of muscle contraction. This article is protected by copyright. All rights reserved

  October 05, 2017   doi: 10.1113/JP275186   open full text
• Chronic beta2‐adrenoceptor agonist treatment alters muscle proteome and functional adaptations induced by high intensity training in young men.
  Morten Hostrup, Johan Onslev, Glenn Jacobson, Richard Wilson, Jens Bangsbo.
  The Journal of Physiology. October 05, 2017
  Although the effects of training have been studied for decades, data on muscle proteome signature remodelling induced by high intensity training in relation to functional changes in humans remains incomplete. Likewise, β2‐agonists are frequently used to counteract exercise‐induced bronchoconstriction, but the effects β2‐agonist treatment on muscle remodelling and adaptations to training are unknown. In a placebo‐controlled parallel study, we randomized 21 trained men to four weeks of high intensity training with (HIT + β2A) or without (HIT) daily inhalation of β2‐agonist (terbutaline, 4 mg d−1). Of 486 proteins identified by mass‐spectrometry proteomics of muscle biopsies sampled before and after the intervention, 32 and 85 were changing (FDR ≤ 5%) with the intervention in HIT and HIT + β2A. Proteome signature changes were different in HIT and HIT + β2A (P = 0.005), wherein β2‐agonist caused a repression of 25 proteins in HIT + β2A compared to HIT, and an upregulation of 7 proteins compared to HIT. β2‐agonist repressed or even downregulated training‐induced enrichment of pathways related to oxidative phosphorylation and glycogen metabolism, but upregulated pathways related to histone trimethylation and the nucleosome. Muscle contractile phenotype changed differently in HIT and HIT + β2A (P ≤ 0.001), with a fast‐to‐slow twitch transition in HIT and a slow‐to‐fast twitch transition in HIT + β2A. β2‐agonist attenuated training‐induced enhancements in maximal oxygen consumption (P ≤ 0.01) and exercise performance (11.6 vs. 6.1%, P ≤ 0.05) in HIT + β2A compared to HIT. These findings indicate that daily β2‐agonist treatment attenuates the beneficial effects of high intensity training on exercise performance and oxidative capacity, and causes remodelling of muscle proteome signature towards a fast‐twitch phenotype. This article is protected by copyright. All rights reserved

  October 05, 2017   doi: 10.1113/JP274970   open full text
• Post‐exercise recovery of contractile function and endurance in humans and mice is accelerated by heating and slowed by cooling skeletal muscle.
  Arthur J. Cheng, Sarah J. Willis, Christoph Zinner, Thomas Chaillou, Niklas Ivarsson, Niels Ørtenblad, Johanna T. Lanner, Hans‐Christer Holmberg, Håkan Westerblad.
  The Journal of Physiology. October 04, 2017
  Manipulation of muscle temperature is believed to improve post‐exercise recovery, with cooling being especially popular among athletes. However, it is unclear whether such temperature manipulations actually have positive effects. Accordingly, we studied the effect of muscle temperature on the acute recovery of force and fatigue resistance after endurance exercise. One hour of moderate‐intensity arm cycling exercise in humans was followed by two hours recovery in which the upper arms were either heated to 38°C, not treated (33°C), or cooled to ∼15°C. Fatigue resistance after the recovery period was assessed by performing 3 × 5 min sessions of all‐out arm cycling at physiological temperature for all conditions (i.e. not heated or cooled). Power output during the all‐out exercise was better maintained when muscles were heated during recovery, whereas cooling had the opposite effect. Mechanisms underlying the temperature‐dependent effect on recovery were tested in mouse intact single muscle fibres, which were exposed to ∼12 min of glycogen‐depleting fatiguing stimulation (350 ms tetani given at 10 s interval until force decreased to 30% of the starting force). Fibres were subsequently exposed to the same fatiguing stimulation protocol after 1–2 h of recovery at 16–36°C. Recovery of submaximal force (30 Hz), the tetanic myoplasmic free [Ca2+] (measured with the fluorescent indicator indo‐1), and fatigue resistance were all impaired by cooling (16‐26°C) and improved by heating (36°C). In addition, glycogen resynthesis was faster at 36°C than 26°C in whole FDB muscles. We conclude that recovery from exhaustive endurance exercise is accelerated by raising and slowed by lowering muscle temperature. This article is protected by copyright. All rights reserved

  October 04, 2017   doi: 10.1113/JP274870   open full text
• Rapid decline in MyHC I(β) mRNA expression in rat soleus during hindlimb unloading is associated with the AMPK dephosphorylation.
  Natalia A. Vilchinskaya, Ekaterina P. Mochalova, Tatiana L. Nemirovskaya, Timur M. Mirzoev, Olga V. Turtikova, Boris S. Shenkman.
  The Journal of Physiology. October 03, 2017
  One of the key events that occurs during skeletal muscle inactivation is a change in myosin phenotype, i.e. increased expression of fast isoforms and decreased expression of slow isoform of myosin heavy chain (MyHC). It is known that calcineurin/NFAT and AMP‐activated protein kinase (AMPK) can regulate the expression of genes encoding MyHC slow isoform. Earlier, we found a significant decrease in phosphorylated AMPK in rat soleus after 24 h of hindlimb unloading (HU). We hypothesized that a decrease in AMPK phosphorylation and subsequent histone deacetylase (HDAC) nuclear translocation can be one of the triggering events leading to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with AMPK activator (AICAR) for 6 d before HU as well as during 24‐h HU. We discovered that AICAR treatment prevented a decrease in pre‐mRNA and mRNA expression of MyHC I as well as MyHC IIa mRNA expression. 24‐h HS resulted in HDAC4 accumulation in the nuclei of rat soleus but AICAR pretreatment prevented such an accumulation. The results of the study indicate that AMPK dephosphorylation after 24‐h HU had a significant impact on the MyHC I and MyHC IIa mRNA expression in rat soleus. AMPK dephosphorylation also contributed to the HDAC4 translocation to the nuclei of soleus muscle fibres, suggesting an important role of HDAC4 as an epigenetic regulator in the process of myosin phenotype transformation. This article is protected by copyright. All rights reserved

  October 03, 2017   doi: 10.1113/JP275184   open full text
• δ‐ and β‐cells are electrically coupled and regulate α‐cell activity via somatostatin.
  L. J. B. Briant, T. M. Reinbothe, I. Spiliotis, C. Miranda, B. Rodriguez, P. Rorsman.
  The Journal of Physiology. October 03, 2017
  Glucagon, the body's principal hyperglycaemic hormone, is released from the α‐cells of the pancreatic islet. The secretion of this hormone is dysregulated in type 2 diabetes mellitus but the mechanisms controlling secretion are not well understood. Regulation of glucagon secretion by factors secreted by neighbouring β‐ and δ‐cells (paracrine regulation) have been proposed to be important. In this study, we explored the importance of paracrine regulation by using an optogenetic strategy. Specific light‐induced activation of β‐cells in mouse islets expressing the light‐gated channelrhodopsin‐2 resulted in stimulation of electrical activity in δ‐cells but suppression of α‐cell activity. The activation of the δ‐cells was rapid and sensitive to the gap junction inhibitor carbenoxolone, whereas the effect on electrical activity in α‐cells was blocked by CYN 154806, an antagonist of the somatostatin‐2 receptor. These observations indicate that optogenetic activation of the β‐cells propagates to the δ‐cells via gap junctions, and the consequential stimulation of somatostatin secretion inhibits α‐cell electrical activity by a paracrine mechanism. To explore whether this pathway is important for regulating α‐cell activity and glucagon secretion in human islets, we constructed computational models of human islets. These models had detailed architectures based on human islets and consisted of a collection of >500 α‐, β‐ and δ‐cells. Simulations of these models revealed that this gap junctional/paracrine mechanism accounts for up to 23% of the suppression of glucagon secretion by high glucose. This article is protected by copyright. All rights reserved

  October 03, 2017   doi: 10.1113/JP274581   open full text
• Maternal exercise modifies body composition and energy substrates handling in high‐fat/high‐sucrose diet fed male offspring.
  Charline Quiclet, Hervé Dubouchaud, Phanélie Berthon, Hervé Sanchez, Guillaume Vial, Farida Siti, Eric Fontaine, Cécile Batandier, Karine Couturier.
  The Journal of Physiology. October 03, 2017
  Maternal exercise during gestation has been reported to modify offspring metabolism and health. Whether these effects are exacerbated when offspring is under high‐fat diet remains rather unclear. Our purpose was to evaluate the effect of maternal exercise before and during gestation on the offspring fed a high‐fat/high‐sucrose diet (HF), by assessing its body composition, pancreatic function and the energy substrates handling by two major glucose‐utilizing tissues: liver and muscle. Fifteen week‐old nulliparous female Wistar rats exercised 4 weeks before as well as during gestation at a constant submaximal intensity (TR) or remained sedentary (CT). At weaning, pups from each group were fed either a standard diet (TRCD or CTCD) or a high‐fat/high‐sucrose diet (TRHF or CTHF) for 10 weeks. Offspring from TR dams gained less weight compared to those from CT dams. Selected fat depots were larger with HF diet compared to CD but significantly smaller in TRHF compared to CTHF. Surprisingly, insulin secretion index was higher in islets from HF offspring compared to CD. TR offspring showed a higher muscle insulin sensitivity estimated by the pPKB/PKB ratio compared with CT offspring (+48%, P < 0.05). With CD, permeabilized isolated muscle fibres from TR rats displayed a lower apparent affinity constant (Km) for pyruvate and palmitoyl Co‐A as substrates compared to the CT group (−46% and −58% respectively, P < 0.05). These results suggest that maternal exercise has positive effects on young adult offspring body composition and on muscle carbohydrate and lipid metabolism depending on the nutritional status. This article is protected by copyright. All rights reserved

  October 03, 2017   doi: 10.1113/JP274739   open full text
• Visuo‐manual tracking: does intermittent control with aperiodic sampling explain linear power and non‐linear remnant without sensorimotor noise?
  Henrik Gollee, Peter J. Gawthrop, Martin Lakie, Ian D. Loram.
  The Journal of Physiology. October 01, 2017
  Key points A human controlling an external system is described most easily and conventionally as linearly and continuously translating sensory input to motor output, with the inevitable output remnant, non‐linearly related to the input, attributed to sensorimotor noise. Recent experiments show sustained manual tracking involves repeated refractoriness (insensitivity to sensory information for a certain duration), with the temporary 200–500 ms periods of irresponsiveness to sensory input making the control process intrinsically non‐linear. This evidence calls for re‐examination of the extent to which random sensorimotor noise is required to explain the non‐linear remnant. This investigation of manual tracking shows how the full motor output (linear component and remnant) can be explained mechanistically by aperiodic sampling triggered by prediction error thresholds. Whereas broadband physiological noise is general to all processes, aperiodic sampling is associated with sensorimotor decision making within specific frontal, striatal and parietal networks; we conclude that manual tracking utilises such slow serial decision making pathways up to several times per second. Abstract The human operator is described adequately by linear translation of sensory input to motor output. Motor output also always includes a non‐linear remnant resulting from random sensorimotor noise from multiple sources, and non‐linear input transformations, for example thresholds or refractory periods. Recent evidence showed that manual tracking incurs substantial, serial, refractoriness (insensitivity to sensory information of 350 and 550 ms for 1st and 2nd order systems respectively). Our two questions are: (i) What are the comparative merits of explaining the non‐linear remnant using noise or non‐linear transformations? (ii) Can non‐linear transformations represent serial motor decision making within the sensorimotor feedback loop intrinsic to tracking? Twelve participants (instructed to act in three prescribed ways) manually controlled two systems (1st and 2nd order) subject to a periodic multi‐sine disturbance. Joystick power was analysed using three models, continuous‐linear‐control (CC), continuous‐linear‐control with calculated noise spectrum (CCN), and intermittent control with aperiodic sampling triggered by prediction error thresholds (IC). Unlike the linear mechanism, the intermittent control mechanism explained the majority of total power (linear and remnant) (77–87% vs. 8–48%, IC vs. CC). Between conditions, IC used thresholds and distributions of open loop intervals consistent with, respectively, instructions and previous measured, model independent values; whereas CCN required changes in noise spectrum deviating from broadband, signal dependent noise. We conclude that manual tracking uses open loop predictive control with aperiodic sampling. Because aperiodic sampling is inherent to serial decision making within previously identified, specific frontal, striatal and parietal networks we suggest that these structures are intimately involved in visuo‐manual tracking.

  October 01, 2017   doi: 10.1113/JP274288   open full text
• Inducible satellite cell depletion attenuates skeletal muscle regrowth following a scald‐burn injury.
  Celeste C. Finnerty, Colleen F. McKenna, Lauren A. Cambias, Camille R. Brightwell, Anesh Prasai, Ye Wang, Amina El Ayadi, David N. Herndon, Oscar E. Suman, Christopher S. Fry.
  The Journal of Physiology. October 01, 2017
  Key points Severe burns result in significant skeletal muscle cachexia that impedes recovery. Activity of satellite cells, skeletal muscle stem cells, is altered following a burn injury and likely hinders regrowth of muscle. Severe burn injury induces satellite cell proliferation and fusion into myofibres with greater activity in muscles proximal to the injury site. Conditional depletion of satellite cells attenuates recovery of myofibre area and volume following a scald burn injury in mice. Skeletal muscle regrowth following a burn injury requires satellite cell activity, underscoring the therapeutic potential of satellite cells in the prevention of prolonged frailty in burn survivors. Abstract Severe burns result in profound skeletal muscle atrophy; persistent muscle atrophy and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma, and we have previously shown concurrent activation and apoptosis of muscle satellite cells following a burn injury in paediatric patients. To determine the necessity of satellite cells during muscle recovery following a burn injury, we utilized a genetically modified mouse model (Pax7CreER‐DTA) that allows for the conditional depletion of satellite cells in skeletal muscle. Additionally, mice were provided 5‐ethynyl‐2′‐deoxyuridine to determine satellite cell proliferation, activation and fusion. Juvenile satellite cell‐wild‐type (SC‐WT) and satellite cell‐depleted (SC‐Dep) mice (8 weeks of age) were randomized to sham or burn injury consisting of a dorsal scald burn injury covering 30% of total body surface area. Both hindlimb and dorsal muscles were studied at 7, 14 and 21 days post‐burn. SC‐Dep mice had >93% depletion of satellite cells compared to SC‐WT (P < 0.05). Burn injury induced robust atrophy in muscles located both proximal and distal to the injury site (∼30% decrease in fibre cross‐sectional area, P < 0.05). Additionally, burn injury induced skeletal muscle regeneration, satellite cell proliferation and fusion. Depletion of satellite cells impaired post‐burn recovery of both muscle fibre cross‐sectional area and volume (P < 0.05). These findings support an integral role for satellite cells in the aetiology of lean tissue recovery following a severe burn injury.

  October 01, 2017   doi: 10.1113/JP274841   open full text
• Defining the neural fulcrum for chronic vagus nerve stimulation: implications for integrated cardiac control.
  Jeffrey L. Ardell, Heath Nier, Matthew Hammer, E. Marie Southerland, Christopher L. Ardell, Eric Beaumont, Bruce H. KenKnight, J. Andrew Armour.
  The Journal of Physiology. September 30, 2017
  Key points The evoked cardiac response to bipolar cervical vagus nerve stimulation (VNS) reflects a dynamic interaction between afferent mediated decreases in central parasympathetic drive and suppressive effects evoked by direct stimulation of parasympathetic efferent axons to the heart. The neural fulcrum is defined as the operating point, based on frequency–amplitude–pulse width, where a null heart rate response is reproducibly evoked during the on‐phase of VNS. Cardiac control, based on the principal of the neural fulcrum, can be elicited from either vagus. Beta‐receptor blockade does not alter the tachycardia phase to low intensity VNS, but can increase the bradycardia to higher intensity VNS. While muscarinic cholinergic blockade prevented the VNS‐induced bradycardia, clinically relevant doses of ACE inhibitors, beta‐blockade and the funny channel blocker ivabradine did not alter the VNS chronotropic response. While there are qualitative differences in VNS heart control between awake and anaesthetized states, the physiological expression of the neural fulcrum is maintained. Abstract Vagus nerve stimulation (VNS) is an emerging therapy for treatment of chronic heart failure and remains a standard of therapy in patients with treatment‐resistant epilepsy. The objective of this work was to characterize heart rate (HR) responses (HRRs) during the active phase of chronic VNS over a wide range of stimulation parameters in order to define optimal protocols for bidirectional bioelectronic control of the heart. In normal canines, bipolar electrodes were chronically implanted on the cervical vagosympathetic trunk bilaterally with anode cephalad to cathode (n = 8, ‘cardiac’ configuration) or with electrode positions reversed (n = 8, ‘epilepsy’ configuration). In awake state, HRRs were determined for each combination of pulse frequency (2–20 Hz), intensity (0–3.5 mA) and pulse widths (130–750 μs) over 14 months. At low intensities and higher frequency VNS, HR increased during the VNS active phase owing to afferent modulation of parasympathetic central drive. When functional effects of afferent and efferent fibre activation were balanced, a null HRR was evoked (defined as ‘neural fulcrum’) during which HRR ≈ 0. As intensity increased further, HR was reduced during the active phase of VNS. While qualitatively similar, VNS delivered in the epilepsy configuration resulted in more pronounced HR acceleration and reduced HR deceleration during VNS. At termination, under anaesthesia, transection of the vagi rostral to the stimulation site eliminated the augmenting response to VNS and enhanced the parasympathetic efferent‐mediated suppressing effect on electrical and mechanical function of the heart. In conclusion, VNS activates central then peripheral aspects of the cardiac nervous system. VNS control over cardiac function is maintained during chronic therapy.

  September 30, 2017   doi: 10.1113/JP274678   open full text
• Pre‐clinical evaluation of N‐acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy.
  Gavin J. Pinniger, Jessica R. Terrill, Evanna B. Assan, Miranda D. Grounds, Peter G. Arthur.
  The Journal of Physiology. September 30, 2017
  Key points Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N‐acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. Abstract Duchenne muscular dystrophy (DMD) is a fatal X‐linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N‐acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non‐dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC‐treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw)−1; P < 0.001] and C57 mice [3.90 (0.32) vs 5.32 (0.60) g bw−1; P < 0.001]. Maximum specific force was significantly greater in NAC‐treated mdx muscles [9.80 (2.27) vs 13.07 (3.37) N cm−2; P = 0.038]. Increased force in mdx mice was associated with reduced thiol oxidation and inflammation in fast muscles, and increased citrate synthase activity in slow muscle. Importantly, NAC significantly impaired body weight gain in both strains of young growing mice, and reduced liver weight in C57 mice and muscle weight in mdx mice. These potentially adverse effects of NAC emphasise the need for caution when interpreting improvements in muscle function based on normalised force measures, and that careful consideration be given to these effects when proposing NAC as a potential treatment for young DMD boys.

  September 30, 2017   doi: 10.1113/JP274229   open full text
• Interstitial IgG antibody pharmacokinetics assessed by combined in vivo‐ and physiologically‐based pharmacokinetic modelling approaches.
  Miro J. Eigenmann, Tine V. Karlsen, Ben‐Fillippo Krippendorff, Olav Tenstad, Ludivine Fronton, Michael B. Otteneder, Helge Wiig.
  The Journal of Physiology. September 27, 2017
  For most therapeutic antibodies, the interstitium is the target space. Although experimental methods for measuring antibody pharmacokinetics (PK) in this space are not well established, making quantitative assessment difficult, the interstitial antibody concentration is assumed to be low. Here, we combined direct quantification of antibodies in the interstitial fluid with a physiologically‐based PK (PBPK) modelling approach with the goal of better describing the PK of monoclonal antibodies in the interstitial space of different tissues. We isolated interstitial fluid by tissue centrifugation, and conducted an antibody biodistribution study in mice, measuring total tissue‐ and interstitial concentrations in selected tissues. Residual plasma, interstitial volumes and lymph flows, which are important PBPK model parameters, were assessed in vivo. We could thereby refine PBPK modelling of monoclonal antibodies, better interpret antibody biodistribution data and more accurately predict their PK in the different tissue spaces. Our results indicate that in tissues with discontinuous capillaries (liver and spleen), interstitial concentrations are reflected by plasma concentration. In tissues with continuous capillaries (e.g. skin and muscle), ∼50‐60% of plasma concentration is found in the interstitial space. In brain and kidney, on the other hand, antibodies are restricted to the vascular space. Our data may significantly impact the interpretation of biodistribution data of monoclonal antibodies and might be important when relating measured concentrations to a therapeutic effect. Opposing the view that antibodies distribution to the interstitial space is limited, we show by direct measurements and model‐based data interpretation that high antibody interstitial concentrations are reached in most tissues. This article is protected by copyright. All rights reserved

  September 27, 2017   doi: 10.1113/JP274819   open full text
• The α2A adrenoceptor suppresses excitatory synaptic transmission to both excitatory and inhibitory neurons in layer 4 barrel cortex.
  Minoru Ohshima, Chiaki Itami, Fumitaka Kimura.
  The Journal of Physiology. September 26, 2017
  The mammalian neocortex is widely innervated by noradrenergic (NA) fibres from the locus coeruleus. To determine the effects of NA on vertical synaptic inputs to layer 4 (L4) cells from the ventrobasal (VB) thalamus and layer 2/3 (L2/3), thalamocortical slices were prepared and whole‐cell recordings were made from L4 cells. Excitatory synaptic responses were evoked by electrical stimulation of the thalamus or L2/3 immediately above. Recorded cells were identified as regular spiking (RS), regular spiking non‐pyramidal (RSNP) or fast spiking (FS) cells through their firing patterns in response to current injections. NA suppressed (∼50% of control) excitatory vertical inputs to all cell types in a dose‐dependent manner. The presynaptic site of action of NA was suggested by three independent studies. First, responses caused by iontophoretically applied glutamate were not suppressed by NA. Second, paired pulse ratio was increased during NA suppression. Finally, a CV−2 (CV: coefficient of variation) analysis was performed. The resultant diagonal alignment of the ratio of CV−2 plotted against the ratio of the amplitude of postsynaptic responses suggests a presynaptic mechanism for the suppression. Experiments with phenylephrine (α1 agonist), prazosin (α1 antagonist), yohimbine (α2 antagonist) and propranolol (β antagonist) indicated that suppression was mediated by α2 adrenoceptor. To determine whether the α2A adrenoceptor subtype was involved, α2A adrenoceptor knockout mice were used. NA failed to suppress EPSCs in all cell types, suggesting an involvement of α2A adrenoceptor. Altogether, we concluded that NA suppresses vertical excitatory synaptic connections in L4 excitatory and inhibitory cells through presynaptic α2A adrenoceptor. This article is protected by copyright. All rights reserved

  September 26, 2017   doi: 10.1113/JP275142   open full text
• Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells.
  Tam T. T. Phuong, Sarah N. Redmon, Oleg Yarishkin, Jacob M. Winter, Dean Y. Li, David Križaj.
  The Journal of Physiology. September 26, 2017
  The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Taking advantage of immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+/cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVECs (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F‐actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE‐cadherin) and β−catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type, but not in TRPV4 knockout mice. Agonist‐evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells (CECs) showed lower TRPV4 mRNA/protein levels and less pronounced agonist‐evoked calcium signals compared to MVECs. These findings demonstrate a major role for TRPV4 in Ca2+ homeostasis and barrier function in the human retinal microvascular endothelial barrier and suggest TRPV4 may differentially contribute to the inner vis à vis outer blood‐retinal barrier function. This article is protected by copyright. All rights reserved

  September 26, 2017   doi: 10.1113/JP275052   open full text
• Cortical thickness is associated with altered autonomic function in cognitively impaired and non‐impaired older adults.
  Feng Lin, Ping Ren, Xixi Wang, Mia Anthony, Duje Tadin, Kathi L. Heffner.
  The Journal of Physiology. September 26, 2017
  Background Parasympathetic nervous system (PNS) is critical for adaptation to environment demands. PNS can reflect an individual's regulatory capacity of frontal brain regions and has been linked to cognitive capacity. Yet, the relationship of PNS function with cognitive decline and abnormal frontal function that characterize preclinical progression toward Alzheimer's disease (AD) is unclear. Here, we aimed to elucidate the relationship between PNS function and AD‐associated neurodegeneration by testing two competing hypotheses involving frontal regions’ activity (neurodegeneration vs. compensation). Methods In 38 older human adults with amnestic mild cognitive impairment (aMCI) or normative cognition, we measured AD‐associated neurodegeneration (AD signature cortical thickness; ADSCT), resting‐state fMRI of frontal regions’ spontaneous activation, and an electrocardiography measure of PNS (high frequency heart rate variability; HF‐HRV). HF‐HRV was assessed at rest and during a cognitive task protocol designed to capture HF‐HRV reactivity. Results Higher HF‐HRV at rest was significantly related to both more severe AD‐associated neurodegeneration (lower ADSCT scores) and worse cognitive ability. Cognitive impairments were also related to greater suppression of HF‐HRV reactivity. High activities of the anterior cingulate cortex significantly mediated relationships between ADSCT and both HF‐HRV at rest and HF‐HRV reactivity. Notably, these relationships were not affected by the clinical phenotype. Conclusions We show that AD‐associated neurodegeneration is associated with altered PNS regulation and that compensatory processes linked to frontal overactivation might be responsible for those alterations. This finding provides the first line of evidence in a new framework for understanding how early‐stage AD‐associated neurodegeneration affects autonomic regulation. This article is protected by copyright. All rights reserved

  September 26, 2017   doi: 10.1113/JP274714   open full text
• An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular type I hair cells.
  Paolo Spaiardi, Elisa Tavazzani, Marco Manca, Veronica Milesi, Giancarlo Russo, Ivo Prigioni, Walter Marcotti, Jacopo Magistretti, Sergio Masetto.
  The Journal of Physiology. September 24, 2017
  Key points Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low‐voltage‐activated outward rectifying K+ current, IK,L, whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L. We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L. Abstract Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low‐voltage‐activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch‐clamp whole‐cell technique, we examined the voltage‐ and time‐dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half‐activation at –79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The ‘early’ open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the ‘native’ biophysical properties of IK,L in adult mouse vestibular type I hair cells.

  September 24, 2017   doi: 10.1113/JP274202   open full text
• Sex differences in the role of phospholipase A2‐dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.
  Abdulla A. Ahmad, Michael D. Randall, Richard E. Roberts.
  The Journal of Physiology. September 24, 2017
  Key points The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT‐induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT‐induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. Abstract Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase‐derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re‐added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re‐addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT‐induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT‐induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT‐induced vasoconstriction in male and female PCAs, respectively.

  September 24, 2017   doi: 10.1113/JP274831   open full text
• Do right‐ventricular trabeculae gain energetic advantage from having a greater velocity of shortening?
  Toan Pham, June‐Chiew Han, Andrew Taberner, Denis Loiselle.
  The Journal of Physiology. September 24, 2017
  Key points We designed a study to test whether velocity of shortening in right‐ventricular tissue preparations is greater than that of the left side under conditions mimicking those encountered by the heart in vivo. Our experiments allowed us to explore whether greater velocity of shortening results in any energetic advantage. We found that velocity of shortening was higher in the rat right‐ventricular trabeculae. These results at the tissue level seem paradoxical to the velocity of ventricular ejection at the organ level, and are not always in accord with shortening of unloaded cells. Despite greater velocity of shortening in right‐ventricular trabeculae, they neither gained nor lost advantage with respect to both mechanical efficiency and the heat generated during shortening. Abstract Our study aimed to ascertain whether the interventricular difference of shortening velocity, reported for isolated cardiac tissues in vitro, affects interventricular mechano‐energetic performance when tested under physiological conditions using a shortening protocol designed to mimic those in vivo. We isolated trabeculae from both ventricles of the rat, mounted them in a calorimeter, and performed experiments at 37°C and 5 Hz stimulus frequency to emulate conditions of the rat heart in vivo. Each trabecula was subjected to two experimental protocols: (i) isotonic work‐loop contractions at a variety of afterloads, and (ii) isometric contractions at a variety of preloads. Velocity of shortening was calculated from the former protocol during the isotonic shortening phase of the contraction. Simultaneous measurements of force–length work and heat output allowed calculation of mechanical efficiency. The shortening‐dependent thermal component was quantified from the difference in heat output between the two protocols. Our results show that both extent of shortening and velocity of shortening were higher in trabeculae from the right ventricle. Despite these differences, trabeculae from both ventricles developed the same stress, performed the same work, liberated the same amount of heat, and hence operated at the same mechanical efficiency. Shortening heat was also ventricle independent. The interventricular differences in velocity of shortening and extent of shortening of isolated trabeculae were not manifested in any index of energetics. These collective results underscore the absence of any mechano‐energetic advantage or disadvantage conferred on right‐ventricular trabeculae arising from their superior velocity of shortening.

  September 24, 2017   doi: 10.1113/JP274837   open full text
• Elevated resting H+ current in the R1239H type 1 hypokalaemic periodic paralysis mutated Ca2+ channel.
  Clarisse Fuster, Jimmy Perrot, Christine Berthier, Vincent Jacquemond, Bruno Allard.
  The Journal of Physiology. September 24, 2017
  Key points Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage‐gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. Acute expression of human wild‐type and R1239H HypoPP1 mutant α1 subunits in mature mouse muscles showed that R1239H fibres displayed Ca2+ currents of reduced amplitude and larger resting leak inward current increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data suggest that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore and could explain why paralytic attacks preferentially occur during the recovery period following muscle exercise. Abstract Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage‐gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. The present study aimed at identifying the changes in muscle fibre electrical properties induced by acute expression of the R1239H hypokalaemic periodic paralysis human mutant α1 subunit of Ca2+ channels in a mature muscle environment to better understand the pathophysiological mechanisms involved in this disorder. We transferred genes encoding wild‐type and R1239H mutant human Ca2+ channels into hindlimb mouse muscle by electroporation and combined voltage‐clamp and intracellular pH measurements on enzymatically dissociated single muscle fibres. As compared to fibres expressing wild‐type α1 subunits, R1239H mutant‐expressing fibres displayed Ca2+ currents of reduced amplitude and a higher resting leak inward current that was increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data indicate that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore created by the mutation and that external acidification favours onset of muscle paralysis by potentiating H+ depolarizing currents and inhibiting resting inward rectifier K+ currents. Our results could thus explain why paralytic attacks preferentially occur during the recovery period following intense muscle exercise.

  September 24, 2017   doi: 10.1113/JP274638   open full text
• Feto‐ and utero‐placental vascular adaptations to chronic maternal hypoxia in the mouse.
  Lindsay S. Cahill, Monique Y. Rennie, Johnathan Hoggarth, Lisa X. Yu, Anum Rahman, John C. Kingdom, Mike Seed, Christopher K. Macgowan, John G. Sled.
  The Journal of Physiology. September 24, 2017
  Key points Chronic fetal hypoxia is one of the most common complications of pregnancy and is known to cause fetal growth restriction. The structural adaptations of the placental vasculature responsible for growth restriction with chronic hypoxia are not well elucidated. Using a mouse model of chronic maternal hypoxia in combination with micro‐computed tomography and scanning electron microscopy, we found several placental adaptations that were beneficial to fetal growth including capillary expansion, thinning of the interhaemal membrane and increased radial artery diameters, resulting in a large drop in total utero‐placental vascular resistance. One of the mechanisms used to achieve the rapid increase in capillaries was intussusceptive angiogenesis, a strategy used in human placental development to form terminal gas‐exchanging villi. These results contribute to our understanding of the structural mechanisms of the placental vasculature responsible for fetal growth restriction and provide a baseline for understanding adaptive physiological responses of the placenta to chronic hypoxia. Abstract The fetus and the placenta in eutherian mammals have a unique set of compensatory mechanisms to respond to several pregnancy complications including chronic maternal hypoxia. This study examined the structural adaptations of the feto‐ and utero‐placental vasculature in an experimental mouse model of chronic maternal hypoxia (11% O2 from embryonic day (E) 14.5–E17.5). While placental weights were unaffected by exposure to chronic hypoxia, using micro‐computed tomography, we found a 44% decrease in the absolute feto‐placental arterial vascular volume and a 30% decrease in total vessel segments in the chronic hypoxia group compared to control group. Scanning electron microscopy imaging showed significant expansion of the capillary network; consequently, the interhaemal membrane was 11% thinner to facilitate maternal–fetal exchange in the chronic hypoxia placentas. One of the mechanisms for the rapid capillary expansion was intussusceptive angiogenesis. Analysis of the utero‐placental arterial tree showed significant increases (24%) in the diameter of the radial arteries, resulting in a decrease in the total utero‐placental resistance by 2.6‐fold in the mice exposed to chronic maternal hypoxia. Together these adaptations acted to preserve placental weight whereas fetal weight was decreased.

  September 24, 2017   doi: 10.1113/JP274845   open full text
• Volume loading augments cutaneous vasodilatation and cardiac output of heat stressed older adults.
  Daniel Gagnon, Steven A. Romero, Hai Ngo, Satyam Sarma, William K. Cornwell, Paula Y. S. Poh, Douglas Stoller, Benjamin D. Levine, Craig G. Crandall.
  The Journal of Physiology. September 22, 2017
  Key points Age‐related changes in cutaneous microvascular and cardiac functions limit the extent of cutaneous vasodilatation and the increase in cardiac output that healthy older adults can achieve during passive heat stress. However, it is unclear if these age‐related changes in microvascular and cardiac functions maximally restrain the levels of cutaneous vasodilatation and cardiac output that healthy older adults can achieve during heat stress. We observed that rapid volume loading, performed during passive heat stress, augments both cutaneous vasodilatation and cardiac output in healthy older humans. These findings demonstrate that the microcirculation of healthy aged skin can further dilate during passive heat exposure, despite peripheral limitations to vasodilatation. Furthermore, healthy older humans can augment cardiac output when cardiac pre‐load is increased during heat stress. Abstract Primary ageing markedly attenuates cutaneous vasodilatation and the increase in cardiac output during passive heating. However, it remains unclear if these responses are maximally restrained by age‐related changes in cutaneous microvascular and cardiac functions. We hypothesized that rapid volume loading performed during heat stress would increase cardiac output in older adults without parallel increases in cutaneous vasodilatation. Twelve young (Y: 26 ± 5 years) and ten older (O: 69 ± 3 years) healthy adults were passively heated until core temperature increased by 1.5°C. Cardiac output (thermodilution), forearm vascular conductance (FVC, venous occlusion plethysmography) and cutaneous vascular conductance (CVC, laser‐Doppler) were measured before and after rapid infusion of warmed saline (15 mL kg−1, ∼7 min). While heat stressed, but prior to saline infusion, cardiac output (O: 6.8 ± 0.4 vs. Y: 9.4 ± 0.6 L min−1), FVC (O: 0.08 ± 0.01 vs. Y: 0.17 ± 0.02 mL (100 mL min−1 mmHg−1)−1), and CVC (O: 1.29 ± 0.34 vs. Y: 1.93 ± 0.30 units mmHg−1) were lower in older adults (all P < 0.01). Rapid saline infusion increased cardiac output (O: +1.9 ± 0.3, Y: +1.8 ± 0.7 L min−1), FVC (O: +0.015 ± 0.007, Y: +0.048 ± 0.013 mL (100 mL min−1 mmHg−1)−1), and CVC (O: +0.28 ± 0.10, Y: +0.29 ± 0.16 units mmHg−1) in both groups (all P < 0.01). The absolute increase in cardiac output and CVC were similar between groups, whereas FVC increased to a greater extent in young adults (P < 0.01). These results demonstrate that healthy older adults can achieve greater levels of cutaneous vasodilatation and cardiac output during passive heating.

  September 22, 2017   doi: 10.1113/JP274742   open full text
• Subunit‐dependent oxidative stress sensitivity of LRRC8 volume‐regulated anion channels.
  Antonella Gradogna, Paola Gavazzo, Anna Boccaccio, Michael Pusch.
  The Journal of Physiology. September 22, 2017
  Key points Swelling‐activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel‐forming proteins or on regulatory factors. We found that LRRC8A–LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A–LRRC8C and LRRC8A–LRRC8D heteromers are inhibited by oxidation. Volume‐regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells. Our results show that LRRC8 channel proteins are directly modulated by oxidation in a subunit‐specific manner. Abstract The volume‐regulated anion channel (VRAC) is formed by heteromers of LRRC8 proteins containing the essential LRRC8A subunit and at least one among the LRRC8B–E subunits. Reactive oxygen species (ROS) play physiological and pathophysiological roles and VRAC channels are highly ROS sensitive. However, it is unclear if ROS act directly on the channels or on molecules involved in the activation pathway. We used fluorescently tagged LRRC8 proteins that yield large constitutive currents to test direct effects of oxidation. We found that 8A/8E heteromers are dramatically potentiated (more than 10‐fold) by oxidation of intracellular cysteine residues by chloramine‐T or tert‐butyl hydroperoxide. Oxidation was, however, not necessary for hypotonicity‐induced activation. In contrast, 8A/8C and 8A/8D heteromers were strongly inhibited by oxidation. Endogenous VRAC currents in Jurkat T lymphocytes were similarly inhibited by oxidation, in agreement with the finding that LRRC8C and LRRC8D subunits were more abundantly expressed than LRRC8E in Jurkat cells. Our results show that LRRC8 channels are directly modulated by oxidation in a subunit‐dependent manner.

  September 22, 2017   doi: 10.1113/JP274795   open full text
• Co‐ordination of the upper and lower limbs for vestibular control of balance.
  Craig P. Smith, Jonathan E. Allsop, Michael Mistry, Raymond F. Reynolds.
  The Journal of Physiology. September 22, 2017
  Key points When standing and holding an earth‐fixed object, galvanic vestibular stimulation (GVS) can evoke upper limb responses to maintain balance. In the present study, we determined how these responses are affected by grip context (no contact, light grip and firm grip), as well as how they are co‐ordinated with the lower limbs to maintain balance. When GVS was applied during firm grip, hand and ground reaction forces were generated. The directions of these force vectors were co‐ordinated such that the overall body sway response was always aligned with the inter‐aural axis (i.e. craniocentric). When GVS was applied during light grip (< 1 N), hand forces were secondary to body movement, suggesting that the arm performed a mostly passive role. These results demonstrate that a minimum level of grip is required before the upper limb becomes active in balance control and also that the upper and lower limbs co‐ordinate for an appropriate whole‐body sway response. Abstract Vestibular stimulation can evoke responses in the arm when it is used for balance. In the present study, we determined how these responses are affected by grip context, as well as how they are co‐ordinated with the rest of the body. Galvanic vestibular stimulation (GVS) was used to evoke balance responses under three conditions of manual contact with an earth‐fixed object: no contact, light grip (< 1 N) (LG) and firm grip (FG). As grip progressed along this continuum, we observed an increase in GVS‐evoked hand force, with a simultaneous reduction in ground reaction force (GRF) through the feet. During LG, hand force was secondary to the GVS‐evoked body sway response, indicating that the arm performed a mostly passive role. By contrast, during FG, the arm became actively involved in driving body sway, as revealed by an early force impulse in the opposite direction to that seen in LG. We then examined how the direction of this active hand vector was co‐ordinated with the lower limbs. Consistent with previous findings on sway anisotropy, FG skewed the direction of the GVS‐evoked GRF vector towards the axis of baseline postural instability. However, this was effectively cancelled by the hand force vector, such that the whole‐body sway response remained aligned with the inter‐aural axis, maintaining the craniocentric principle. These results show that a minimum level of grip is necessary before the upper limb plays an active role in vestibular‐evoked balance responses. Furthermore, they demonstrate that upper and lower‐limb forces are co‐ordinated to produce an appropriate whole‐body sway response.

  September 22, 2017   doi: 10.1113/JP274272   open full text
• Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth restricted fetal sheep.
  Paul J. Rozance, Laura Zastoupil, Stephanie R. Wesolowski, David A. Goldstrohm, Brittany Strahan, Melanie Cree‐Green, Melinda Sheffield‐Moore, Giacomo Meschia, William W. Hay, Randall B. Wilkening, Laura D. Brown.
  The Journal of Physiology. September 22, 2017
  Reduced skeletal muscle mass in the IUGR fetus persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake, and protein accretion rates in skeletal muscle, late gestation CON (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate, and fractional synthetic rate were lower in IUGR compared to CON (P < 0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9 ± 5.6, CON: 60.9 ± 6.5 ml·min−1; P < 0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4 ± 1.4, CON: 14.7 ± 1.3 μmol·min−1·100 g−1; P < 0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3 ± 0.5, CON: 2.9 ± 0.2 μmol·min−1·100 g−1; P < 0.05). Blood O2 saturation (R2 = 0.80, P < 0.0001) and plasma glucose (R2 = 0.68, P < 0.0001), insulin (R2 = 0.40, P < 0.005), and IGF‐1 (R2 = 0.80, P < 0.0001) were positively associated and norepinephrine (R2 = 0.59, P < 0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are likely hormonally mediated by mechanisms that include increased fetal norepinephrine and reduced IGF‐1 and insulin. This article is protected by copyright. All rights reserved

  September 22, 2017   doi: 10.1113/JP275230   open full text
• Kindlin‐2 interacts with endothelial adherens junctions to support vascular barrier integrity.
  Elzbieta Pluskota, Kamila M. Bledzka, Katarzyna Bialkowska, Dorota Szpak, Dmitry A. Soloviev, Sidney V. Jones, Dmitriy Verbovetskiy, Edward F. Plow.
  The Journal of Physiology. September 21, 2017
  Key points A reduction in Kindlin‐2 levels in endothelial cells compromises vascular barrier function. Kindlin‐2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with β‐ or γ‐catenin and cortical actin filaments, Kindlin‐2 stabilizes adherens junctions. The Kindlin‐2 binding sites for β‐ and γ‐catenin reside within its F1 and F3 subdomains. Although Kindlin‐2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin‐2+/− mice. Abstract Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin‐2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin‐2 in regulating vascular integrity using several different approaches to decrease Kindlin‐2 levels in EC. Reduced levels of Kindlin‐2 in Kindlin‐2+/– mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin‐2 siRNA showed enhanced basal and platelet‐activating factor (PAF) or lipopolysaccharide‐stimulated vascular leakage compared to wild‐type (WT) counterparts. PAF preferentially disrupted the Kindlin‐2+/− MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin‐2 co‐localized and co‐immunoprecipitated with vascular endothelial cadherin‐based complexes, including β‐ and γ‐catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin‐2 with β‐ and γ‐catenin and actin was demonstrated in co‐immunoprecipitation and surface plasmon resonance experiments. In thrombin‐stimulated HUVECs, Kindlin‐2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The β‐ and γ‐catenin binding site resides within the F1 and F3 subdomains of Kindlin‐2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin‐2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin‐based complexes to cortical actin and thereby stabilizing AJ.

  September 21, 2017   doi: 10.1113/JP274380   open full text
• Adenosine and dopamine oppositely modulate a hyperpolarization‐activated current Ih in chemosensory neurons of the rat carotid body in co‐culture.
  Min Zhang, Cathy Vollmer, Colin A. Nurse.
  The Journal of Physiology. September 21, 2017
  Key points Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co‐cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization‐activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih. Abstract Adenosine and dopamine (DA) act as neurotransmitters or neuromodulators at the carotid body (CB) chemosensory synapse, but their mechanisms of action are not fully understood. Using a functional co‐culture model of rat CB chemoreceptor (type I) cell clusters and juxtaposed afferent petrosal neurons (PNs), we tested the hypothesis that adenosine and DA act postsynaptically to modulate a hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) cation current (Ih). In whole‐cell recordings from hypoxia‐responsive PNs, cAMP mimetics enhanced Ih whereas the HCN blocker ZD7288 (2 μm) reversibly inhibited Ih. Adenosine caused a potentiation of Ih (EC50 ∼ 35 nm) that was sensitive to the A2a blocker SCH58261 (5 nm), and an ∼16 mV depolarizing shift in V½ for voltage dependence of Ih activation. By contrast, DA (10 μm) caused an inhibition of Ih that was sensitive to the D2 blocker sulpiride (1–10 μm), and an ∼11 mV hyperpolarizing shift in V½. Sulpiride potentiated Ih in neurons adjacent to, but not distant from, type I cell clusters. DA also decreased PN action potential frequency whereas adenosine had the opposite effect. During simultaneous paired recordings, SCH58261 inhibited both the presynaptic hypoxia‐induced receptor potential in type I cells and the postsynaptic PN response. By contrast, SCH58261 inhibited only the postsynaptic PN response induced by isohydric hypercapnia. Confocal immunofluorescence confirmed the localization of HCN4 subunits in tyrosine hydroxylase‐positive chemoafferent neurons in tissue sections of rat petrosal ganglia. These data suggest that adenosine and DA, acting through A2a and D2 receptors respectively, regulate PN excitability via their opposing actions on Ih.

  September 21, 2017   doi: 10.1113/JP274743   open full text
• Synaptic excitation by climbing fibre collaterals in the cerebellar nuclei of juvenile and adult mice.
  Marion Najac, Indira M. Raman.
  The Journal of Physiology. September 20, 2017
  Key points The inferior olive sends instructive motor signals to the cerebellum via the climbing fibre projection, which sends collaterals directly to large premotor neurons of the mouse cerebellar nuclei (CbN cells). Optogenetic activation of inferior olivary axons in vitro evokes EPSCs in CbN cells of several hundred pA to more than 1 nA. The inputs are three‐fold larger at younger ages, 12 to 14 days old, than at 2 months old, suggesting a strong functional role for this pathway earlier in development. The EPSCs are multipeaked, owing to burst firing in several olivary afferents that fire asynchronously. The convergence of climbing fibre collaterals onto CbN cells decreases from ∼40 to ∼8, which is consistent with the formation of closed‐loop circuits in which each CbN neuron receives input from 4–7 collaterals from inferior olivary neurons as well as from all 30–50 Purkinje cells that are innervated by those olivary neurons. Abstract The inferior olive conveys instructive signals to the cerebellum that drive sensorimotor learning. Inferior olivary neurons transmit their signals via climbing fibres, which powerfully excite Purkinje cells, evoking complex spikes and depressing parallel fibre synapses. Additionally, however, these climbing fibres send collaterals to the cerebellar nuclei (CbN). In vivo and in vitro data suggest that climbing fibre collateral excitation is weak in adult mice, raising the question of whether the primary role of this pathway may be developmental. We therefore examined climbing fibre collateral input to large premotor CbN cells over development by virally expressing channelrhodopsin in the inferior olive. In acute cerebellar slices from postnatal day (P)12–14 mice, light‐evoked EPSCs were large (> 1 nA at −70 mV). The amplitude of these EPSCs decreased over development, reaching a plateau of ∼350 pA at P20–60. Trains of EPSCs (5 Hz) depressed strongly throughout development, whereas convergence estimates indicated that the total number of functional afferents decreased with age. EPSC waveforms consisted of multiple peaks, probably resulting from action potential bursts in single collaterals and variable times to spike threshold in converging afferents. Activating climbing fibre collaterals evoked well‐timed increases in firing probability in CbN neurons, especially in younger mice. The initially strong input, followed by the decrement in synaptic strength coinciding with the pruning of climbing fibres in the cerebellar cortex, implicates the climbing fibre collateral pathway in early postnatal development. Additionally, the persistence of substantial synaptic input at least to P60 suggests that this pathway may function in cerebellar processing into adulthood.

  September 20, 2017   doi: 10.1113/JP274598   open full text
• Persistent aberrant cortical phase–amplitude coupling following seizure treatment in absence epilepsy models.
  Atul Maheshwari, Abraham Akbar, Mai Wang, Rachel L. Marks, Katherine Yu, Suhyeorn Park, Brett L. Foster, Jeffrey L. Noebels.
  The Journal of Physiology. September 19, 2017
  Key points In two monogenic models of absence epilepsy, interictal beta/gamma power is augmented in homozygous stargazer (stg/stg) but not homozygous tottering (tg/tg) mice. There are distinct gene‐linked patterns of aberrant phase–amplitude coupling in the interictal EEG of both stg/stg and tg/tg mice, compared to +/+ and stg/+ mice. Treatment with ethosuximide significantly blocks seizures in both genotypes, but the abnormal phase–amplitude coupling remains. Seizure‐free stg/+ mice have normal power and phase–amplitude coupling, but beta/gamma power is significantly reduced with NMDA receptor blockade, revealing a latent cortical network phenotype that is separable from, and therefore not a result of, seizures. Altogether, these findings reveal gene‐linked quantitative electrographic biomarkers free from epileptiform activity, and provide a potential network correlate for persistent cognitive deficits in absence epilepsy despite effective treatment. Abstract In childhood absence epilepsy, cortical seizures are brief and intermittent; however there are extended periods without behavioural or electrographic ictal events. This genetic disorder is associated with variable degrees of cognitive dysfunction, but no consistent functional biomarkers that might provide insight into interictal cortical function have been described. Previous work in monogenic mouse models of absence epilepsy have shown that the interictal EEG displays augmented beta/gamma power in homozygous stargazer (stg/stg) mice bearing a presynaptic AMPA receptor defect, but not homozygous tottering (tg/tg) mice with a P/Q type calcium channel mutation. To further evaluate the interictal EEG, we quantified phase–amplitude coupling (PAC) in stg/stg, stg/+, tg/tg and wild‐type (+/+) mice. We found distinct gene‐linked patterns of aberrant PAC in stg/stg and tg/tg mice compared to +/+ and stg/+ mice. Treatment with ethosuximide significantly blocks seizures in both stg/stg and tg/tg, but the abnormal PAC remains. Stg/+ mice are seizure free with normal baseline beta/gamma power and normal theta‐gamma PAC, but like stg/stg mice, beta/gamma power is significantly reduced by NMDA receptor blockade, a treatment that paradoxically enhances seizures in stg/stg mice. Stg/+ mice, therefore, have a latent cortical network phenotype that is veiled by NMDA‐mediated neurotransmission. Altogether, these findings reveal gene‐linked quantitative electrographic biomarkers in the absence of epileptiform activity and provide a potential network correlate for persistent cognitive deficits in absence epilepsy despite effective treatment.

  September 19, 2017   doi: 10.1113/JP274696   open full text
• Median preoptic glutamatergic neurons promote thermoregulatory heat loss and water consumption in mice.
  Stephen B. G. Abbott, Clifford B. Saper.
  The Journal of Physiology. September 13, 2017
  Key points Glutamatergic neurons in the median preoptic area were stimulated using genetically targeted Channelrhodopsin 2 in transgenic mice. Stimulation of glutamatergic median preoptic area neurons produced a profound hypothermia due to cutaneous vasodilatation. Stimulation also produced drinking behaviour that was inhibited as water was ingested, suggesting pre‐systemic feedback gating of drinking. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap. Abstract The median preoptic nucleus (MnPO) serves an important role in the integration of water/electrolyte homeostasis and thermoregulation, but we have a limited understanding these functions at a cellular level. Using Cre–Lox genetic targeting of Channelrhodospin 2 in VGluT2 transgenic mice, we examined the effect of glutamatergic MnPO neuron stimulation in freely behaving mice while monitoring drinking behaviour and core temperature. Stimulation produced a strong hypothermic response in 62% (13/21) of mice (core temperature: −4.6 ± 0.5°C, P = 0.001 vs. controls) caused by cutaneous vasodilatation. Stimulating glutamatergic MnPO neurons also produced robust drinking behaviour in 82% (18/22) of mice. Mice that drank during stimulation consumed 912 ± 163 μl (n = 8) during a 20 min trial in the dark phase, but markedly less during the light phase (421 ± 83 μl, P = 0.0025). Also, drinking during stimulation was inhibited as water was ingested, suggesting pre‐systemic feedback gating of drinking. Both hypothermia and drinking during stimulation occurred in 50% of mice tested. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap. Thus, activation of separate but overlapping populations of glutamatergic MnPO neurons produces effects on drinking and autonomic thermoregulatory mechanisms, providing a structural basis for their frequently being coordinated (e.g. during hyperthermia).

  September 13, 2017   doi: 10.1113/JP274667   open full text
• Molecular composition and heterogeneity of the LRRC8‐containing swelling‐activated osmolyte channels in primary rat astrocytes.
  Alexandra L. Schober, Corinne S. Wilson, Alexander A. Mongin.
  The Journal of Physiology. September 12, 2017
  Key points The volume‐regulated anion channel (VRAC) is a swelling‐activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners. We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling‐activated release of diverse organic osmolytes in rat astrocytes. Efflux of uncharged osmolytes (myo‐inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E. Conversely, release of charged osmolytes (d‐aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D. Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D‐containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules. Abstract The volume‐regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl−/anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d‐[14C]aspartate and [3H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [3H]taurine and myo‐[3H]inositol, without major impact on the simultaneously measured efflux of the charged d‐[14C]aspartate. In contrast, the release of d‐[14C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D‐containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules.

  September 12, 2017   doi: 10.1113/JP275053   open full text
• Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.
  Natalie M. Hawken, Elena I. Zaika, Terunaga Nakagawa.
  The Journal of Physiology. September 12, 2017
  Key points The AMPA‐type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid‐accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex. Abstract During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid‐exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR–CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg‐ and CNIH3‐bound AMPARs were compared. Specifically, in detergent the GluA2‐A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2‐A793F–CNIH3 complex expressed a gain of function. In contrast, the GluA2‐A793F–Stg complex was stable, but had diminished gating modulation. GluA2‐C528L destabilized the AMPAR–CNIH3 complex but stabilized the AMPAR–Stg complex, with overall loss of function in gating modulation. Furthermore, loss‐of‐function mutations in this TMD region cancelled the effects of a gain‐of‐function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit‐specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant‐phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.

  September 12, 2017   doi: 10.1113/JP274897   open full text
• Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.
  Jessica D. Clarke, Jessica L. Caldwell, Charles M. Pearman, David A. Eisner, Andrew W. Trafford, Katharine M. Dibb.
  The Journal of Physiology. September 11, 2017
  Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. Abstract Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via ICa‐L but decreased peak ICa‐L. Decreased peak ICa‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak ICa‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

  September 11, 2017   doi: 10.1113/JP274053   open full text
• Pulmonary artery wave propagation and reservoir function in conscious man: impact of pulmonary vascular disease, respiration and dynamic stress tests.
  Junjing Su, Charlotte Manisty, Ulf Simonsen, Luke S. Howard, Kim H. Parker, Alun D. Hughes.
  The Journal of Physiology. September 11, 2017
  Key points Wave travel plays an important role in cardiovascular physiology. However, many aspects of pulmonary arterial wave behaviour remain unclear. Wave intensity and reservoir‐excess pressure analyses were applied in the pulmonary artery in subjects with and without pulmonary hypertension during spontaneous respiration and dynamic stress tests. Arterial wave energy decreased during expiration and Valsalva manoeuvre due to decreased ventricular preload. Wave energy also decreased during handgrip exercise due to increased heart rate. In pulmonary hypertension patients, the asymptotic pressure at which the microvascular flow ceases, the reservoir pressure related to arterial compliance and the excess pressure caused by waves increased. The reservoir and excess pressures decreased during Valsalva manoeuvre but remained unchanged during handgrip exercise. This study provides insights into the influence of pulmonary vascular disease, spontaneous respiration and dynamic stress tests on pulmonary artery wave propagation and reservoir function. Abstract Detailed haemodynamic analysis may provide novel insights into the pulmonary circulation. Therefore, wave intensity and reservoir‐excess pressure analyses were applied in the pulmonary artery to characterize changes in wave propagation and reservoir function during spontaneous respiration and dynamic stress tests. Right heart catheterization was performed using a pressure and Doppler flow sensor tipped guidewire to obtain simultaneous pressure and flow velocity measurements in the pulmonary artery in control subjects and patients with pulmonary arterial hypertension (PAH) at rest. In controls, recordings were also obtained during Valsalva manoeuvre and handgrip exercise. The asymptotic pressure at which the flow through the microcirculation ceases, the reservoir pressure related to arterial compliance and the excess pressure caused by arterial waves increased in PAH patients compared to controls. The systolic and diastolic rate constants also increased, while the diastolic time constant decreased. The forward compression wave energy decreased by ∼8% in controls and ∼6% in PAH patients during expiration compared to inspiration, while the wave speed remained unchanged throughout the respiratory cycle. Wave energy decreased during Valsalva manoeuvre (by ∼45%) and handgrip exercise (by ∼27%) with unaffected wave speed. Moreover, the reservoir and excess pressures decreased during Valsalva manoeuvre but remained unaltered during handgrip exercise. In conclusion, reservoir‐excess pressure analysis applied to the pulmonary artery revealed distinctive differences between controls and PAH patients. Variations in the ventricular preload and afterload influence pulmonary arterial wave propagation as demonstrated by changes in wave energy during spontaneous respiration and dynamic stress tests.

  September 11, 2017   doi: 10.1113/JP274385   open full text
• Mouse retinal ganglion cell signalling is dynamically modulated through parallel anterograde activation of cannabinoid and vanilloid pathways.
  Andrew O. Jo, Jennifer M. Noel, Monika Lakk, Oleg Yarishkin, Daniel A. Ryskamp, Koji Shibasaki, Maureen A. McCall, David Križaj.
  The Journal of Physiology. September 07, 2017
  Key points Retinal cells use vanilloid transient receptor potential (TRP) channels to integrate light‐evoked signals with ambient mechanical, chemical and temperature information. Localization and function of the polymodal non‐selective cation channel TRPV1 (transient receptor potential vanilloid isoform 1) remains elusive. TRPV1 is expressed in a subset of mouse retinal ganglion cells (RGCs) with peak expression in the mid‐peripheral retina. Endocannabinoids directly activate TRPV1 and inhibit it through cannabinoid type 1 receptors (CB1Rs) and cAMP pathways. Activity‐dependent endocannabinoid release may modulate signal gain in RGCs through simultaneous manipulation of calcium and cAMP signals mediated by TRPV1 and CB1R. Abstract How retinal ganglion cells (RGCs) process and integrate synaptic, mechanical, swelling stimuli with light inputs is an area of intense debate. The nociceptive cation channel TRPV1 (transient receptor potential vanilloid type 1) modulates RGC Ca2+ signals and excitability yet the proportion of RGCs that express it remains unclear. Furthermore, TRPV1's response to endocannabinoids (eCBs), the putative endogenous retinal activators, is unknown, as is the potential modulation by cannabinoid receptors (CBRs). The density of TRPV1‐expressing RGCs in the Ai9:Trpv1 reporter mouse peaked in the mid‐peripheral retina. TRPV1 agonists including capsaicin (CAP) and the eCBs anandamide and N‐arachidonoyl‐dopamine elevated [Ca2+]i in 30–40% of wild‐type RGCs, with effects suppressed by TRPV1 antagonists capsazepine (CPZ) and BCTC ((4‐(3‐chloro‐2‐pyridinyl)‐N‐[4‐(1,1‐dimethylethyl)phenyl]‐1‐piperazinecarboxamide), and lacking in Trpv1−/− cells. The cannabinoid receptor type 1 (CB1R) colocalized with TRPV1:tdTomato expression. Its agonists 2‐arachidonoylglycerol (2‐AG) and WIN55,122 inhibited CAP‐induced [Ca2+]i signals in adult, but not early postnatal, RGCs. The suppressive effect of 2‐AG on TRPV1 activation was emulated by positive modulators of the protein kinase A (PKA) pathway, inhibited by the CB1R antagonist rimonabant and Gi uncoupler pertussis toxin, and absent in Cnr1−/− RGCs. We conclude that TRPV1 is a modulator of Ca2+ homeostasis in a subset of RGCs that show non‐uniform distribution across the mouse retina. Non‐retrograde eCB‐mediated modulation of RGC signalling involves a dynamic push–pull between direct TRPV1 activation and PKA‐dependent regulation of channel inactivation, with potential functions in setting the bandwidth of postsynaptic responses, sensitivity to mechanical/excitotoxic stress and neuroprotection.

  September 07, 2017   doi: 10.1113/JP274562   open full text
• Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.
  Adrianne R. Wilson‐Poe, Hyo‐Jin Jeong, Christopher W. Vaughan.
  The Journal of Physiology. September 07, 2017
  Key points Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. Abstract The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ‐opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO‐induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ‐opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

  September 07, 2017   doi: 10.1113/JP274157   open full text
• Hypercapnia‐induced active expiration increases in sleep and enhances ventilation in unanaesthetized rats.
  Isabela P. Leirão, Carlos A. Silva, Luciane H. Gargaglioni, Glauber S. F. da Silva.
  The Journal of Physiology. September 02, 2017
  Key points Expiratory muscles (abdominal and thoracic) can be recruited when respiratory drive increases under conditions of increased respiratory demand such as hypercapnia. Studying hypercapnia‐induced active expiration in unanaesthetized rats importantly contributes to the understanding of how the control system is integrated in vivo in freely moving animals. In unanaesthetized rats, hypercapnia‐induced active expiration was not always recruited either in wakefulness or in sleep, suggesting that additional factors influence the recruitment of active expiration. The pattern of abdominal muscle recruitment varied in a state‐dependent manner with active expiration being more predominant in the sleep state than in quiet wakefulness. Pulmonary ventilation was enhanced in periods with active expiration compared to periods without it. Abstract Expiration is passive at rest but becomes active through recruitment of abdominal muscles under increased respiratory drive. Hypercapnia‐induced active expiration has not been well explored in unanaesthetized rats. We hypothesized that (i) CO2‐evoked active expiration is recruited in a state‐dependent manner, i.e. differently in sleep or wakefulness, and (ii) recruitment of active expiration enhances ventilation, hence having an important functional role in meeting metabolic demand. To test these hypotheses, Wistar rats (280–330 g) were implanted with electrodes for EEG and electromyography EMG of the neck, diaphragm (DIA) and abdominal (ABD) muscles. Active expiratory events were considered as rhythmic ABDEMG activity interposed to DIAEMG. Animals were exposed to room air followed by hypercapnia (7% CO2) with EEG, EMG and ventilation (V̇E) recorded throughout the experimental protocol. No active expiration was observed during room air exposure. During hypercapnia, CO2‐evoked active expiration was predominantly recruited during non‐rapid eye movement sleep. Its increased occurrence during sleep was evidenced by the decreased DIA‐to‐ADB ratio (1:1 ratio means that each DIA event is followed by an ABD event, indicating a high occurrence of ABD activity). Moreover, V̇E was also enhanced (P < 0.05) in periods with active expiration. V̇E had a positive correlation (P < 0.05) with the peak amplitude of ABDEMG activity. The data demonstrate strongly that hypercapnia‐induced active expiration increases during sleep and provides an important functional role to support V̇E in conditions of increased respiratory demand.

  September 02, 2017   doi: 10.1113/JP274726   open full text
• Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons.
  Cedric Yapo, Anu G. Nair, Lorna Clement, Liliana R. Castro, Jeanette Hellgren Kotaleski, Pierre Vincent.
  The Journal of Physiology. September 02, 2017
  Key points Brief dopamine events are critical actors of reward‐mediated learning in the striatum; the intracellular cAMP–protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub‐micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA‐dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA‐dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase‐1 (PP1) inhibition by DARPP‐32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. Abstract The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA‐dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium‐sized spiny neurons (MSNs), which express either D1 or D2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub‐micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D2 than on D1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA‐dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP‐32. Simulations further suggested that D2 MSNs can also operate in a ‘tone‐sensing’ mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought.

  September 02, 2017   doi: 10.1113/JP274475   open full text
• Dissociating external power from intramuscular exercise intensity during intermittent bilateral knee‐extension in humans.
  Matthew J. Davies, Alan P. Benson, Daniel T. Cannon, Simon Marwood, Graham J. Kemp, Harry B. Rossiter, Carrie Ferguson.
  The Journal of Physiology. September 02, 2017
  Key points Continuous high‐intensity constant‐power exercise is unsustainable, with maximal oxygen uptake (V̇O2 max ) and the limit of tolerance attained after only a few minutes. Performing the same power intermittently reduces the O2 cost of exercise and increases tolerance. The extent to which this dissociation is reflected in the intramuscular bioenergetics is unknown. We used pulmonary gas exchange and 31P magnetic resonance spectroscopy to measure whole‐body V̇O2, quadriceps phosphate metabolism and pH during continuous and intermittent exercise of different work:recovery durations. Shortening the work:recovery durations (16:32 s vs. 32:64 s vs. 64:128 s vs. continuous) at a work rate estimated to require 110% peak aerobic power reduced V̇O2, muscle phosphocreatine breakdown and muscle acidification, eliminated the glycolytic‐associated contribution to ATP synthesis, and increased exercise tolerance. Exercise intensity (i.e. magnitude of intramuscular metabolic perturbations) can be dissociated from the external power using intermittent exercise with short work:recovery durations. Abstract Compared with work‐matched high‐intensity continuous exercise, intermittent exercise dissociates pulmonary oxygen uptake (V̇O2) from the accumulated work. The extent to which this reflects differences in O2 storage fluctuations and/or contributions from oxidative and substrate‐level bioenergetics is unknown. Using pulmonary gas‐exchange and intramuscular 31P magnetic resonance spectroscopy, we tested the hypotheses that, at the same power: ATP synthesis rates are similar, whereas peak V̇O2 amplitude is lower in intermittent vs. continuous exercise. Thus, we expected that: intermittent exercise relies less upon anaerobic glycolysis for ATP provision than continuous exercise; shorter intervals would require relatively greater fluctuations in intramuscular bioenergetics than in V̇O2 compared to longer intervals. Six men performed bilateral knee‐extensor exercise (estimated to require 110% peak aerobic power) continuously and with three different intermittent work:recovery durations (16:32, 32:64 and 64:128 s). Target work duration (576 s) was achieved in all intermittent protocols; greater than continuous (252 ± 174 s; P < 0.05). Mean ATP turnover rate was not different between protocols (∼43 mm min−1 on average). However, the intramuscular phosphocreatine (PCr) component of ATP generation was greatest (∼30 mm min−1), and oxidative (∼10 mm min−1) and anaerobic glycolytic (∼1 mm min−1) components were lowest for 16:32 and 32:64 s intermittent protocols, compared to 64:128 s (18 ± 6, 21 ± 10 and 10 ± 4 mm min−1, respectively) and continuous protocols (8 ± 6, 20 ± 9 and 16 ± 14 mm min−1, respectively). As intermittent work duration increased towards continuous exercise, ATP production relied proportionally more upon anaerobic glycolysis and oxidative phosphorylation, and less upon PCr breakdown. However, performing the same high‐intensity power intermittently vs. continuously reduced the amplitude of fluctuations in V̇O2 and intramuscular metabolism, dissociating exercise intensity from the power output and work done.

  September 02, 2017   doi: 10.1113/JP274589   open full text
• Cortical control of object‐specific grasp relies on adjustments of both activity and effective connectivity: a common marmoset study.
  Banty Tia, Mitsuaki Takemi, Akito Kosugi, Elisa Castagnola, Alberto Ansaldo, Takafumi Nakamura, Davide Ricci, Junichi Ushiba, Luciano Fadiga, Atsushi Iriki.
  The Journal of Physiology. September 02, 2017
  Key points The cortical mechanisms of grasping have been extensively studied in macaques and humans; here, we investigated whether common marmosets could rely on similar mechanisms despite strong differences in hand morphology and grip diversity. We recorded electrocorticographic activity over the sensorimotor cortex of two common marmosets during the execution of different grip types, which allowed us to study cortical activity (power spectrum) and physiologically inferred connectivity (phase‐slope index). Analyses were performed in beta (16–35 Hz) and gamma (75–100 Hz) frequency bands and our results showed that beta power varied depending on grip type, whereas gamma power displayed clear epoch‐related modulation. Strength and direction of inter‐area connectivity varied depending on grip type and epoch. These findings suggest that fundamental control mechanisms are conserved across primates and, in future research, marmosets could represent an adequate model to investigate primate brain mechanisms. Abstract The cortical mechanisms of grasping have been extensively studied in macaques and humans. Here, we investigated whether common marmosets could rely on similar mechanisms despite striking differences in manual dexterity. Two common marmosets were trained to grasp‐and‐pull three objects eliciting different hand configurations: whole‐hand, finger and scissor grips. The animals were then chronically implanted with 64‐channel electrocorticogram arrays positioned over the left premotor, primary motor and somatosensory cortex. Power spectra, reflecting predominantly cortical activity, and phase‐slope index, reflecting the direction of information flux, were studied in beta (16–35 Hz) and gamma (75–100 Hz) bands. Differences related to grip type, epoch (reach, grasp) and cortical area were statistically assessed. Results showed that whole‐hand and scissor grips triggered stronger beta desynchronization than finger grip. Task epochs clearly modulated gamma power, especially for finger and scissor grips. Considering effective connectivity, finger and scissor grips evoked stronger outflow from primary motor to premotor cortex, whereas whole‐hand grip displayed the opposite pattern. These findings suggest that fundamental control mechanisms, relying on adjustments of cortical activity and connectivity, are conserved across primates. Consistently, marmosets could represent a good model to investigate primate brain mechanisms.

  September 02, 2017   doi: 10.1113/JP274629   open full text
• Rearing‐environment‐dependent hippocampal local field potential differences in wild‐type and inositol trisphosphate receptor type 2 knockout mice.
  Mika Tanaka, Xiaowen Wang, Katsuhiko Mikoshiba, Hajime Hirase, Yoshiaki Shinohara.
  The Journal of Physiology. August 27, 2017
  Key points Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca2+ surges are involved in this experience‐dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP3R2)‐knockout (KO) mice, in which IP3/Ca2+ signalling in astrocytes is largely diminished. We found that this experience‐dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP3R2 deficiency. Abstract Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression‐like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane‐anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non‐theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP3R2)‐knockout (KO) mice, in which astrocytic Ca2+ elevations are largely diminished. We found that the gamma power was also higher in IP3R2‐KO‐ENR mice compared to IP3R2‐KO‐ISO mice, suggesting that the rearing‐environment‐dependent gamma power alteration does not necessarily require the astrocytic IP3/Ca2+ pathway. By contrast, ripple events showed genotype‐dependent changes, as well as rearing condition‐dependent changes: ISO housing and IP3R2 deficiency both lead to longer inter‐ripple intervals. Moreover, we found that ripple magnitude in the right CA1 tended to be smaller in IP3R2‐KO. Because IP3R2‐KO mice have been reported to have depression phenotypes, our results suggest that ripple events and the mood of animals may be broadly correlated.

  August 27, 2017   doi: 10.1113/JP274573   open full text
• Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.
  Ning Gao, Ming‐Ho Tsai, Audrey N. Chang, Weiqi He, Cai‐Ping Chen, Minsheng Zhu, Kristine E. Kamm, James T. Stull.
  The Journal of Physiology. August 24, 2017
  Key points Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+/calmodulin‐dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve‐mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock‐in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Abstract Ca2+/calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation‐dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI‐17 (PKC‐potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI‐17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal‐force responses were similar in trachea from MYPT1SM+/+, MYPT1SM‐/− and the knock‐in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

  August 24, 2017   doi: 10.1113/JP274715   open full text
• Differential effects of late gestation maternal overnutrition on the regulation of surfactant maturation in fetal and postnatal life.
  Mitchell C. Lock, Erin V. McGillick, Sandra Orgeig, I. Caroline McMillen, Beverly S. Mühlhäusler, Song Zhang, Janna L. Morrison.
  The Journal of Physiology. August 24, 2017
  Key points Offspring of overweight and obese women are at greater risk for respiratory complications at birth. We determined the effect of late gestation maternal overnutrition (LGON) in sheep on surfactant maturation, glucose transport and fatty acid metabolism in the lung in fetal and postnatal life. There were significant decreases in surfactant components and numerical density of surfactant producing cells in the alveolar epithelium due to LGON in the fetal lung. However, there were no differences in the levels of these surfactant components between control and LGON lambs at 30 days of age. The reduced capacity for surfactant production in fetuses as a result of LGON may affect the transition to air breathing at birth. There was altered glucose transport and fatty acid metabolism in the lung as a result of LGON in postnatal life. However, there is a normalisation of surfactant components that suggests accelerated maturation in the lungs after birth. Abstract With the increasing incidence of obesity worldwide, the proportion of women entering pregnancy overweight or obese has increased dramatically. The fetus of an overnourished mother experiences numerous metabolic changes that may modulate lung development and hence successful transition to air breathing at birth. We used a sheep model of maternal late gestation overnutrition (LGON; from 115 days’ gestation, term 147 ± 3 days) to determine the effect of exposure to an increased plane of nutrition in late gestation on lung development in the fetus (at 141 days’ gestation) and the lamb (30 days after birth). We found a decrease in the numerical density of surfactant protein positive cells, as well as a reduction in mRNA expression of surfactant proteins (SFTP‐A, ‐B and ‐C), a rate limiting enzyme in surfactant phospholipid synthesis (phosphate cytidylyltransferase 1, choline, α; PCYT1A), and glucose transporters (SLC2A1 and SLC2A4) in the fetal lung. In lambs at 30 days after birth, there were no differences between Control and LGON groups in the surfactant components that were downregulated in the LGON fetuses. However, mRNA expression of SFTP‐A, PCYT1A, peroxisome proliferator activated receptor‐γ, fatty acid synthase and fatty acid transport protein were increased in LGON lambs compared to controls. These results indicate a reduced capacity for surfactant production in late gestation. While these deficits are normalised by 30 days after birth, the lungs of LGON lambs exhibited altered glucose transport and fatty acid metabolism, which is consistent with an enhanced capacity for surfactant synthesis and restoration of surfactant maturity in these animals.

  August 24, 2017   doi: 10.1113/JP274528   open full text
• Experimental and modelling evidence of shortening heat in cardiac muscle.
  Kenneth Tran, June‐Chiew Han, Edmund John Crampin, Andrew James Taberner, Denis Scott Loiselle.
  The Journal of Physiology. August 22, 2017
  Key points Heat associated with muscle shortening has been repeatedly demonstrated in skeletal muscle, but its existence in cardiac muscle remains contentious after five decades of study. By iterating between experiments and computational modelling, we show compelling evidence for the existence of shortening heat in cardiac muscle and reveal, mechanistically, the source of this excess heat. Our results clarify a long‐standing uncertainty in the field of cardiac muscle energetics. We provide a revised partitioning of cardiac muscle energy expenditure to include this newly revealed thermal component. Abstract When a muscle shortens against an afterload, the heat that it liberates is greater than that produced by the same muscle contracting isometrically at the same level of force. This excess heat is defined as ‘shortening heat’, and has been repeatedly demonstrated in skeletal muscle but not in cardiac muscle. Given the micro‐structural similarities between these two muscle types, and since we imagine that shortening heat is the thermal accompaniment of cross‐bridge cycling, we have re‐examined this issue. Using our flow‐through microcalorimeter, we measured force and heat generated by isolated rat trabeculae undergoing isometric contractions at different muscle lengths and work‐loop (shortening) contractions at different afterloads. We simulated these experimental protocols using a thermodynamically constrained model of cross‐bridge cycling and probed the mechanisms underpinning shortening heat. Predictions generated by the model were subsequently validated by a further set of experiments. Both our experimental and modelling results show convincing evidence for the existence of shortening heat in cardiac muscle. Its magnitude is inversely related to the afterload or, equivalently, directly related to the extent of shortening. Computational simulations reveal that the heat of shortening arises from the cycling of cross‐bridges, and that the rate of ATP hydrolysis is more sensitive to change of muscle length than to change of afterload. Our results clarify a long‐standing uncertainty in the field of cardiac muscle energetics.

  August 22, 2017   doi: 10.1113/JP274680   open full text
• Differential calcium sensitivity in NaV1.5 mixed syndrome mutants.
  Mena Abdelsayed, Alban‐Elouen Baruteau, Karen Gibbs, Shubhayan Sanatani, Andrew D. Krahn, Vincent Probst, Peter C. Ruben.
  The Journal of Physiology. August 20, 2017
  Key points SCN5a mutations may express gain‐of‐function (Long QT Syndrome‐3), loss‐of‐function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium‐sensitive regions. Elevated cytosolic calcium attenuates gain‐of‐function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain‐of‐function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium‐to‐calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. Abstract Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage‐gated sodium channel, NaV1.5. Mutants in NaV1.5 result in Brugada Syndrome (BrS1), Long‐QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome NaV1.5 mutants. We used whole‐cell patch clamp to assess the biophysical properties of NaV1.5 wild‐type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the NaV1.5 α subunit (WT or mutants), β1 subunit and enhanced green fluorescent protein. Voltage‐dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500 nm. In silico, action potential (AP) model simulations were performed using a modified O'Hara Rudy model. Elevated cytosolic calcium attenuates the late sodium current in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Elevated cytosolic calcium restores steady‐state slow inactivation (SSSI) to the WT‐form in Q1909R, but depolarized SSSI in E1784K. Our AP simulations showed a frequency‐dependent reduction of AP duration in ∆KPQ, 1795insD and Q1909R carriers. In E1784K, AP duration is relatively prolonged at both low and high heart rates, resulting in a sodium overload. Cellular perturbations during exercise may affect BrS1/LQT3 patients differently depending on their individual genetic signature. Thus, exercise may be therapeutic or may be an arrhythmogenic trigger in some SCN5a patients.

  August 20, 2017   doi: 10.1113/JP274536   open full text
• Paired motor cortex and cervical epidural electrical stimulation timed to converge in the spinal cord promotes lasting increases in motor responses.
  Asht M. Mishra, Ajay Pal, Disha Gupta, Jason B. Carmel.
  The Journal of Physiology. August 20, 2017
  Key points Pairing motor cortex stimulation and spinal cord epidural stimulation produced large augmentation in motor cortex evoked potentials if they were timed to converge in the spinal cord. The modulation of cortical evoked potentials by spinal cord stimulation was largest when the spinal electrodes were placed over the dorsal root entry zone. Repeated pairing of motor cortex and spinal cord stimulation caused lasting increases in evoked potentials from both sites, but only if the time between the stimuli was optimal. Both immediate and lasting effects of paired stimulation are likely mediated by convergence of descending motor circuits and large diameter afferents onto common interneurons in the cervical spinal cord. Abstract Convergent activity in neural circuits can generate changes at their intersection. The rules of paired electrical stimulation are best understood for protocols that stimulate input circuits and their targets. We took a different approach by targeting the interaction of descending motor pathways and large diameter afferents in the spinal cord. We hypothesized that pairing stimulation of motor cortex and cervical spinal cord would strengthen motor responses through their convergence. We placed epidural electrodes over motor cortex and the dorsal cervical spinal cord in rats; motor evoked potentials (MEPs) were measured from biceps. MEPs evoked from motor cortex were robustly augmented with spinal epidural stimulation delivered at an intensity below the threshold for provoking an MEP. Augmentation was critically dependent on the timing and position of spinal stimulation. When the spinal stimulation was timed to coincide with the descending volley from motor cortex stimulation, MEPs were more than doubled. We then tested the effect of repeated pairing of motor cortex and spinal stimulation. Repetitive pairing caused strong augmentation of cortical MEPs and spinal excitability that lasted up to an hour after just 5 min of pairing. Additional physiology experiments support the hypothesis that paired stimulation is mediated by convergence of descending motor circuits and large diameter afferents in the spinal cord. The large effect size of this protocol and the conservation of the circuits being manipulated between rats and humans makes it worth pursuing for recovery of sensorimotor function after injury to the central nervous system.

  August 20, 2017   doi: 10.1113/JP274663   open full text
• Cortical contributions to sensory gating in the ipsilateral somatosensory cortex during voluntary activity.
  Yuming Lei, Monica A. Perez.
  The Journal of Physiology. August 18, 2017
  Key points It has long been known that the somatosensory cortex gates sensory inputs from the contralateral side of the body. Here, we examined the contribution of the ipsilateral somatosensory cortex (iS1) to sensory gating during index finger voluntary activity. The amplitude of the P25/N33, but not other somatosensory evoked potential (SSEP) components, was reduced during voluntary activity compared with rest. Interhemispheric inhibition between S1s and intracortical inhibition in the S1 modulated the amplitude of the P25/N33. Note that changes in interhemispheric inhibition between S1s correlated with changes in cortical circuits in the ipsilateral motor cortex. Our findings suggest that cortical circuits, probably from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans. Abstract An important principle in the organization of the somatosensory cortex is that it processes afferent information from the contralateral side of the body. The role of the ipsilateral somatosensory cortex (iS1) in sensory gating in humans remains largely unknown. Using electroencephalographic (EEG) recordings over the iS1 and electrical stimulation of the ulnar nerve at the wrist, we examined somatosensory evoked potentials (SSEPs; P14/N20, N20/P25 and P25/N33 components) and paired‐pulse SSEPs between S1s (interhemispheric inhibition) and within (intracortical inhibition) the iS1 at rest and during tonic index finger voluntary activity. We found that the amplitude of the P25/N33, but not other SSEP components, was reduced during voluntary activity compared with rest. Interhemispheric inhibition increased the amplitude of the P25/N33 and intracortical inhibition reduced the amplitude of the P25/N33, suggesting a cortical origin for this effect. The P25/N33 receives inputs from the motor cortex, so we also examined the contribution of distinct sets of cortical interneurons by testing the effect of ulnar nerve stimulation on motor‐evoked potentials (MEPs) elicited by transcranial magnetic stimulation over the ipsilateral motor cortex with the coil in the posterior–anterior (PA) and anterior–posterior (AP) orientation. Afferent input attenuated PA, but not AP, MEPs during voluntary activity compared with rest. Notably, changes in interhemispheric inhibition correlated with changes in PA MEPs. Our novel findings suggest that interhemispheric projections between S1s and intracortical circuits, probably from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans.

  August 18, 2017   doi: 10.1113/JP274504   open full text
• The role of T‐type calcium channels in the subiculum: to burst or not to burst?
  Srdjan M. Joksimovic, Pierce Eggan, Yukitoshi Izumi, Sonja Lj. Joksimovic, Vesna Tesic, Robert M. Dietz, James E. Orfila, Michael R. DiGruccio, Paco S. Herson, Vesna Jevtovic‐Todorovic, Charles F. Zorumski, Slobodan M. Todorovic.
  The Journal of Physiology. August 18, 2017
  Key points Pharmacological, molecular and genetic data indicate a prominent role of low‐voltage‐activated T‐type calcium channels (T‐channels) in the firing activity of both pyramidal and inhibitory interneurons in the subiculum. Pharmacological inhibition of T‐channels switched burst firing with lower depolarizing stimuli to regular spiking, and fully abolished hyperpolarization‐induced burst firing. Our molecular studies showed that CaV3.1 is the most abundantly expressed isoform of T‐channels in the rat subiculum. Consistent with this finding, both regular‐spiking and burst firing patterns were profoundly depressed in the mouse with global deletion of CaV3.1 isoform of T‐channels. Selective inhibition of T‐channels and global deletion of CaV3.1 channels completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway. Abstract Several studies suggest that voltage‐gated calcium currents are involved in generating high frequency burst firing in the subiculum, but the exact nature of these currents remains unknown. Here, we used selective pharmacology, molecular and genetic approaches to implicate Cav3.1‐containing T‐channels in subicular burst firing, in contrast to several previous reports discounting T‐channels as major contributors to subicular neuron physiology. Furthermore, pharmacological antagonism of T‐channels, as well as global deletion of CaV3.1 isoform, completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway. Our results indicate that excitability and synaptic plasticity of subicular neurons relies heavily on T‐channels. Hence, T‐channels may be a promising new drug target for different cognitive deficits.

  August 18, 2017   doi: 10.1113/JP274565   open full text
• Decline in cellular function of aged mouse c‐kit+ cardiac progenitor cells.
  Alessandra Castaldi, Ramsinh Mansinh Dodia, Amabel M. Orogo, Cristina M. Zambrano, Rita H. Najor, Åsa B. Gustafsson, Joan Heller Brown, Nicole H. Purcell.
  The Journal of Physiology. August 18, 2017
  Key points While autologous stem cell‐based therapies are currently being tested on elderly patients, there are limited data on the function of aged stem cells and in particular c‐kit+ cardiac progenitor cells (CPCs). We isolated c‐kit+ cells from young (3 months) and aged (24 months) C57BL/6 mice to compare their biological properties. Aged CPCs have increased senescence, decreased stemness and reduced capacity to proliferate or to differentiate following dexamethasone (Dex) treatment in vitro, as evidenced by lack of cardiac lineage gene upregulation. Aged CPCs fail to activate mitochondrial biogenesis and increase proteins involved in mitochondrial oxidative phosphorylation in response to Dex. Aged CPCs fail to upregulate paracrine factors that are potentially important for proliferation, survival and angiogenesis in response to Dex. The results highlight marked differences between young and aged CPCs, which may impact future design of autologous stem cell‐based therapies. Abstract Therapeutic use of c‐kit+ cardiac progenitor cells (CPCs) is being evaluated for regenerative therapy in older patients with ischaemic heart failure. Our understanding of the biology of these CPCs has, however, largely come from studies of young cells and animal models. In the present study we examined characteristics of CPCs isolated from young (3 months) and aged (24 months) mice that could underlie the diverse outcomes reported for CPC‐based therapeutics. We observed morphological differences and altered senescence indicated by increased senescence‐associated markers β‐galactosidase and p16 mRNA in aged CPCs. The aged CPCs also proliferated more slowly than their young counterparts and expressed lower levels of the stemness marker LIN28. We subsequently treated the cells with dexamethasone (Dex), routinely used to induce commitment in CPCs, for 7 days and analysed expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs also failed to increase mitochondrial biogenesis and expression of the mitochondrial proteins Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. Dex‐treated aged CPCs also had impaired ability to upregulate expression of paracrine factor genes and the conditioned media from these cells had reduced ability to induce angiogenesis in vitro. These findings could impact the design of future CPC‐based therapeutic approaches for the treatment of older patients suffering from cardiac injury.

  August 18, 2017   doi: 10.1113/JP274775   open full text
• Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle.
  Edward S. A. Humphries, Tomoko Kamishima, John M. Quayle, Caroline Dart.
  The Journal of Physiology. August 14, 2017
  Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain. In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII. The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown. In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII. To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Abstract Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.

  August 14, 2017   doi: 10.1113/JP274754   open full text
• Post‐translational palmitoylation controls the voltage gating and lipid raft association of the CALHM1 channel.
  Akiyuki Taruno, Hongxin Sun, Koichi Nakajo, Tatsuro Murakami, Yasuyoshi Ohsaki, Mizuho A. Kido, Fumihito Ono, Yoshinori Marunaka.
  The Journal of Physiology. August 14, 2017
  Key points Calcium homeostasis modulator 1 (CALHM1), a new voltage‐gated ATP‐ and Ca2+‐permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post‐translationally regulated through the process of protein S‐palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine‐tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. Abstract Emerging roles of CALHM1, a recently discovered voltage‐gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post‐translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S‐palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance–voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent‐resistant membranes. Our results comprehensively uncover a post‐translational regulation of the voltage‐dependent gating of CALHM1 by palmitoylation.

  August 14, 2017   doi: 10.1113/JP274164   open full text
• Depletion of Pax7+ satellite cells does not affect diaphragm adaptations to running in young or aged mice.
  Kevin A. Murach, Amy L. Confides, Angel Ho, Janna R. Jackson, Lina S. Ghazala, Charlotte A. Peterson, Esther E. Dupont‐Versteegden.
  The Journal of Physiology. August 14, 2017
  Key points Satellite cell depletion does not affect diaphragm adaptations to voluntary wheel running in young or aged mice. Satellite cell depletion early in life (4 months of age) has minimal effect on diaphragm phenotype by old age (24 months). Prolonged satellite cell depletion in the diaphragm does not result in excessive extracellular matrix accumulation, in contrast to what has been reported in hind limb muscles. Up‐regulation of Pax3 mRNA+ cells after satellite cell depletion in young and aged mice suggests that Pax3+ cells may compensate for a loss of Pax7+ satellite cells in the diaphragm. Future investigations should focus on the role of Pax3+ cells in the diaphragm during adaptation to exercise and ageing. Abstract Satellite cell contribution to unstressed diaphragm is higher compared to hind limb muscles, which is probably attributable to constant activation of this muscle to drive ventilation. Whether satellite cell depletion negatively impacts diaphragm quantitative and qualitative characteristics under stressed conditions in young and aged mice is unknown. We therefore challenged the diaphragm with prolonged running activity in the presence and absence of Pax7+ satellite cells in young and aged mice using an inducible Pax7CreER‐R26RDTA model. Mice were vehicle (Veh, satellite cell‐replete) or tamoxifen (Tam, satellite cell‐depleted) treated at 4 months of age and were then allowed to run voluntarily at 6 months (young) and 22 months (aged). Age‐matched, cage‐dwelling, Veh‐ and Tam‐treated mice without wheel access served as activity controls. Diaphragm muscles were analysed from young (8 months) and aged (24 months) mice. Satellite cell depletion did not alter diaphragm mean fibre cross‐sectional area, fibre type distribution or extracellular matrix content in young or aged mice, regardless of running activity. Resting in vivo diaphragm function was also unaffected by satellite cell depletion. Myonuclear density was maintained in young satellite cell‐depleted mice regardless of running, although it was modestly reduced in aged sedentary (–7%) and running (–19%) mice without satellite cells (P < 0.05). Using fluorescence in situ hybridization, we detected higher Pax3 mRNA+ cell density in both young and aged satellite cell‐depleted diaphragm muscle (P < 0.05), which may compensate for the loss of Pax7+ satellite cells.

  August 14, 2017   doi: 10.1113/JP274611   open full text
• ATP and astrocytes play a prominent role in the control of the respiratory pattern generator in the lamprey.
  Elenia Cinelli, Ludovica Iovino, Donatella Mutolo.
  The Journal of Physiology. August 08, 2017
  Key points The paratrigeminal respiratory group (pTRG) is responsible for the respiratory pattern generation in the lamprey. The role of ATP and astrocytes, known to control respiratory activity in mammals, was investigated in the lamprey respiratory network. ATP microinjected into the pTRG induces a biphasic response consisting of marked increases in respiratory frequency mediated by P2X receptors followed by a decrease in the respiratory motor output due to the ATP metabolite adenosine. We provide evidence that astrocytes are involved in the genesis of the normal respiratory pattern, ATP‐induced responses and acidification‐induced increases of the respiratory activity. The function of astrocytes in rhythmic networks appears to be phylogenetically conserved. Abstract The role of ATP and astrocytes in respiratory rhythm modulation has been recently investigated in neonatal rodents. However, no information on the role of ATP and astrocytes within the respiratory network of the lamprey is available, particularly within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator. To address these issues, the present study was carried out on isolated brainstems of the adult lamprey. Bath application of ATP caused marked increases in respiratory frequency followed by decreases in the respiratory motor output, mediated by the ATP metabolite adenosine at the level of the pTRG. Bath applications and microinjections of agonists and antagonists of purinergic receptors showed that ATP increased respiratory activity through an action on pTRG P2X receptors. To disclose the respiratory role of astrocytes, we used bath application of the gliotoxin aminoadipic acid, which dramatically depressed the respiratory motor output that, however, promptly recovered following glutamine application. Furthermore, the excitatory responses to ATP‐γ‐S (a non‐hydrolysable ATP analogue), but not to substance P, microinjected into the pTRG, were abolished. Finally, we also demonstrated that acidification‐induced increases in respiratory activity were ATP‐independent, but mediated by the astrocytes’ glutamate–glutamine cycle. The results show for the first time that ATP and especially astrocytes strongly contribute to the modulation of the lamprey respiratory pattern. Their role in the modulation or maintenance of rhythmic neuronal activities appears to be phylogenetically conserved.

  August 08, 2017   doi: 10.1113/JP274749   open full text
• Distinct temporal filters in mitral cells and external tufted cells of the olfactory bulb.
  Christopher E. Vaaga, Gary L. Westbrook.
  The Journal of Physiology. August 08, 2017
  Short‐term synaptic plasticity is a critical regulator of neural circuits, and largely determines how information is temporally processed. In the olfactory bulb, afferent olfactory receptor neurons respond to increasing concentrations of odorants with barrages of action potentials, and their terminals have an extraordinarily high release probability (Sicard, 1986; Murphy et al. 2004). These features suggest that during naturalistic stimuli, afferent input to the olfactory bulb is subject to strong synaptic depression, presumably truncating the postsynaptic response to afferent stimuli. To examine this issue, we used single glomerular stimulation in mouse olfactory bulb slices to measure the synaptic dynamics of afferent‐evoked input at physiological stimulus frequencies. In cell‐attached recordings, mitral cells responded to high frequency stimulation with sustained responses, whereas external tufted cells responded transiently. Consistent with previous reports (Murphy et al. 2004), olfactory nerve terminals onto both cell types had a high release probability (0.7), from a single pool of slowly recycling vesicles, indicating that the distinct responses of mitral and external tufted cells to high frequency stimulation did not originate presyaptically. Rather, distinct temporal response profiles in mitral cells and external tufted cells could be attributed to slow dendrodendritic responses in mitral cells, as blocking this slow current in mitral cells converted mitral cell responses to a transient response profile, typical of external tufted cells. Our results suggest that despite strong axodendritic synaptic depression, the balance of axodendritic and dendrodendritic circuitry in external tufted cells and mitral cells, respectively, tunes the postsynaptic responses to high frequency, naturalistic stimulation. This article is protected by copyright. All rights reserved

  August 08, 2017   doi: 10.1113/JP274608   open full text
• Gene expression analyses reveal metabolic specifications in acute O2‐sensing chemoreceptor cells.
  Lin Gao, Victoria Bonilla‐Henao, Paula García‐Flores, Ignacio Arias‐Mayenco, Patricia Ortega‐Sáenz, José López‐Barneo.
  The Journal of Physiology. August 08, 2017
  Key points Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2‐sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2‐sensitive, with superior cervical ganglion neurons, which are practically O2‐insensitive. In O2‐sensitive cells, we found a characteristic prolyl hydroxylase 3 down‐regulation and hypoxia inducible factor 2α up‐regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a ‘signature metabolic profile’. Abstract Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2‐sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2‐sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells, which are O2‐sensitive (glomus cells > chromaffin cells), and superior cervical ganglion neurons, which are practically O2‐insensitive. In the O2‐sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up‐regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a ‘signature metabolic profile’ in chemoreceptor cells.

  August 08, 2017   doi: 10.1113/JP274684   open full text
• NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.
  Brian J. Kinsman, Kirsteen N. Browning, Sean D. Stocker.
  The Journal of Physiology. August 02, 2017
  Key points Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi‐osmotic mannitol/sorbitol did not alter any variable. In vitro whole‐cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi‐osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi‐osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl‐ versus osmo‐sensing. Abstract Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi‐osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague‐Dawley rats. First, intracerebroventricular (icv) infusion (5 μl/10 min) of 1.0 m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi‐osmotic sorbitol (2.0 osmol l−1). Second, OVLT microinjection (20 nl) of 1.0 m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi‐osmotic sorbitol did not alter any variable. Third, in vitro whole‐cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5 mm) and mannitol (+15 mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single‐unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration‐dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi‐osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5 m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi‐osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.

  August 02, 2017   doi: 10.1113/JP274537   open full text
• A reduction in SK channels contributes to increased activity of hypothalamic magnocellular neurons during heart failure.
  Hildebrando C. Ferreira‐Neto, Vinicia C. Biancardi, Javier E. Stern.
  The Journal of Physiology. August 02, 2017
  Key points Small conductance Ca2+‐activated K+ (SK) channels play an important role in regulating the excitability of magnocellular neurosecretory cells (MNCs). Although an increased SK channel function contributes to adaptive physiological responses, it remains unknown whether changes in SK channel function/expression contribute to exacerbated MNC activity under disease conditions. We show that the input–output function of MNCs in heart failure (HF) rats is enhanced. Moreover, the SK channel blocker apamin enhanced the input–output function in sham, although not in HF rats. We found that both the after‐hyperpolarizing potential magnitude and the underlying apamin‐sensitive IAHP are blunted in MNCs from HF rats. The magnitude of spike‐induced increases in intracellular Ca2+ levels was not affected in MNCs of HF rats. We found a diminished expression of SK2/SK3 channel subunit mRNA expression in the supraoptic nucleus of HF rats. Our studies suggest that a reduction in SK channel expression, but not changes in Ca2+‐mediated activation of SK channels, contributes to exacerbated MNC activity in HF rats. Abstract Small conductance Ca2+‐activated K+ channels (SK) play an important role in regulating the activity of magnocellular neurosecretory cells (MNCs) and hormone release from the posterior pituitary. Moreover, enhanced SK activity contributes to the adaptive responses of MNCs to physiological challenge, such as lactation. Nevertheless, whether changes in SK function/expression contribute to exacerbated MNC activity during diseases such as heart failure (HF) remains unknown. In the present study, we used a combination of patch clamp electrophysiology, confocal Ca2+ imaging and molecular biology in a rat model of ischaemic HF. We found that the input–output function of MNCs was enhanced in HF compared to sham rats. Moreover, although the SK blocker apamin (200 nm) strengthened the input–output function in sham rats, it failed to have an effect in HF rats. The magnitude of the after‐hyperpolarizing potential (AHP) following a train of spikes and the underlying apamin‐sensitive IAHP were blunted in MNCs from HF rats. However, spike‐induced increases in intracellular Ca2+ were not affected in the MNCs of HF rats. Real‐time PCR measurements of SK channel subunits mRNA in supraoptic nucleus punches revealed a diminished expression of SK2/SK3 subunits in HF compared to sham rats. Together, our studies demonstrate that MNCs from HF rats exhibit increased membrane excitability and an enhanced input–output function, and also that a reduction in SK channel‐mediated, apamin‐sensitive AHP is a critical contributing mechanism. Moreover, our results suggest that the reduced AHP is related to a down‐regulation of SK2/SK3 channel subunit expression but not the result of a blunted activity‐dependent intracellular Ca2+ increase following a burst of action potentials.

  August 02, 2017   doi: 10.1113/JP274730   open full text
• Release of ATP by pre‐Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+‐dependent P2Y1 receptor mechanism.
  Vishaal Rajani, Yong Zhang, Venkatesh Jalubula, Vladimir Rancic, Shahriar SheikhBahaei, Jennifer D. Zwicker, Silvia Pagliardini, Clayton T. Dickson, Klaus Ballanyi, Sergey Kasparov, Alexander V. Gourine, Gregory D. Funk.
  The Journal of Physiology. July 27, 2017
  Key points The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre‐Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor‐mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. Abstract The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady‐state phase II) that can be life‐threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre‐Bötzinger Complex (preBötC) inspiratory rhythm‐generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia‐induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor‐mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+]i) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

  July 27, 2017   doi: 10.1113/JP274727   open full text
• In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury.
  Andreas Patsalos, Attila Pap, Tamas Varga, Gyorgy Trencsenyi, Gerardo Alvarado Contreras, Ildiko Garai, Zoltan Papp, Balazs Dezso, Eva Pintye, Laszlo Nagy.
  The Journal of Physiology. July 17, 2017
  Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for necrotic debris clearance and for coordination of tissue regeneration. This highly dynamic process is characterized by an in‐situ transition of infiltrating monocytes from an inflammatory (Ly6Chigh) to a repair (Ly6Clow) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross‐talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging we show that preserved muscle integrity and cell composition prior to the injury is necessary for repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross‐talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions are shaping the macrophages in‐situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics. This article is protected by copyright. All rights reserved

  July 17, 2017   doi: 10.1113/JP274361   open full text
• Effect of dietary salt intake on Epithelial Na+ Channels (ENaC) in vasopressin magnocellular neurosecretory neurons in the rat supraoptic nucleus.
  Kaustubh Sharma, Masudul Haque, Richard Guidry, Yoichi Ueta, Ryoichi Teruyama.
  The Journal of Physiology. July 17, 2017
  All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) were located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na+ leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β‐ and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch‐clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high‐salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high‐salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaC which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs. This article is protected by copyright. All rights reserved

  July 17, 2017   doi: 10.1113/JP274856   open full text
• Acetylcholine‐dependent upregulation of TASK‐1 channels in thalamic interneurons by a smooth muscle‐like signalling pathway.
  Michael Leist, Susanne Rinné, Maia Datunashvili, Ania Aissaoui, Hans‐Christian Pape, Niels Decher, Sven G. Meuth, Thomas Budde.
  The Journal of Physiology. July 17, 2017
  The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state‐dependent transmission of visual information. Non‐retinal inputs from the ascending arousal system and inhibition provided by γ‐aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay (TC) neurons by inhibiting two‐pore domain potassium (K2P) channels. Conversely, ACh also hyperpolarizes INs via an as‐yet‐unknown mechanism. By using whole cell patch‐clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors (M2AChRs) induces IN hyperpolarization by recruiting the G beta‐gamma complex (Gβγ), class‐1A phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K), and cellular and sarcoma (c‐Src) tyrosine kinase (TK), leading to activation of two‐pore domain weakly inwardly rectifying K+ channel (TWIK)‐related acid‐sensitive K+ (TASK)‐1 channels. The latter was confirmed by the use of TASK‐1 deficient mice. Furthermore inhibition of phospholipase Cβ (PLCβ) as well as an increase in the intracellular level of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c‐Src TK in regulating IN function in the brain and identified a novel mechanism by which TASK‐1 channels are activated in neurons. This article is protected by copyright. All rights reserved

  July 17, 2017   doi: 10.1113/JP274527   open full text
• N‐glycan content modulates kainate receptor functional properties.
  Claire G. Vernon, Bryan A. Copits, Jacob R. Stolz, Yomayra F. Guzmán, Geoffrey T. Swanson.
  The Journal of Physiology. July 17, 2017
  Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between 4 and 12 consensus sites for N‐glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post‐translational modification. N‐glycosylation is required for proper folding of iGluRs in mammalian cells, but the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged, and mediate many protein‐protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. We show here that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide HNK‐1. Glycosylation patterns likely differ between cell types, across development, or with pathologies, and thus our findings reveal a potential mechanism for context‐specific fine‐tuning of KAR function through diversity in glycan structure. This article is protected by copyright. All rights reserved

  July 17, 2017   doi: 10.1113/JP274790   open full text
• Ivermectin activates GIRK channels in a PIP2‐dependent, Gβγ‐independent manner and an amino acid residue at the slide helix governs the activation.
  I‐Shan Chen, Michihiro Tateyama, Yuko Fukata, Motonari Uesugi, Yoshihiro Kubo.
  The Journal of Physiology. July 17, 2017
  Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate‐gated Cl− channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand‐gated channels, such as Cys‐loop receptors and P2X receptors. In this study, we found that the G‐protein‐gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. By electrophysiological recordings in Xenopus oocytes, we observed that IVM activates GIRK channel in a phosphatidylinositol‐4,5‐biphosphate (PIP2)‐dependent manner, and that the IVM‐mediated GIRK activation is independent of Gβγ. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. By chimeric and mutagenesis analyses, we identified a unique amino acid residue of GIRK2 among GIRK family, Ile82, located in the slide helix between the TM1 and the N‐terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM‐CTD interface in GIRK channel, rather than the TMs, governs IVM‐mediated activation. These findings provide us with novel insights on the action mode of IVM in ion channels, and information toward identification of new pharmacophores which activate GIRK channel. This article is protected by copyright. All rights reserved

  July 17, 2017   doi: 10.1113/JP274871   open full text
• Muscle carnitine availability plays a central role in regulating fuel metabolism in the rodent.
  Craig Porter, Dumitru Constantin‐Teodosiu, Despina Constantin, Brendan Leighton, Simon M. Poucher, Paul L. Greenhaff.
  The Journal of Physiology. July 16, 2017
  Key points Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium‐mediated muscle carnitine depletion on whole‐body fuel selection, and muscle fuel metabolism and its molecular regulation is under‐investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole‐body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole‐body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low‐density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. Abstract The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3‐(2,2,2‐trimethylhydrazinium)‐propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole‐body fuel selection, muscle fuel metabolism and its molecular regulation is under‐investigated. Male lean Zucker rats received water (control, n = 8) or meldonium‐supplemented water (meldonium, n = 8) for 10 days [1.6 g kg−1 body mass (BM) day−1 days 1–2, 0.8 g kg−1 BM day−1 thereafter]. From days 7–10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole‐body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium‐supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo.

  July 16, 2017   doi: 10.1113/JP274415   open full text
• Pronounced limb and fibre type differences in subcellular lipid droplet content and distribution in elite skiers before and after exhaustive exercise.
  Han‐Chow E. Koh, Joachim Nielsen, Bengt Saltin, Hans‐Christer Holmberg, Niels Ørtenblad.
  The Journal of Physiology. July 16, 2017
  Key points Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle – one beneath the sarcolemma and the other between myofibrils. At rest, well‐trained leg muscles of cross‐country skiers contain 4‐ to 6‐fold more lipid droplets than equally well‐trained arm muscles, with a 3‐fold higher content in type 1 than in type 2 fibres. During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres. These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres. Abstract Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85–97% of the total volume fraction, while the subsarcolemmal pool made up 3–15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4‐ to 6‐fold higher in leg than in arm muscles (P < 0.001). Furthermore, the volume fraction of intermyofibrillar lipid droplets was 3‐fold higher in type 1 than in type 2 fibres (P < 0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise, intermyofibrillar lipid droplet volume fraction was 53% lower (P = 0.0082) in both fibre types in arm, but not leg muscles. This reduction was positively associated with the corresponding volume fraction prior to exercise (R2 = 0.84, P < 0.0001). No exercise‐induced change in the subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular distribution of lipid droplets in the type 1 and 2 fibres of well‐trained arm and leg muscles, as well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise. Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.

  July 16, 2017   doi: 10.1113/JP274462   open full text
• Calcium signalling in medial intercalated cell dendrites and spines.
  Cornelia Strobel, Robert K. P. Sullivan, Peter Stratton, Pankaj Sah.
  The Journal of Physiology. July 16, 2017
  Key points Dendritic and spine calcium imaging in combination with electrophysiology in acute slices revealed that in medial intercalated cells of the amygdala: Action potentials back‐propagate into the dendritic tree, but due to the presence of voltage‐dependent potassium channels, probably Kv4.2 channels, attenuate over distance. A mixed population of AMPA receptors with rectifying and linear I–V relations are present at individual spines of a single neuron. Decay kinetics and pharmacology suggest tri‐heteromeric NMDA receptors at basolateral–intercalated cell synapses. NMDA receptors are the main contributors to spine calcium entry in response to synaptic stimulation. Calcium signals in response to low‐ and high‐frequency stimulation, and in combination with spontaneous action potentials are locally restricted to the vicinity of active spines. Together, these data show that calcium signalling in these GABAergic neurons is tightly controlled and acts as a local signal. Abstract The amygdala plays a central role in fear conditioning and extinction. The medial intercalated (mITC) neurons are GABAergic cell clusters interspaced between the basolateral (BLA) and central amygdala (CeA). These neurons are thought to play a key role in fear and extinction, controlling the output of the CeA by feed‐forward inhibition. BLA to mITC cell inputs are thought to undergo synaptic plasticity, a mechanism underlying learning, which is mediated by NMDA receptor‐dependent mechanisms that require changes in cytosolic calcium. Here, we studied the electrical and calcium signalling properties of mITC neurons in GAD67‐eGFP mice using whole‐cell patch clamp recordings and two‐photon calcium imaging. We show that action potentials back‐propagate (bAP) into dendrites, and evoke calcium transients in both the shaft and the dendritic spine. However, bAP‐mediated calcium rises in the dendrites attenuate with distance due to shunting by voltage‐gated potassium channels. Glutamatergic inputs make dual component synapses on spines. At these synapses, postsynaptic AMPA receptors can have linear or rectifying I–V relationships, indicating that some synapses express GluA2‐lacking AMPA receptors. Synaptic NMDA receptors had intermediate decay kinetics, and were only partly blocked by GuN2B selective blockers, indicating these receptors are GluN1/GluN2A/GluN2B trimers. Low‐ or high‐frequency synaptic stimulation raised spine calcium, mediated by calcium influx via NMDA receptors, was locally restricted and did not invade neighbouring spines. Our results show that in mITC neurons, postsynaptic calcium is tightly controlled, and acts as a local signal.

  July 16, 2017   doi: 10.1113/JP274261   open full text
• Lack of linear correlation between dynamic and steady‐state cerebral autoregulation.
  Daan L. K. Jong, Takashi Tarumi, Jie Liu, Rong Zhang, Jurgen A. H. R. Claassen.
  The Journal of Physiology. July 14, 2017
  Key points For correct application and interpretation of cerebral autoregulation (CA) measurements in research and in clinical care, it is essential to understand differences and similarities between dynamic and steady‐state CA. The present study found no correlation between dynamic and steady‐state CA indices in healthy older adults. There was variability between individuals in all (steady‐state and dynamic) autoregulatory indices, ranging from low (almost absent) to highly efficient CA in this healthy population. These findings challenge the assumption that assessment of a single CA parameter or a single set of parameters can be generalized to overall CA functioning. Therefore, depending on specific research purposes, the choice for either steady‐state or dynamic measures or both should be weighed carefully. Abstract The present study aimed to investigate the relationship between dynamic (dCA) and steady‐state cerebral autoregulation (sCA). In 28 healthy older adults, sCA was quantified by a linear regression slope of proportionate (%) changes in cerebrovascular resistance (CVR) in response to proportionate (%) changes in mean blood pressure (BP) induced by stepwise sodium nitroprusside (SNP) and phenylephrine (PhE) infusion. Cerebral blood flow (CBF) was measured at the internal carotid artery (ICA) and vertebral artery (VA) and CBF velocity at the middle cerebral artery (MCA). With CVR = BP/CBF, Slope‐CVRICA, Slope‐CVRVA and Slope‐CVRiMCA were derived. dCA was assessed (i) in supine rest, analysed with transfer function analysis (gain and phase) and autoregulatory index (ARI) fit from spontaneous oscillations (ARIBaseline), and (ii) with transient changes in BP using a bolus injection of SNP (ARISNP) and PhE (ARIPhE). Comparison of sCA and dCA parameters (using Pearson's r for continuous and Spearman's ρ for ordinal parameters) demonstrated a lack of linear correlations between sCA and dCA measures. However, comparisons of parameters within dCA and within sCA were correlated. For sCA slope‐CVRVA with Slope‐CVRiMCA (r = 0.45, P < 0.03); for dCA ARISNP with ARIPhE (ρ = 0.50, P = 0.03), ARIBaseline (ρ = 0.57, P = 0.03) and PhaseLF (ρ = 0.48, P = 0.03); and for GainVLF with GainLF (r = 0.51, P = 0.01). By contrast to the commonly held assumption based on an earlier study, there were no linear correlations between sCA and dCA. As an additional observation, there was strong inter‐individual variability, both in dCA and sCA, in this healthy group of elderly, in a range from low to high CA efficiency.

  July 14, 2017   doi: 10.1113/JP274304   open full text
• EEA1 restores homeostatic synaptic plasticity in hippocampal neurons from Rett syndrome mice.
  Xin Xu, Lucas Pozzo‐Miller.
  The Journal of Physiology. July 12, 2017
  Key points Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Mecp2 deletion in mice results in an imbalance of excitation and inhibition in hippocampal neurons, which affects ‘Hebbian’ synaptic plasticity. We show that Mecp2‐deficient neurons also lack homeostatic synaptic plasticity, likely due to reduced levels of EEA1, a protein involved in AMPA receptor endocytosis. Expression of EEA1 restored homeostatic synaptic plasticity in Mecp2‐deficient neurons, providing novel targets of intervention in Rett syndrome. Abstract Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Deletion of Mecp2 in mice results in an imbalance of synaptic excitation and inhibition in hippocampal pyramidal neurons, which affects ‘Hebbian’ long‐term synaptic plasticity. Since the excitatory–inhibitory balance is maintained by homeostatic mechanisms, we examined the role of MeCP2 in homeostatic synaptic plasticity (HSP) at excitatory synapses. Negative feedback HSP, also known as synaptic scaling, maintains the global synaptic strength of individual neurons in response to sustained alterations in neuronal activity. Hippocampal neurons from Mecp2 knockout (KO) mice do not show the characteristic homeostatic scaling up of the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and of synaptic levels of the GluA1 subunit of AMPA‐type glutamate receptors after 48 h silencing with the Na+ channel blocker tetrodotoxin. This deficit in HSP is bidirectional because Mecp2 KO neurons also failed to scale down mEPSC amplitudes and GluA1 synaptic levels after 48 h blockade of type A GABA receptor (GABAAR)‐mediated inhibition with bicuculline. Consistent with the role of synaptic trafficking of AMPA‐type of glutamate receptors in HSP, Mecp2 KO neurons have lower levels of early endosome antigen 1 (EEA1), a protein involved in AMPA‐type glutamate receptor endocytosis. In addition, expression of EEA1 in Mecp2 KO neurons reduced mEPSC amplitudes to wild‐type levels, and restored synaptic scaling down of mEPSC amplitudes after 48 h blockade of GABAAR‐mediated inhibition with bicuculline. The identification of a molecular deficit in HSP in Mecp2 KO neurons provides potentially novel targets of intervention for improving hippocampal function in Rett syndrome individuals.

  July 12, 2017   doi: 10.1113/JP274450   open full text
• Heterotypic endosomal fusion as an initial trigger for insulin‐induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.
  Hiroyasu Hatakeyama, Makoto Kanzaki.
  The Journal of Physiology. July 10, 2017
  Key points Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot‐based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving ‘static retention’ and ‘stimulus‐dependent liberation’. Vital imaging analyses and super‐resolution microscopy‐based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4‐containing vesicles in skeletal myofibres. Prior exposure to exercise‐mimetic stimuli potentiated this insulin‐responsive endomembrane fusion event involving GLUT4‐containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Abstract Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin‐induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4‐containing vesicles with a subset of transferrin receptor‐containing endosomes. Importantly, pretreatment with exercise‐mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe‐like localization close to sarcomeric α‐actinin and that insulin induced a reduction of the stripe‐like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.

  July 10, 2017   doi: 10.1113/JP273985   open full text
• Mechanisms underlying vestibulo‐cerebellar motor learning in mice depend on movement direction.
  Kai Voges, Bin Wu, Laura Post, Martijn Schonewille, Chris I. Zeeuw.
  The Journal of Physiology. July 10, 2017
  Key points Directionality, inherent to movements, has behavioural and neuronal correlates. Direction of vestibular stimulation determines motor learning efficiency. Vestibulo‐ocular reflex gain–increase correlates with Purkinje cell simple spike potentiation. The locus of neural correlates for vestibulo‐ocular reflex adaptation is paradigm specific. Abstract Compensatory eye movements elicited by head rotation, also known as vestibulo‐ocular reflex (VOR), can be adapted with the use of visual feedback. The cerebellum is essential for this type of movement adaptation, although its neuronal correlates remain to be clarified. In the present study, we show that the direction of vestibular input determines the magnitude of eye movement adaptation induced by mismatched visual input in mice, with larger changes during contraversive head rotation. Moreover, the location of the neural correlate of this changed behaviour depends on the type of paradigm. Gain–increase paradigms induce increased simple spike (SS) activity in ipsilateral cerebellar Purkinje cells (PC), which is in line with eye movements triggered by optogenetic PC activation. By contrast, gain–decrease paradigms do not induce changes in SS activity, indicating that the murine vestibulo‐cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements.

  July 10, 2017   doi: 10.1113/JP274346   open full text
• Ecto‐5′‐nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia.
  Andrew P. Holmes, Clare J. Ray, Selina A. Pearson, Andrew M. Coney, Prem Kumar.
  The Journal of Physiology. July 09, 2017
  Key points Carotid body dysfunction is recognized as a cause of hypertension in a number of cardiorespiratory diseases states and has therefore been identified as a potential therapeutic target. Purinergic transmission is an important element of the carotid body chemotransduction pathway. We show that inhibition of ecto‐5′‐nucleotidase (CD73) in vitro reduces carotid body basal discharge and responses to hypoxia and mitochondrial inhibition. Additionally, inhibition of CD73 in vivo decreased the hypoxic ventilatory response, reduced the hypoxia‐induced heart rate elevation and exaggerated the blood pressure decrease in response to hypoxia. Our data show CD73 to be a novel regulator of carotid body sensory function and therefore suggest that this enzyme may offer a new target for reducing carotid body activity in selected cardiovascular diseases. Abstract Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies, including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The present study investigated whether ecto‐5′‐nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of CD73 by α,β‐methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8‐(p‐sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (ΔV̇E control 74 ± 6%, ΔV̇E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as indicated by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify CD73 as a novel regulator of CB sensory activity. Future investigations are warranted to clarify whether inhibition of CD73 can effectively reduce CB activity in CB‐mediated cardiovascular pathology.

  July 09, 2017   doi: 10.1113/JP274498   open full text
• Sympatholytic effect of intravascular ATP is independent of nitric oxide, prostaglandins, Na+/K+‐ATPase and KIR channels in humans.
  Christopher M. Hearon, Jennifer C. Richards, Mathew L. Racine, Gary J. Luckasen, Dennis G. Larson, Michael J. Joyner, Frank A. Dinenno.
  The Journal of Physiology. July 09, 2017
  Key points Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia. Similar to exercise, ATP‐mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR), and synthesis of nitric oxide (NO) and prostaglandins (PG). However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1‐adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR, NO, PGs and Na+/K+‐ATPase activity. Blockade of KIR channels alone or in combination with NO, PGs and Na+/K+‐ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1‐adrenergic vasoconstriction. This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans. Abstract Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α‐adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+/K+‐ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+/K+‐ATPase would not alter the ability of ATP to blunt α1‐adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n = 7; Protocol 1); NO (l‐NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+/K+‐ATPase (ouabain) and KIR channel inhibition (n = 6; Protocol 2). ATP attenuated PE‐mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE‐mediated ΔFVC: ATP: −16 ± 2; ADO: −38 ± 6; SNP: −59 ± 6%; P < 0.05 vs. ADO and SNP). Blockade of KIR channels alone or combined with NO, PGs and Na+/K+‐ATPase, attenuated ATP‐mediated vasodilatation (∼35 and ∼60% respectively; P < 0.05 vs. control). However, ATP maintained the ability to blunt PE‐mediated vasoconstriction (PE‐mediated ΔFVC: KIR blockade alone: −6 ± 5%; combined blockade:−4 ± 14%; P > 0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1‐adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+/K+‐ATPase in humans, consistent with a role for endothelium‐derived hyperpolarization in functional sympatholysis.

  July 09, 2017   doi: 10.1113/JP274532   open full text
• Transcriptomic analysis identifies a role of PI3K/Akt signalling in the responses of skeletal muscle to acute hypoxia in vivo.
  Zhuohui Gan, Frank L. Powell, Alexander C. Zambon, Kyle S. Buchholz, Zhenxing Fu, Karen Ocorr, Rolf Bodmer, Esteban A. Moya, Jennifer C. Stowe, Gabriel G. Haddad, Andrew D. McCulloch.
  The Journal of Physiology. July 08, 2017
  The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia‐tolerant tissues like skeletal muscles. We used RNA‐seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down‐regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down‐regulated genes had mRNA half‐lives longer than 2 h, suggesting a role for post‐transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K/Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K/Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K/Akt targets, 75% could be explained by a deactivation of ARE‐binding protein BRF1, a target of PI3K/Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K/Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the de‐phosphorylation of ARE‐binding protein BRF1. This article is protected by copyright. All rights reserved

  July 08, 2017   doi: 10.1113/JP274556   open full text
• Sensory feedback from the urethra evokes state‐dependent lower urinary tract reflexes in rat.
  Zachary C. Danziger, Warren M. Grill.
  The Journal of Physiology. July 07, 2017
  Key points The lower urinary tract is regulated by reflexes responsible for maintaining continence and producing efficient voiding. It is unclear how sensory information from the bladder and urethra engages differential, state‐dependent reflexes to either maintain continence or promote voiding. Using a new in vivo experimental approach, we quantified how sensory information from the bladder and urethra are integrated to switch reflex responses to urethral sensory feedback from maintaining continence to producing voiding. The results demonstrate how sensory information regulates state‐dependent reflexes in the lower urinary tract and contribute to our understanding of the pathophysiology of urinary retention and incontinence where sensory feedback may engage these reflexes inappropriately. Abstract Lower urinary tract reflexes are mediated by peripheral afferents from the bladder (primarily in the pelvic nerve) and the urethra (in the pudendal and pelvic nerves) to maintain continence or initiate micturition. If fluid enters the urethra at low bladder volumes, reflexes relax the bladder and evoke external urethral sphincter (EUS) contraction (guarding reflex) to maintain continence. Conversely, urethral flow at high bladder volumes, excites the bladder (micturition reflex) and relaxes the EUS (augmenting reflex). We conducted measurements in a urethane‐anaesthetized in vivo rat preparation to characterize systematically the reflexes evoked by fluid flow through the urethra. We used a novel preparation to manipulate sensory feedback from the bladder and urethra independently by controlling bladder volume and urethral flow. We found a distinct bladder volume threshold (74% of bladder capacity) above which flow‐evoked bladder contractions were 252% larger and evoked phasic EUS activation 2.6 times as often as responses below threshold, clearly demonstrating a discrete transition between continence (guarding) and micturition (augmenting) reflexes. Below this threshold urethral flow evoked tonic EUS activity, indicative of the guarding reflex, that was proportional to the urethral flow rate. These results demonstrate the complementary roles of sensory feedback from the bladder and urethra in regulating reflexes in the lower urinary tract that depend on the state of the bladder. Understanding the neural control of functional reflexes and how they are mediated by sensory information in the bladder and urethra will open new opportunities, especially in neuromodulation, to treat pathologies of the lower urinary tract.

  July 07, 2017   doi: 10.1113/JP274191   open full text
• Impact of ageing on postsynaptic neuronal nicotinic neurotransmission in auditory thalamus.
  Sarah Y. Sottile, Lynne Ling, Brandon C. Cox, Donald M. Caspary.
  The Journal of Physiology. July 07, 2017
  Key points Neuronal nicotinic acetylcholine receptors (nAChRs) play a fundamental role in the attentional circuitry throughout the mammalian CNS. In the present study, we report a novel finding that ageing negatively impacts nAChR efficacy in auditory thalamus, and this is probably the result of a loss of nAChR density (Bmax) and changes in the subunit composition of nAChRs. Our data support the hypothesis that age‐related maladaptive changes involving nAChRs within thalamocortical circuits partially underpin the difficulty that elderly adults experience with respect to attending to speech and other salient acoustic signals. Abstract The flow of auditory information through the medial geniculate body (MGB) is regulated, in part, by cholinergic projections from the pontomesencephalic tegmentum. The functional significance of these projections is not fully established, although they have been strongly implicated in the allocation of auditory attention. Using in vitro slice recordings, we have analysed postsynaptic function and pharmacology of neuronal nicotinic ACh receptors (nAChRs) in young adult and the aged rat MGB. We find that ACh produces significant excitatory postsynaptic actions on young MGB neurons, probably mediated by β2‐containing heteromeric nAChRs. Radioligand binding studies show a significant age‐related loss of heteromeric nAChR receptor number, which supports patch clamp data showing an age‐related loss in ACh efficacy in evoking postsynaptic responses. Use of the β2‐selective nAChR antagonist, dihydro‐β‐erythroidine, suggests that loss of cholinergic efficacy may also be the result of an age‐related subunit switch from high affinity β2‐containing nAChRs to low affinity β4‐containing nAChRs, in addition to the loss of total nAChR number. This age‐related nAChR dysfunction may partially underpin the attentional deficits that contribute to the loss of speech understanding in the elderly.

  July 07, 2017   doi: 10.1113/JP274467   open full text
• Promotion of endocytosis efficiency through an ATP‐independent mechanism at rat calyx of Held terminals.
  Hai‐Yuan Yue, Erhard Bieberich, Jianhua Xu.
  The Journal of Physiology. July 05, 2017
  Key points At rat calyx of Held terminals, ATP was required not only for slow endocytosis, but also for rapid phase of compensatory endocytosis. An ATP‐independent form of endocytosis was recruited to accelerate membrane retrieval at increased activity and temperature. ATP‐independent endocytosis primarily involved retrieval of pre‐existing membrane, which depended on Ca2+ and the activity of neutral sphingomyelinase but not clathrin‐coated pit maturation. ATP‐independent endocytosis represents a non‐canonical mechanism that can efficiently retrieve membrane at physiological conditions without competing for the limited ATP at elevated neuronal activity. Abstract Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP‐dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP‐independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at rat calyx of Held terminals by whole‐cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis but only partially inhibited excess endocytosis following intense stimulation. The ATP‐independent endocytosis occurred at calyces from postnatal days 8–15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin‐coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre‐existing at the surface, and was impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP‐independent endocytosis became more significant at 34–35°C, and recovered membrane by an amount that, on average, was close to exocytosis. The results of the present study suggest that activity and temperature recruit ATP‐independent endocytosis of pre‐existing membrane (in addition to ATP‐dependent endocytosis) to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non‐canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.

  July 05, 2017   doi: 10.1113/JP274275   open full text
• Chronic electromyograms in treadmill running SOD1 mice reveal early changes in muscle activation.
  Katharina A. Quinlan, Elma Kajtaz, Jody D. Ciolino, Rebecca D. Imhoff‐Manuel, Matthew C. Tresch, Charles J. Heckman, Vicki M. Tysseling.
  The Journal of Physiology. July 05, 2017
  Key points The present study demonstrates that electromyograms (EMGs) obtained during locomotor activity in mice were effective for identification of early physiological markers of amyotrophic lateral sclerosis (ALS). These measures could be used to evaluate therapeutic intervention strategies in animal models of ALS. Several parameters of locomotor activity were shifted early in the disease time course in SOD1G93A mice, especially when the treadmill was inclined, including intermuscular phase, burst skew and amplitude of the locomotor bursts. The results of the present study indicate that early compensatory changes may be taking place within the neural network controlling locomotor activity, including spinal interneurons. Locomotor EMGs could have potential use as a clinical diagnostic tool. Abstract To improve our understanding of early disease mechanisms and to identify reliable biomarkers of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, we measured electromyogram (EMG) activity in hind limb muscles of SOD1G93A mice. By contrast to clinical diagnostic measures using EMGs, which are performed on quiescent patients, we monitored activity during treadmill running aiming to detect presymptomatic changes in motor patterning. Chronic EMG electrodes were implanted into vastus lateralis, biceps femoris posterior, lateral gastrocnemius and tibialis anterior in mice from postnatal day 55 to 100 and the results obtained were assessed using linear mixed models. We evaluated differences in parameters related to EMG amplitude (peak and area) and timing (phase and skew, a measure of burst shape) when animals ran on level and inclined treadmills. There were significant changes in both the timing of activity and the amplitude of EMG bursts in SOD1G93A mice. Significant differences between wild‐type and SOD1G93A mice were mainly observed when animals locomoted on inclined treadmills. All muscles had significant effects of mutation that were independent of age. These novel results indicate (i) locomotor EMG activity might be an early measure of disease onset; (ii) alterations in locomotor patterning may reflect changes in neuronal drive and compensation at the network level including altered activity of spinal interneurons; and (iii) the increased power output necessary on an inclined treadmill was important in revealing altered activity in SOD1G93A mice.

  July 05, 2017   doi: 10.1113/JP274170   open full text
• Altered post‐capillary and collecting venular reactivity in skeletal muscle with metabolic syndrome.
  Kent A. Lemaster, Zahra Farid, Robert W. Brock, Carl D. Shrader, Daniel Goldman, Dwayne N. Jackson, Jefferson C. Frisbee.
  The Journal of Physiology. July 05, 2017
  Key points With the development of the metabolic syndrome, both post‐capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired. The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress. Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level. For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation. Abstract While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17 weeks of age, skeletal muscle post‐capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25 μm (post‐capillary) and ∼75 μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress‐based loss of nitric oxide bioavailability (post‐capillary) and increased α1‐ (and α2‐) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1‐adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3 Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3 Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi‐faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.

  July 05, 2017   doi: 10.1113/JP274291   open full text
• Glial EAAT2 regulation of extracellular nTS glutamate critically controls neuronal activity and cardiorespiratory reflexes.
  Michael P. Matott, David D. Kline, Eileen M. Hasser.
  The Journal of Physiology. July 05, 2017
  Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, their influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2 specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK‐induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex‐mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function. This article is protected by copyright. All rights reserved

  July 05, 2017   doi: 10.1113/JP274620   open full text
• Inhibitory modulation of medial prefrontal cortical activation on lateral orbitofrontal cortex‐amygdala information flow.
  Chun‐hui Chang, Ta‐wen Ho.
  The Journal of Physiology. July 05, 2017
  Several neocortical projections converge onto the basolateral complex of the amygdala (BLA), including the lateral orbitofrontal cortex (lOFC) and medial prefrontal cortex (mPFC). Lateral orbitofrontal input to BLA is important for cue‐outcome contingencies, while medial prefrontal input is essential for emotion control. In this study, we examined how mPFC, specifically the infralimbic (IL) division of mPFC, modulates the lOFC‐BLA information flow, using combined in vivo extracellular single‐unit recordings and pharmacological manipulations in anesthetized rats. We found that the majority (over 95%) of BLA neurons that responded to lOFC stimulation also responded to mPFC stimulation. Compared to basal condition, pharmacological (N‐Methyl‐D‐aspartate, NMDA) or electrical activation of the mPFC exerted an inhibitory modulation of the lOFC‐BLA pathway, which was reversed with intra‐amygdala blockade of GABAergic receptors with combined GABAA and GABAB antagonists (bicuculline and saclofen). Moreover, mPFC tetanus potentiated the lOFC‐BLA pathway, but mPFC tetanus or low‐frequency stimulation (LFS) did not alter its inhibitory modulatory gating on the lOFC‐BLA pathway. These results show that the mPFC potently inhibits lOFC drive of BLA neurons in a GABA‐dependent manner. Our result is informative in understanding the normal and potential pathophysiological state of emotion and contingency associations regulating behaviour. This article is protected by copyright. All rights reserved

  July 05, 2017   doi: 10.1113/JP274568   open full text
• Kir2.1 and K2P1 channels reconstitute two levels of resting membrane potential in cardiomyocytes.
  Dongchuan Zuo, Kuihao Chen, Min Zhou, Zheng Liu, Haijun Chen.
  The Journal of Physiology. July 04, 2017
  Key points Outward and inward background currents across the cell membrane balance, determining resting membrane potential. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the resting membrane potential of cardiomyocytes. Human cardiomyocytes exhibit two levels of resting membrane potential at subphysiological extracellular K+ concentrations or pathological hypokalaemia, however, the underlying mechanism is unclear. In the present study, we show that human cardiomyocytes derived from induced pluripotent stem cells with enhanced expression of isoform 1 of Kir2 (Kir2.1) channels and mouse HL‐1 cardiomyocytes with ectopic expression of two pore‐domain K+ channel isoform 1 (K2P1) recapitulate two levels of resting membrane potential, indicating the contributions of Kir2.1 and K2P1 channels to the phenomenon. In Chinese hamster ovary cells that express the channels, Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced K2P1 leak cation currents, reconstituting two levels of resting membrane potential. These findings support the hypothesis that Kir2 currents non‐linearly counterbalance inward background cation currents, such as K2P1 currents, accounting for two levels of resting membrane potential in human cardiomyocytes and demonstrating a novel mechanism that regulates excitability. Abstract Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K+ concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K+ channel subfamily 2 isoform 1 (Kir2.1) currents non‐linearly counterbalance hypokalaemia‐induced two pore‐domain K+ channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL‐1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N‐shaped current–voltage relationships that cross the voltage axis three times and the first and third zero‐current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced K2P1 leak cation currents, yielding the N‐shaped current–voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero‐current potential to depolarization at the third zero‐current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non‐linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.

  July 04, 2017   doi: 10.1113/JP274268   open full text
• A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions.
  Gregory K. Potts, Rachel M. McNally, Rocky Blanco, Jae‐Sung You, Alexander S. Hebert, Michael S. Westphall, Joshua J. Coon, Troy A. Hornberger.
  The Journal of Physiology. July 04, 2017
  Key points Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill‐defined. Here we report a mass spectrometry‐based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal‐intensity contractions (MICs). Our dataset provides the first comprehensive map of the MIC‐regulated phosphoproteome. Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well‐known MIC‐regulated signalling pathways, as well as to a plethora of novel MIC‐regulated events. We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field. Abstract The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal‐intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal‐intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC‐regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation‐based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase‐substrate predictions indicated that the MIC‐regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z‐disc, with over 74% of the Z‐disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z‐disc kinases (striated muscle‐specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.

  July 04, 2017   doi: 10.1113/JP273904   open full text
• VEGF‐A165b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF‐A splice isoforms from the kidney.
  Megan Stevens, Christopher R. Neal, Andrew H. J. Salmon, David O. Bates, Steven J. Harper, Sebastian Oltean.
  The Journal of Physiology. July 03, 2017
  Key points Progressive depletion of all vascular endothelial growth factor A (VEGF‐A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over‐expression of VEGF‐A165b only. VEGF‐A165b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub‐podocyte space coverage, produced by VEGF‐A depletion. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF‐A165b has opposite effects to VEGF‐A165 on the expression of genes involved in endothelial cell migration and proliferation. Abstract Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF‐A). However, little is known about the contribution of VEGF‐A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF‐A165b (resulting from alternative usage of a 3′ splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad‐transgenic model, that over‐expression of VEGF‐A165b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF‐A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub‐podocyte space coverage is reduced when VEGF‐A is depleted, all of which are rescued in VEGF‐A165b over‐expressors. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule‐1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down‐regulates genes involved in migration and proliferation of endothelial cells, otherwise up‐regulated by the canonical isoform VEGF‐A165. The results of the present study indicate that manipulation of VEGF‐A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

  July 03, 2017   doi: 10.1113/JP274481   open full text
• Impaired activity of adherens junctions contributes to endothelial dilator dysfunction in ageing rat arteries.
  Fumin Chang, Sheila Flavahan, Nicholas A. Flavahan.
  The Journal of Physiology. June 30, 2017
  Key points Ageing‐induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE‐cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium‐dependent dilatation. Ageing increased internalization and degradation of VE‐cadherin, resulting in impaired activity of adherens junctions. Inhibition of VE‐cadherin clustering at adherens junctions (function‐blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries. Src tyrosine kinase activity and tyrosine phosphorylation of VE‐cadherin were increased in old arteries. Src inhibition increased VE‐cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE‐cadherin FBA. Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing. Abstract Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3–4 months) and old (22–24 months) F344 rats. VE‐cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE‐cadherin species was present on the cell surface and in TTX‐insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE‐cadherin were decreased, whereas levels of a TTX‐soluble 115 kDa VE‐cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium‐dependent dilatation that was decreased in old compared to young arteries. Disruption of VE‐cadherin clustering at AJs (function‐blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE‐cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE‐cadherin in the TTX‐insoluble fraction, (ii) VE‐cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src‐induced phosphorylation, internalization and degradation of VE‐cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing.

  June 30, 2017   doi: 10.1113/JP274189   open full text
• Enhancement of synchronized activity between hippocampal CA1 neurons during initial storage of associative fear memory.
  Yu‐Zhang Liu, Yao Wang, Weida Shen, Zhiru Wang.
  The Journal of Physiology. June 30, 2017
  Key points Learning and memory storage requires neuronal plasticity induced in the hippocampus and other related brain areas, and this process is thought to rely on synchronized activity in neural networks. We used paired whole‐cell recording in vivo to examine the synchronized activity that was induced in hippocampal CA1 neurons by associative fear learning. We found that both membrane potential synchronization and spike synchronization of CA1 neurons could be transiently enhanced after task learning, as observed on day 1 but not day 5. On day 1 after learning, CA1 neurons showed a decrease in firing threshold and rise times of suprathreshold membrane potential changes as well as an increase in spontaneous firing rates, possibly contributing to the enhancement of spike synchronization. The transient enhancement of CA1 neuronal synchronization may play important roles in the induction of neuronal plasticity for initial storage and consolidation of associative memory. Abstract The hippocampus is critical for memory acquisition and consolidation. This function requires activity‐ and experience‐induced neuronal plasticity. It is known that neuronal plasticity is largely dependent on synchronized activity. As has been well characterized, repetitive correlated activity of presynaptic and postsynaptic neurons can lead to long‐term modifications at their synapses. Studies on network activity have also suggested that memory processing in the hippocampus may involve learning‐induced changes of neuronal synchronization, as observed in vivo between hippocampal CA3 and CA1 networks as well as between the rhinal cortex and the hippocampus. However, further investigation of learning‐induced synchronized activity in the hippocampus is needed for a full understanding of hippocampal memory processing. In this study, by performing paired whole‐cell recording in vivo on CA1 pyramidal cells (PCs) in anaesthetized adult rats, we examined CA1 neuronal synchronization before and after associative fear learning. We first found in naive animals that there was a low level of membrane potential (MP) synchronization and spike synchronization of CA1 PCs. In conditioned animals, we found a significant enhancement of both MP synchronization and spike synchronization, as observed on day 1 after learning, and this enhancement was transient and not observed on day 5. Accompanying learning‐induced synchronized activity was a decreased firing threshold and rise time of suprathreshold MP changes as well as an increased spontaneous firing rate, possibly contributing to the enhanced spike synchronization. The transiently enhanced CA1 neuronal synchronization may have important roles in generating neuronal plasticity for hippocampal storage and consolidation of associative memory traces.

  June 30, 2017   doi: 10.1113/JP274212   open full text
• Threshold position control of anticipation in humans: a possible role of corticospinal influences.
  Lei Zhang, Nicolas A. Turpin, Anatol G. Feldman.
  The Journal of Physiology. June 28, 2017
  Key points Sudden unloading of preloaded wrist muscles elicits motion to a new wrist position. Such motion is prevented if subjects unload muscles using the contralateral arm (self‐unloading). Corticospinal influences originated from the primary motor cortex maintain tonic influences on motoneurons of wrist muscles before sudden unloading but modify these influences prior to the onset and until the end of self‐unloading. Results are interpreted based on the previous finding that intentional actions are caused by central, particularly corticospinal, shifts in the spatial thresholds at which wrist motoneurons are activated, thus predetermining the attractor point at which the neuromuscular periphery achieves mechanical balance with environment forces. By maintaining or shifting the thresholds, descending systems let body segments go to the equilibrium position in the respective unloading tasks without the pre‐programming of kinematics or muscle activation patterns. The study advances the understanding of how motor actions in general, and anticipation in particular, are controlled. Abstract The role of corticospinal (CS) pathways in anticipatory motor actions was evaluated using transcranial magnetic stimulation (TMS) of the primary motor cortex projecting to motoneurons (MNs) of wrist muscles. Preloaded wrist flexors were suddenly unloaded by the experimenter or by the subject using the other hand (self‐unloading). After sudden unloading, the wrist joint involuntarily flexed to a new position. In contrast, during self‐unloading the wrist remained almost motionless, implying that an anticipatory postural adjustment occurred. In the self‐unloading task, anticipation was manifested by a decrease in descending facilitation of pre‐activated flexor MNs starting ∼72 ms before changes in the background EMG activity. Descending facilitation of extensor MNs began to increase ∼61 ms later. Conversely, these influences remained unchanged before sudden unloading, implying the absence of anticipation. We also tested TMS responses during EMG silent periods produced by brief muscle shortening, transiently resulting in similar EMG levels before the onset and after the end of self‐unloading. We found reduced descending facilitation of flexor MNs after self‐unloading. To explain why the wrist excursion was minimized in self‐unloading due to these changes in descending influences, we relied on previous demonstrations that descending systems pre‐set the threshold positions of body segments at which muscles begin to be activated, thus predetermining the equilibrium point to which the system is attracted. Based on this notion, a more consistent explanation of the kinematic, EMG and descending patterns in the two types of unloading is proposed compared to the alternative notion of direct pre‐programming of kinematic and/or EMG patterns.

  June 28, 2017   doi: 10.1113/JP274309   open full text
• Skeletal myofiber vascular endothelial growth factor is required for the exercise training‐induced increase in dentate gyrus neuronal precursor cells.
  Benjamin Rich, Miriam Scadeng, Masahiro Yamaguchi, Peter D. Wagner, Ellen C. Breen.
  The Journal of Physiology. June 28, 2017
  Key points Peripheral vascular endothelial growth factor (VEGF) is necessary for exercise to stimulate hippocampal neurogenesis. Here we report that skeletal myofiber VEGF directly or indirectly regulates exercise‐signalled proliferation of neuronal precursor cells. Our results found skeletal myofiber VEGF to be necessary for maintaining blood flow through hippocampal regions independent of exercise training state. This study demonstrates that skeletal myofiber VEGF is required for the hippocampal VEGF response to acute exercise. These results help to establish the mechanisms by which exercise, through skeletal myofiber VEGF, affects the hippocampus. Abstract Exercise signals neurogenesis in the dentate gyrus of the hippocampus. This phenomenon requires vascular endothelial growth factor (VEGF) originating from outside the blood–brain barrier, but no cellular source has been identified. Thus, we hypothesized that VEGF produced by skeletal myofibers plays a role in regulating hippocampal neuronal precursor cell proliferation following exercise training. This was tested in adult conditional skeletal myofiber‐specific VEGF gene‐ablated mice (VEGFHSA−/−) by providing VEGFHSA−/− and non‐ablated (VEGFf/f) littermates with running wheels for 14 days. Following this training period, hippocampal cerebral blood flow (CBF) was measured by functional magnetic resonance imaging (fMRI), and neuronal precursor cells (BrdU+/Nestin+) were detected by immunofluorescence. The VEGFf/f trained group showed improvements in both speed and endurance capacity in acute treadmill running tests (P < 0.05). The VEGFHSA−/− group did not. The number of proliferating neuronal precursor cells was increased with training in VEGFf/f (P < 0.05) but not in VEGFHSA−/− mice. Endothelial cell (CD31+) number did not change in this region with exercise training or skeletal myofiber VEGF gene deletion. However, resting blood flow through the hippocampal region was lower in VEGFHSA−/− mice, both untrained and trained, than untrained VEGFf/f mice (P < 0.05). An acute hypoxic challenge decreased CBF (P < 0.05) in untrained VEGFf/f, untrained VEGFHSA−/− and trained VEGFHSA−/− mice, but not trained VEGFf/f mice. VEGFf/f, but not VEGFHSA−/−, mice were able to acutely run on a treadmill at an intensity sufficient to increase hippocampus VEGF levels. These data suggest that VEGF expressed by skeletal myofibers may directly or indirectly regulate both hippocampal blood flow and neurogenesis.

  June 28, 2017   doi: 10.1113/JP273994   open full text
• Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome.
  Chih‐Jen Cheng, Yi‐Fen Lo, Jen‐Chi Chen, Chou‐Long Huang, Shih‐Hua Lin.
  The Journal of Physiology. June 27, 2017
  Key points The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype–phenotype association has not been established. Low‐level expression of the human ClC‐Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC‐Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C‐terminal in‐frame green fluorescent protein fusion may slow down the proteasome‐mediated proteolysis. Barttin co‐expression necessarily improves the stability, membrane trafficking and gating of ClC‐Kb. CLCNKB mutations in barttin‐binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC‐Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. Abstract Mutations in the CLCNKB gene encoding the human voltage‐gated chloride ClC‐Kb (hClC‐Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC‐Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype–phenotype association has not been established. In this study, we found that hClC‐Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In‐frame fusion of green fluorescent protein (GFP) to the C‐terminus of the channel may ameliorate proteasome degradation. Co‐expression of barttin increased protein abundance and membrane trafficking of hClC‐Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC‐Kb‐GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well‐described functional consequences and clinical presentations for genotype–phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC‐Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C‐terminus of hClC‐Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

  June 27, 2017   doi: 10.1113/JP274344   open full text
• Training alters the distribution of perilipin proteins in muscle following acute free fatty acid exposure.
  S. O. Shepherd, J. A. Strauss, Q. Wang, J. J. Dube, B. Goodpaster, D. G. Mashek, L. S. Chow.
  The Journal of Physiology. June 27, 2017
  Key points The lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown. We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only. We conclude that trained individuals are able to redistribute the pre‐existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG. Abstract Because the lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic–euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type‐specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P < 0.05) but did not affect PLIN protein content. At baseline, PLIN2 (+65%), PLIN3 (+105%) and PLIN5 (+53%; all P < 0.05) protein content was higher in trained compared to sedentary individuals. In trained individuals, lipid infusion increased the number of LDs associated with PLIN2 (+27%), PLIN3 (+73%) and PLIN5 (+40%; all P < 0.05) in type I fibres. By contrast, in sedentary individuals, lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute free fatty acid elevation therefore induces a redistribution of PLIN proteins to an expanded LD pool in trained individuals only and this may be part of the mechanism that enables fatty acids to be stored in IMTG.

  June 27, 2017   doi: 10.1113/JP274374   open full text
• Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis.
  E. Matthew Morris, Colin S. McCoin, Julie A. Allen, Michelle L. Gastecki, Lauren G. Koch, Steven L. Britton, Justin A. Fletcher, Xiarong Fu, Wen‐Xing Ding, Shawn C. Burgess, R. Scott Rector, John P. Thyfault.
  The Journal of Physiology. June 27, 2017
  Key points Low intrinsic aerobic capacity is associated with increased all‐cause and liver‐related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high‐fat/high‐sucrose diet‐induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high‐fat/high‐sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non‐alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. Abstract Low aerobic capacity increases risk for non‐alcoholic fatty liver disease and liver‐related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet‐induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16‐week ‘western diet’ (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD‐fed low‐fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP‐1, TLR4, TLR2 and IL‐1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD‐induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD‐induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

  June 27, 2017   doi: 10.1113/JP274281   open full text
• Spike threshold dynamics in spinal motoneurons during scratching and swimming.
  Ramunas Grigonis, Aidas Alaburda.
  The Journal of Physiology. June 27, 2017
  During functional spinal neural network activity motoneurons receive intense synaptic input, and this could modulate the threshold for action potential generation, providing the ability to dynamically adjust the excitability and recruitment order for functional needs. In the present study we investigated the dynamics of action potential threshold during motor network activity. Intracellular recordings from spinal motoneurons in an ex vivo carapace‐spinal cord preparation from adult turtles were performed during two distinct types of motor behaviour – fictive scratching and fictive swimming. We found that the threshold of the first spike in episodes of scratching and swimming was the lowest. The threshold potential depolarizes by about 10 mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Depolarization of the threshold potential results in decreased excitability of motoneurons. Synaptic inputs do not modulate the threshold of the first action potential during episodes of scratching or of swimming. There is no correlation between changes in spike threshold and interspike intervals within bursts. Slow synaptic integration that results in a wave of membrane potential depolarization rather than fast synaptic events preceding each spike is the factor influencing the threshold potential within firing bursts during motor behaviours. This article is protected by copyright. All rights reserved

  June 27, 2017   doi: 10.1113/JP274434   open full text
• Heterogeneity of Purkinje cell simple spike–complex spike interactions: zebrin‐ and non‐zebrin‐related variations.
  Tianyu Tang, Jianqiang Xiao, Colleen Y. Suh, Amelia Burroughs, Nadia L. Cerminara, Linjia Jia, Sarah P. Marshall, Andrew K. Wise, Richard Apps, Izumi Sugihara, Eric J. Lang.
  The Journal of Physiology. June 26, 2017
  Key points Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist. Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike–complex spike interactions both within and across zebrin populations. Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre‐ and post‐complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions. Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs. The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions. Abstract Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS‐associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre‐CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z−) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre‐CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the amplitudes of the SS modulation phases were generally weak. Division of spikelets into likely axonally propagated and non‐propagated groups (based on their interspikelet interval) showed that the correlation of spikelet number with SS firing rate primarily reflected a relationship with non‐propagated spikelets. In sum, the results show both zebrin‐related and non‐zebrin‐related physiological heterogeneity in SS–CS interactions among PCs, which suggests that the cerebellar cortex is more functionally diverse than is assumed by standard theories of cerebellar function.

  June 26, 2017   doi: 10.1113/JP274252   open full text
• Coupling of excitation to Ca2+ release is modulated by dysferlin.
  Valeriy Lukyanenko, Joaquin M. Muriel, Robert J. Bloch.
  The Journal of Physiology. June 26, 2017
  Key points Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin‐null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L‐type Ca2+ channels protect voltage‐induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+‐free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury‐induced SR Ca2+ leak. Abstract Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage‐induced Ca2+ transients elicited in dysferlin‐null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus‐dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L‐type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI‐induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1–FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus‐dysferlin prevented OSI‐induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1‐mediated Ca2+ leak from the SR into the cytoplasm.

  June 26, 2017   doi: 10.1113/JP274515   open full text
• Blood flow restricted training leads to myocellular macrophage infiltration and upregulation of heat shock proteins, but no apparent muscle damage.
  Jakob L. Nielsen, Per Aagaard, Tatyana A. Prokhorova, Tobias Nygaard, Rune D. Bech, Charlotte Suetta, Ulrik Frandsen.
  The Journal of Physiology. June 23, 2017
  Key points Muscular contractions performed using a combination of low external loads and partial restriction of limb blood flow appear to induce substantial gains in muscle strength and muscle mass. This exercise regime may initially induce muscular stress and damage; however, the effects of a period of blood flow restricted training on these parameters remain largely unknown. The present study shows that short‐term, high‐frequency, low‐load muscle training performed with partial blood flow restriction does not induce significant muscular damage. However, signs of myocellular stress and inflammation that were observed in the early phase of training and after the training intervention, respectively, may be facilitating the previously reported gains in myogenic satellite cell content and muscle hypertrophy. The present results improve our current knowledge about the physiological effects of low‐load muscular contractions performed under blood flow restriction and may provide important information of relevance for future therapeutic treatment of muscular atrophy. Abstract Previous studies indicate that low‐load muscle contractions performed under local blood flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study protocols were conducted, covering 3 weeks (3 wk) or one week (1 wk). Subjects performed BFR exercise (100 mmHg, 20% 1RM) to concentric failure (BFRE) (3 wk/1 wk), while controls performed work‐matched (LLE) (3 wk) or high‐load (HLE; 70% 1RM) (1 wk) free‐flow exercise. Muscle biopsies (3 wk) were obtained at baseline (Pre), 8 days into the intervention (Mid8), and 3 and 10 days after training cessation (Post3, Post10) to examine macrophage (M1/M2) content as well as heat shock protein (HSP27/70) and tenascin‐C expression. Blood samples (1 wk) were collected before and after (0.1–24 h) the first and last training session to examine markers of muscle damage (creatine kinase), oxidative stress (total antibody capacity, glutathione) and inflammation (monocyte chemotactic protein‐1, interleukin‐6, tumour necrosis factor α). M1‐macrophage content increased 108–165% with BFRE and LLE at Post3 (P < 0.05), while M2‐macrophages increased (163%) with BFRE only (P < 0.01). Membrane and intracellular HSP27 expression increased 60–132% at Mid8 with BFRE (P < 0.05–0.01). No or only minor changes were observed in circulating markers of muscle damage, oxidative stress and inflammation. The amplitude, timing and localization of the above changes indicate that only limited muscle damage was evoked with BFRE. This study is the first to show that a period of high‐frequency, low‐load BFR training does not appear to induce general myocellular damage. However, signs of tissue inflammation and focal myocellular membrane stress and/or reorganization were observed that may be involved in the adaptation processes evoked by BFR muscle exercise.

  June 23, 2017   doi: 10.1113/JP273907   open full text
• Presence of ethanol‐sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens.
  B. Förstera, B. Muñoz, M. K. Lobo, R. Chandra, D. M. Lovinger, L. G. Aguayo.
  The Journal of Physiology. June 23, 2017
  Key points The nucleus accumbens (nAc) is involved in addiction‐related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse. Abstract Alcohol abuse causes major social, economic and health‐related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)‐positive MSNs. Interestingly, the glycine‐evoked currents in dissociated DRD1‐positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR‐mediated tonic current by ethanol suggests that they modulate the excitability of DRD1‐positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction‐related and compulsive behaviours.

  June 23, 2017   doi: 10.1113/JP273767   open full text
• Cerebral haemodynamic response to somatosensory stimulation in neonatal lambs.
  Shinji Nakamura, David W. Walker, Flora Y. Wong.
  The Journal of Physiology. June 23, 2017
  The neurovascular coupling response has been defined for the adult brain but in the neonate non‐invasive measurement of local cerebral perfusion using NIRS or BOLD fMRI have yielded variable and inconsistent results, including negative responses suggesting decreased perfusion and localised tissue tissue hypoxia. Also, the impact of permissive hypercapnia (PaCO2 > 50 mmHg) in the management of neonates on cerebrovascular responses to somatosensory input is unknown. Using NIRS to measure changes in cerebral oxy‐ and deoxy‐haemoglobin (ΔoxyHb, ΔdeoxyHb) in 8 anaesthetised newborn lambs, we studied the cerebral haemodynamic functional response to left median nerve stimulation using stimulus trains of 1.8, 4.8 and 7.8 s. Stimulation always produced a somatosensory evoked response, and superficial cortical perfusion measured by Laser Doppler Flowmetry predominantly increased following median nerve stimulation. However, with 1.8 s stimulation, oxyHb responses in the contralateral hemisphere were either positive (i.e. increased oxyHb), negative, or absent; and with 4.8 and 7.8 s stimulations, both positive and negative responses were observed. Hypercapnia increased baseline oxyHb and total Hb consistent with cerebral vasodilatation, and 6 of 7 lambs tested showed increased Δtotal Hb responses after the 7.8 s stimulation; among which 4 lambs also showed increased ΔoxyHb responses. In 2 of 3 lambs, the negative ΔoxyHb response became a positive pattern during hypercapnia. These results show that instead of functional hyperaemia, somatosensory stimulation can evoke negative (decreased oxyHb, total Hb) functional responses in the neonatal brain suggestive of decreased local perfusion and vasoconstriction, and that hypercapnia produces both baseline hyperperfusion and increased functional hyperaemia. This article is protected by copyright. All rights reserved

  June 23, 2017   doi: 10.1113/JP274244   open full text
• Lack of adaptation during prolonged split‐belt locomotion in the intact and spinal cat.
  Victoria Kuczynski, Alessandro Telonio, Yann Thibaudier, Marie‐France Hurteau, Charline Dambreville, Etienne Desrochers, Adam Doelman, Tayler Ross, Alain Frigon.
  The Journal of Physiology. June 23, 2017
  In humans, gait adapts to prolonged walking on a split‐belt treadmill, where one leg steps faster than the other, by gradually restoring the symmetry of interlimb kinematic variables, such as double support periods and step lengths, and by reducing muscle activity (EMG, electromyography). The adaptation is also characterized by reversing the asymmetry of interlimb variables observed during the early split‐belt period when returning to tied‐belt locomotion, termed an after‐effect. To determine if cats adapt to prolonged split‐belt locomotion and to assess if spinal locomotor circuits participate in the adaptation, we measured interlimb variables and EMG in intact and spinal‐transected cats before, during and after 10 min of split‐belt locomotion. In spinal cats only the hindlimbs performed stepping with the forelimbs stationary. In intact and spinal cats, step lengths and double support periods were, on average, symmetric, during tied‐belt locomotion. They became asymmetric during split‐belt locomotion and remained asymmetric throughout the split‐belt period. Upon returning to tied‐belt locomotion, symmetry was immediately restored. In intact cats, the mean EMG amplitude of hindlimb extensors increased during split‐belt locomotion and remained increased throughout the split‐belt period, whereas in spinal cats, EMG amplitude did not change. Therefore, the results indicate that the locomotor pattern of cats does not adapt to prolonged split‐belt locomotion, suggesting an important physiological difference in the control of locomotion between cats and humans. We propose that restoring left‐right symmetry is not required to maintain balance during prolonged asymmetric locomotion in the cat, a quadruped, as opposed to human bipedal locomotion. This article is protected by copyright. All rights reserved

  June 23, 2017   doi: 10.1113/JP274518   open full text
• Intraglomerular gap junctions enhance interglomerular synchrony in a sparsely‐connected olfactory bulb network.
  Frederic Pouille, Thomas S. McTavish, Lawrence E. Hunter, Diego Restrepo, Nathan E. Schoppa.
  The Journal of Physiology. June 22, 2017
  A dominant feature of the olfactory bulb response to odour is fast synchronized oscillations at beta (15–40 Hz) or gamma (40–90 Hz) frequencies, thought to be involved in integration of olfactory signals. Mechanistically, the bulb presents an interesting case study for understanding how beta/gamma oscillations arise. Fast oscillatory synchrony in the activity of output mitral cells (MCs) appears to result from interactions with GABAergic granule cells (GCs), yet the incidence of MC‐GC connections is very low, around 4%. Here, we combined computational and experimental approaches to examine how oscillatory synchrony can nevertheless arise, focusing mainly on activity between “non‐sister” MCs affiliated with different glomeruli (interglomerular synchrony). In a sparsely connected model of MCs and GCs, we found first that interglomerular synchrony was generally quite low, but could be increased by a factor of 4 by physiological‐levels of gap junctional coupling between sister MCs at the same glomerulus. This effect was due to enhanced mutually synchronizing interactions between MC and GC populations. The potent role of gap junctions was confirmed in patch‐clamp recordings in bulb slices from wild‐type and connexin 36‐knockout (KO) mice. KO reduced both beta/gamma local field potential oscillations as well as synchrony of inhibitory signals in pairs of non‐sister MCs. These effects were independent of potential KO‐actions on network excitation. Divergent synaptic connections did not contribute directly to the vast majority of synchronized signals. Thus, in a sparsely connected network, gap junctions between a small subset of cells can, through population‐effects, greatly amplify oscillatory synchrony amongst unconnected cells. This article is protected by copyright. All rights reserved

  June 22, 2017   doi: 10.1113/JP274408   open full text
• Exercise‐induced quadriceps muscle fatigue in men and women: effects of arterial oxygen content and respiratory muscle work.
  Paolo B. Dominelli, Yannick Molgat‐Seon, Donald E. G. Griesdale, Carli M. Peters, Jean‐Sébastien Blouin, Mypinder Sekhon, Giulio S. Dominelli, William R. Henderson, Glen E. Foster, Lee M. Romer, Michael S. Koehle, A. William Sheel.
  The Journal of Physiology. June 19, 2017
  Key points High work of breathing and exercise‐induced arterial hypoxaemia (EIAH) can decrease O2 delivery and exacerbate exercise‐induced quadriceps fatigue in healthy men. Women have a higher work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles and develop EIAH. Despite a greater reduction in men's work of breathing, the attenuation of quadriceps fatigue was similar between the sexes. The degree of EIAH was similar between sexes, and regardless of sex, those who developed the greatest hypoxaemia during exercise demonstrated the most attenuation of quadriceps fatigue. Based on our previous finding that women have a greater relative oxygen cost of breathing, women appear to be especially susceptible to work of breathing‐related changes in quadriceps muscle fatigue. Abstract Reducing the work of breathing or eliminating exercise‐induced arterial hypoxaemia (EIAH) during exercise decreases the severity of quadriceps fatigue in men. Women have a greater work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles, and demonstrate EIAH, suggesting women may be especially susceptible to quadriceps fatigue. Healthy subjects (8 male, 8 female) completed three constant load exercise tests over 4 days. During the first (control) test, subjects exercised at ∼85% of maximum while arterial blood gases and work of breathing were assessed. Subsequent constant load exercise tests were iso‐time and iso‐work rate, but with EIAH prevented by inspiring hyperoxic gas or work of breathing reduced via a proportional assist ventilator (PAV). Quadriceps fatigue was assessed by measuring force in response to femoral nerve stimulation. For both sexes, quadriceps force was equally reduced after the control trial (−27 ± 2% baseline) and was attenuated with hyperoxia and PAV (−18 ± 1 and −17 ± 2% baseline, P < 0.01, respectively), with no sex difference. EIAH was similar between the sexes, and regardless of sex, subjects with the lowest oxyhaemoglobin saturation during the control test had the greatest quadriceps fatigue attenuation with hyperoxia (r2 = 0.79, P < 0.0001). For the PAV trial, despite reducing the work of breathing to a greater degree in men (men: 60 ± 5, women: 75 ± 6% control, P < 0.05), the attenuation of quadriceps fatigue was similar between the sexes (36 ± 4 vs. 37 ± 7%). Owing to a greater relative V̇O2 of the respiratory muscles in women, less of a change in work of breathing is needed to reduce quadriceps fatigue.

  June 19, 2017   doi: 10.1113/JP274068   open full text
• Functional expression of calcium‐permeable Canonical Transient Receptor Potential 4‐containing channels promotes migration of medulloblastoma cells.
  Wei‐Chun Wei, Wan‐Chen Huang, Yu‐Ping Lin, Esther B. E. Becker, Olaf Ansorge, Veit Flockerzi, Daniele Conti, Giovanna Cenacchi, Maike D. Glitsch.
  The Journal of Physiology. June 19, 2017
  Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilised in response to activation of cell surface receptors. OGR1 (Ovarian cancer G protein coupled Receptor 1, aka GPR68) is a proton‐sensing Gq‐coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We find that nine distinct human MB samples all express OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promotes expression of the proton‐potentiated member of the Canonical Transient Receptor Potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we find that all MB samples also express TRPC4. In DAOY cells, activation of TRPC4‐containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration was observed. Our results suggest that OGR1‐dependent increases in TRPC4 expression may favour formation of highly Ca2+‐permeable TRPC4‐containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB. This article is protected by copyright. All rights reserved

  June 19, 2017   doi: 10.1113/JP274659   open full text
• Chemosensitive Phox2b‐expressing neurons are crucial for hypercapnic ventilatory response in the nucleus tractus solitarius.
  Congrui Fu, Jinyu Xue, Ri Wang, Jinting Chen, Lan Ma, Yixian Liu, Xuejiao Wang, Fang Guo, Yi Zhang, Xiangjian Zhang, Sheng Wang.
  The Journal of Physiology. June 16, 2017
  Key points Central hypercapnic hypoventilation is highly prevalent in children suffering from congenital central hypoventilation syndrome (CCHS). Mutations of the gene for paired‐like homeobox 2b (Phox2b) are aetiologically associated with CCHS and Phox2b is present in central components of respiratory chemoreflex, such as the nucleus tractus solitarius (NTS). Injection of the neurotoxin substance P‐saporin into NTS destroys Phox2b‐expressing neurons. Impaired hypercapnic ventilatory response caused by this neurotoxin is attributable to a loss of CO2‐sensitive Phox2b‐expressing NTS neurons. A subgroup of Phox2b‐expressing neurons exhibits intrinsic chemosensitivity. A background K+ channel‐like current is partially responsible for such chemosensitivity in Phox2b‐expressing neurons. The present study helps us better understand the mechanism of respiratory deficits in CCHS and potentially locates a brainstem site for development of precise clinical intervention. Abstract The nucleus tractus solitarius (NTS) neurons have been considered to function as central respiratory chemoreceptors. However, the common molecular marker defined for these neurons remains unknown. The present study investigated whether paired‐like homeobox 2b (Phox2b)‐expressing NTS neurons are recruited in hypercapnic ventilatory response (HCVR) and whether these neurons exhibit intrinsic chemosensitivity. HCVR was assessed using whole body plethysmography and neuronal chemosensitivity was examined by patch clamp recordings in brainstem slices or dissociated neurons from Phox2b‐EGFP transgenic mice. Injection of the neurotoxin substance P‐saporin (SSP‐SAP) into NTS destroyed Phox2b‐expressing neurons. Minute ventilation and tidal volume were both reduced by 13% during exposure to 8% CO2 in inspired air when ∼13% of the Phox2b‐expressing neurons were eliminated. However, a loss of ∼18% of these neurons was associated with considerable decreases in minute ventilation by ≥18% and in tidal volume by≥22% when challenged by ≥4% CO2. In both cases, breathing frequency was unaffected. Most CO2‐activated neurons were immunoreactive to Phox2b. In brainstem slices, ∼43% of Phox2b‐expressing neurons from Phox2b‐EGFP mice displayed a sustained or transient increase in firing rate during physiological acidification (pH 7.0 or 8% CO2). Such a response was also present in dissociated neurons in favour of an intrinsic property. In voltage clamp recordings, a background K+ channel‐like current was found in a subgroup of Phox2b‐expressing neurons. Thus, the respiratory deficits caused by injection of SSP‐SAP into the NTS are attributable to proportional lesions of CO2/H+‐sensitive Phox2b‐expressing neurons.

  June 16, 2017   doi: 10.1113/JP274437   open full text
• An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.
  Karla Droguett, Mariana Rios, Daniela V. Carreño, Camilo Navarrete, Christian Fuentes, Manuel Villalón, Nelson P. Barrera.
  The Journal of Physiology. June 15, 2017
  Key points Extracellular ATP, in association with [Ca2+]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Abstract Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+]i‐dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml−1) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1–100 μm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

  June 15, 2017   doi: 10.1113/JP273996   open full text
• Systolic [Ca2+]i regulates diastolic levels in rat ventricular myocytes.
  R. Sankaranarayanan, K. Kistamas, D. J. Greensmith, L. A. Venetucci, D. A. Eisner.
  The Journal of Physiology. June 15, 2017
  [Ca2+]i must be low enough in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the RyR leaky (with caffeine or ryanodine) or by decreasing SERCA activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Diastolic and time‐averaged [Ca2+]i were decreased by decreasing the L‐type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres which decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+]i. This identifies a novel mechanism whereby changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i. This article is protected by copyright. All rights reserved

  June 15, 2017   doi: 10.1113/JP274366   open full text
• In vivo analysis of synaptic activity in cerebellar nuclei neurons unravels the efficacy of excitatory inputs.
  Yasmin Yarden‐Rabinowitz, Yosef Yarom.
  The Journal of Physiology. June 15, 2017
  It is commonly agreed that the main function of the cerebellar system is to provide well‐timed signals used for the execution of motor commands or prediction of sensory inputs. This function is manifested as a temporal sequence of spiking that should be expressed in the cerebellar nuclei projection neurons (CN). Whether spiking activity is generated by excitation or release from inhibition is still a hotly debated issue. In an attempt to resolve this debate, we recorded intracellularly from CN neurons in anaesthetized mice and performed an analysis of synaptic activity that yielded a number of important observations. First, we demonstrate that CN neurons can be classified into four groups. Second, shape‐index plots of the excitatory events suggest that they are distributed all over the dendritic tree. Third, the rise time of excitatory events is linearly related to the amplitude, suggesting that all excitatory events contribute equally to the generation of action potentials. Fourth, we identified a temporal pattern of spontaneous excitatory events that represent climbing fibre inputs and confirm the results by direct stimulation and analysis on harmaline evoked activity. Finally, we demonstrate that the probability of excitatory inputs to generate an AP is 0.1 yet half of the APs are generated by excitatory events. Moreover, the probability of a presumably spontaneous climbing fibre input to generate an AP is higher, reaching mean population value of 0.15. In view of these results, the mode of synaptic integration at the level of the CN should be re‐considered. This article is protected by copyright. All rights reserved

  June 15, 2017   doi: 10.1113/JP274115   open full text
• Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects.
  Lasse Gliemann, Nicolai Rytter, Mads Lindskrog, Martina H. Lundberg Slingsby, Thorbjörn Åkerström, Lykke Sylow, Erik A. Richter, Ylva Hellsten.
  The Journal of Physiology. June 15, 2017
  Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor‐ and vascular function in humans, twelve young healthy male subjects supplemented their diet with 3 × 75 g sucrose day−1 for 14 days in a randomized cross‐over design. Before and after the intervention period, the hyperemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2 %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM‐1), endothelial nitric oxide synthase, NADPH oxidase and the Rho family GTPase Rac1 protein expression in the muscle tissue as well as an increased basal phosphorylation status of Vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM‐1. The phosphorylation status was not acutely altered with passive leg movement. These findings indicate that regular intake of high levels of sucrose can impair vascular mechanotransduction, increase the oxidative stress potential and suggest that dietary excessive sugar intake may contribute to the development of vascular disease. This article is protected by copyright. All rights reserved

  June 15, 2017   doi: 10.1113/JP274623   open full text
• Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating.
  A. Liavas, G. Lignani, S. Schorge.
  The Journal of Physiology. June 15, 2017
  Voltage‐gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well‐tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals. This article is protected by copyright. All rights reserved

  June 15, 2017   doi: 10.1113/JP274693   open full text
• A multiscale computational modelling approach predicts mechanisms of female sex risk in the setting of arousal‐induced arrhythmias.
  Pei‐Chi Yang, Laura L. Perissinotti, Fernando López‐Redondo, Yibo Wang, Kevin R. DeMarco, Mao‐Tsuen Jeng, Igor Vorobyov, Robert D. Harvey, Junko Kurokawa, Sergei Y. Noskov, Colleen E. Clancy.
  The Journal of Physiology. June 14, 2017
  Key points This study represents a first step toward predicting mechanisms of sex‐based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo‐EM structure for the Eag‐family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. Abstract Female sex is a risk factor for inherited and acquired long‐QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long‐QT syndrome patients. We used a combined experimental and computational approach to predict ‘the perfect storm’ of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long‐QT. More specifically, we developed mathematical models of acquired and inherited long‐QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These ‘male’ and ‘female’ model myocytes and tissues then were used to predict how various sex‐based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long‐QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP‐related blockers in the intra‐cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal‐induced arrhythmias.

  June 14, 2017   doi: 10.1113/JP273142   open full text
• Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with post‐traumatic stress disorder.
  Jeanie Park, Paul J. Marvar, Peizhou Liao, Melanie L. Kankam, Seth D. Norrholm, Ryan M. Downey, S. Ashley McCullough, Ngoc‐Anh Le, Barbara O. Rothbaum.
  The Journal of Physiology. June 14, 2017
  Key points Patients with post‐traumatic stress disorder (PTSD) are at a significantly higher risk of developing hypertension and cardiovascular disease. The mechanisms underlying this increased risk are not known. Studies have suggested that PTSD patients have an overactive sympathetic nervous system (SNS) that could contribute to cardiovascular risk; however, sympathetic function has not previously been rigorously evaluated in PTSD patients. Using direct measurements of sympathetic nerve activity and pharmacological manipulation of blood pressure, we show that veterans with PTSD have augmented SNS and haemodynamic reactivity during both combat‐related and non‐combat related mental stress, impaired sympathetic and cardiovagal baroreflex sensitivity, and increased inflammation. Identifying the mechanisms contributing to increased cardiovascular (CV) risk in PTSD will pave the way for developing interventions to improve sympathetic function and reduce CV risk in these patients. Abstract Post‐traumatic stress disorder (PTSD) is associated with increased cardiovascular (CV) risk. We tested the hypothesis that PTSD patients have augmented sympathetic nervous system (SNS) and haemodynamic reactivity during mental stress, as well as impaired arterial baroreflex sensitivity (BRS). Fourteen otherwise healthy Veterans with combat‐related PTSD were compared with 14 matched Controls without PTSD.  Muscle sympathetic nerve activity (MSNA), continuous blood pressure (BP) and electrocardiography were measured at baseline, as well as during two types of mental stress:  combat‐related mental stress using virtual reality combat exposure (VRCE) and non‐combat related stress using mental arithmetic (MA). A cold pressor test (CPT) was administered for comparison. BRS was tested using pharmacological manipulation of BP via the Modified Oxford technique at rest and during VRCE. Blood samples were analysed for inflammatory biomarkers. Baseline characteristics, MSNA and haemodynamics were similar between the groups. In PTSD vs. Controls, MSNA (+8.2 ± 1.0 vs. +1.2 ± 1.3 bursts min–1, P < 0.001) and heart rate responses (+3.2 ± 1.1 vs. −2.3 ± 1.0 beats min–1, P = 0.003) were significantly augmented during VRCE.  Similarly, in PTSD vs. Controls, MSNA (+21.0 ± 2.6 vs. +6.7 ± 1.5 bursts min–1, P < 0.001) and diastolic BP responses (+6.3 ± 1.0 vs. +3.5 ± 1.0 mmHg, P = 0.011) were significantly augmented during MA but not during CPT (P = not significant). In the PTSD group, sympathetic BRS (–1.2 ± 0.2 vs. –2.0 ± 0.3 burst incidence mmHg−1, P = 0.026) and cardiovagal BRS (9.5 ± 1.4 vs. 23.6 ± 4.3 ms mmHg−1, P = 0.008) were significantly blunted at rest. PTSD patients had significantly higher highly sensitive‐C‐reactive protein levels compared to Controls (2.1 ± 0.4 vs. 1.0 ± 0.3 mg L−1, P = 0.047). Augmented SNS and haemodynamic responses to mental stress, blunted BRS and inflammation may contribute to an increased CV risk in PTSD.

  June 14, 2017   doi: 10.1113/JP274269   open full text
• Facilitation of mossy fibre‐driven spiking in the cerebellar nuclei by the synchrony of inhibition.
  Yeechan Wu, Indira M. Raman.
  The Journal of Physiology. June 11, 2017
  Key points Large premotor neurons of the cerebellar nuclei (CbN cells) integrate synaptic inhibition from Purkinje neurons and synaptic excitation from mossy fibres to generate cerebellar output. We find that mossy fibre inputs to CbN cells generate unitary AMPA receptor EPSCs of ∼1 nS that decay in ∼1 ms and mildly voltage‐dependent NMDA receptor EPSCs of ∼0.6 nS that decay in ∼7 ms. A few hundred mossy fibres active at a few tens of spikes s−1 must converge on CbN cells to generate physiological CbN spike rates (∼60 spikes s−1) during convergent inhibition from spontaneously active Purkinje cells. Dynamic clamp studies in cerebellar slices from weanling mice demonstrate that synaptic excitation from mossy fibres becomes more effective at increasing the rate of CbN cell spiking when the coherence (synchrony) of convergent inhibition is increased. Abstract Large projection neurons of the cerebellar nuclei (CbN cells), whose activity generates movement, are inhibited by Purkinje cells and excited by mossy fibres. The high convergence, firing rates and strength of Purkinje inputs predict powerful suppression of CbN cell spiking, raising the question of what activity patterns favour excitation over inhibition. Recording from CbN cells at near‐physiological temperatures in cerebellar slices from weanling mice, we measured the amplitude, kinetics, voltage dependence and short‐term plasticity of mossy fibre‐mediated EPSCs. Unitary EPSCs were small and brief (AMPA receptor, ∼1 nS, ∼1 ms; NMDA receptor, ∼0.6 nS, ∼7 ms) and depressed moderately. Using these experimentally measured parameters, we applied combinations of excitation and inhibition to CbN cells with dynamic clamp. Because Purkinje cells can fire coincident simple spikes during cerebellar behaviours, we varied the proportion (0–20 of 40) and precision (0–4 ms jitter) of synchrony of inhibitory inputs, along with the rates (0–100 spikes s−1) and number (0–800) of excitatory inputs. Even with inhibition constant, when inhibitory synchrony was higher, excitation increased CbN cell firing rates more effectively. Partial inhibitory synchrony also dictated CbN cell spike timing, even with physiological rates of excitation. These effects were present with ≥10 inhibitory inputs active within 2–4 ms of each other. Conversely, spiking was most effectively suppressed when inhibition was maximally asynchronous. Thus, the rate and relative timing of Purkinje‐mediated inhibition set the rate and timing of cerebellar output. The results suggest that increased coherence of Purkinje cell activity can facilitate mossy fibre‐driven spiking by CbN cells, in turn driving movements.

  June 11, 2017   doi: 10.1113/JP274321   open full text
• Large‐conductance Ca2+‐activated K+ channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium.
  Nathan A. Zaidman, Angela Panoskaltsis‐Mortari, Scott M. O'Grady.
  The Journal of Physiology. June 11, 2017
  Key points Hydrocortisone (HC) is required for activation of large‐conductance Ca2+‐activated K+ current (BK) by purinergic receptor agonists. HC reduces insertion of the stress‐regulated exon (STREX) in the KCNMA1 gene, permitting protein kinase C (PKC)‐dependent channel activation. Overlapping and unique purinergic signalling regions exist at the apical border of differentiated surface cells. BK channels localize in the cilia of surface cells. Abstract In the present study we investigated the role of hydrocortisone (HC) on uridine‐5ʹ‐triphosphate (UTP)‐stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. The presence of a UTP‐stimulated, paxilline‐sensitive large‐conductance Ca2+‐activated K+ (BK) current was demonstrated in control epithelia but was not stimulated in epithelia differentiated in the absence of HC (HC0). Addition of the BK channel opener NS11021 directly activated channels in control epithelia; however, under HC0 conditions, activation only occurred when UTP was added after NS11021. The PKC inhibitors GF109203x and Gö6983 blocked BK activation by UTP in control epithelia, suggesting that PKC‐mediated phosphorylation plays a permissive role in purinoceptor‐stimulated BK activation. Moreover, HC0 epithelia expressed significantly more KCNMA1 containing the stress‐regulated exon (STREX), a splice‐variant of the α‐subunit that displays altered channel regulation by phosphorylation, compared to control epithelia. Furthermore, BK channels as well as purinergic receptors were shown to localize in unique and overlapping domains at the apical membrane of ciliated surface cells. These results establish a previously unrecognized role for glucocorticoids in regulation of BK channels in airway epithelial cells.

  June 11, 2017   doi: 10.1113/JP274200   open full text
• Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice.
  Maximilian Kleinert, Benjamin L. Parker, Andreas M. Fritzen, Jonas R. Knudsen, Thomas E. Jensen, Rasmus Kjøbsted, Lykke Sylow, Markus Ruegg, David E. James, Erik A. Richter.
  The Journal of Physiology. June 11, 2017
  Key points Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle. Abstract Exercise increases glucose uptake into insulin‐resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin‐controlled glucose uptake, has been reported to interact with ras‐related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise‐induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running‐induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild‐type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5′ AMP‐activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N‐myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise‐dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion‐transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

  June 11, 2017   doi: 10.1113/JP274203   open full text
• Evolved changes in the intracellular distribution and physiology of muscle mitochondria in high‐altitude native deer mice.
  Sajeni Mahalingam, Grant B. McClelland, Graham R. Scott.
  The Journal of Physiology. June 07, 2017
  Key points Mitochondrial function changes over time at high altitudes, but the potential benefits of these changes for hypoxia resistance remains unclear. We used high‐altitude‐adapted populations of deer mice, which exhibit enhanced aerobic performance in hypoxia, to examine whether changes in mitochondrial physiology or intracellular distribution in the muscle contribute to hypoxia resistance. Permeabilized muscle fibres from the gastrocnemius muscle had higher respiratory capacities in high‐altitude mice than in low‐altitude mice. Highlanders also had higher mitochondrial volume densities, due entirely to an enriched abundance of subsarcolemmal mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. There were several effects of hypoxia acclimation on mitochondrial function, some of which were population specific, but they differed from the evolved changes in high‐altitude natives, which probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes. Abstract High‐altitude natives that have evolved to live in hypoxic environments provide a compelling system to understand how animals can overcome impairments in oxygen availability. We examined whether these include changes in mitochondrial physiology or intracellular distribution that contribute to hypoxia resistance in high‐altitude deer mice (Peromyscus maniculatus). Mice from populations native to high and low altitudes were born and raised in captivity, and as adults were acclimated to normoxia or hypobaric hypoxia (equivalent to 4300 m elevation). We found that highlanders had higher respiratory capacities in the gastrocnemius (but not soleus) muscle than lowlanders (assessed using permeabilized fibres with single or multiple inputs to the electron transport system), due in large part to higher mitochondrial volume densities in the gastrocnemius. The latter was attributed to an increased abundance of subsarcolemmal (but not intermyofibrillar) mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. Hypoxia acclimation had no significant effect on these population differences, but it did increase mitochondrial cristae surface densities of mitochondria in both populations. Hypoxia acclimation also altered the physiology of isolated mitochondria by affecting respiratory capacities and cytochrome c oxidase activities in population‐specific manners. Chronic hypoxia decreased the release of reactive oxygen species by isolated mitochondria in both populations. There were subtle differences in O2 kinetics between populations, with highlanders exhibiting increased mitochondrial O2 affinity or catalytic efficiency in some conditions. Our results suggest that evolved changes in mitochondrial physiology in high‐altitude natives are distinct from the effects of hypoxia acclimation, and probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes.

  June 07, 2017   doi: 10.1113/JP274130   open full text
• Structure and function of human muscle fibres and muscle proteome in physically active older men.
  Lorenza Brocca, Jamie S. McPhee, Emanuela Longa, Monica Canepari, Olivier Seynnes, Giuseppe Vito, Maria Antonietta Pellegrino, Marco Narici, Roberto Bottinelli.
  The Journal of Physiology. June 05, 2017
  Key points Loss of muscle mass and strength in the growing population of elderly people is a major health concern for modern societies. This condition, termed sarcopenia, is a major cause of falls and of the subsequent increase in morbidity and mortality. Despite numerous studies on the impact of ageing on individual muscle fibres, the contribution of single muscle fibre adaptations to ageing‐induced atrophy and functional impairment is still unsettled. The level of physical function and disuse is often associated with ageing. We studied relatively healthy older adults in order to understand the effects of ageing per se without the confounding impact of impaired physical function. We found that in healthy ageing, structural and functional alterations of muscle fibres occur. Protein post‐translational modifications, oxidation and phosphorylation contribute to such alterations more than loss of myosin and other muscle protein content. Abstract Contradictory results have been reported on the impact of ageing on structure and functions of skeletal muscle fibres, likely to be due to a complex interplay between ageing and other phenomena such as disuse and diseases. Here we recruited healthy, physically and socially active young (YO) and elderly (EL) men in order to study ageing per se without the confounding effects of impaired physical function. In vivo analyses of quadriceps and in vitro analyses of vastus lateralis muscle biopsies were performed. In EL subjects, our results show that (i) quadriceps volume, maximum voluntary contraction isometric torque and patellar tendon force were significantly lower; (ii) muscle fibres went through significant atrophy and impairment of specific force (isometric force/cross‐sectional area) and unloaded shortening velocity; (iii) myosin/actin ratio and myosin content in individual muscle fibres were not altered; (iv) the muscle proteome went through quantitative adaptations, namely an up‐regulation of the content of several groups of proteins among which were myofibrillar proteins and antioxidant defence systems; (v) the muscle proteome went through qualitative adaptations, namely phosphorylation of several proteins, including myosin light chain‐2 slow and troponin T and carbonylation of myosin heavy chains. The present results indicate that impairment of individual muscle fibre structure and function is a major feature of ageing per se and that qualitative adaptations of muscle proteome are likely to be more involved than quantitative adaptations in determining such a phenomenon.

  June 05, 2017   doi: 10.1113/JP274148   open full text
• Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol‐induced proliferation of ovine uterine artery endothelial cells.
  Rosalina Villalon Landeros, Sheikh O. Jobe, Gabrielle Aranda‐Pino, Gladys E. Lopez, Jing Zheng, Ronald R. Magness.
  The Journal of Physiology. June 05, 2017
  Key points The catechol metabolites of 17β‐oestradiol (E2β), 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate proliferation of pregnancy‐derived ovine uterine artery endothelial cells (P‐UAECs) through β‐adrenoceptors (β‐ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2‐OHE2‐ and 4‐OHE2‐induced P‐UAEC proliferation, as well as proliferation induced by the parent hormone E2β and other β‐AR signalling hormones (i.e. catecholamines). Conversely, although 2‐OHE2 and 4‐OHE2 rapidly activate phosphatidylinositol 3‐kinase (PI3K), its activation is not involved in catecholoestradiol‐induced P‐UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol‐induced P‐UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2β‐ and catecholamine‐induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. Abstract Previously we demonstrated that the biologically active metabolites of 17β‐oestradiol, 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate pregnancy‐specific proliferation of uterine artery endothelial cells derived from pregnant (P‐UAECs), but not non‐pregnant ewes. However, unlike 17β‐oestradiol, which induces proliferation via oestrogen receptor‐β (ER‐β), the catecholoestradiols mediate P‐UAEC proliferation via β‐adrenoceptors (β‐AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P‐UAECs. P‐UAECs were treated with 2‐OHE2 and 4‐OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3‐kinase (PI3K)–AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist‐induced P‐UAEC proliferation. 2‐OHE2 and 4‐OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol‐induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β‐oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P‐UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol‐, 17β‐oestradiol‐ and catecholamine‐induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.

  June 05, 2017   doi: 10.1113/JP274119   open full text
• The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.
  Paulo W. Pires, Eun‐A. Ko, Harry A.T. Pritchard, Michael Rudokas, Evan Yamasaki, Scott Earley.
  The Journal of Physiology. June 01, 2017
  Key points The angiotensin II receptor type 1b (AT1Rb) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane‐stretch induced stimulation of AT1Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT1Ra or AT1Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT1Rb mRNA is ∼30‐fold higher than AT1Ra in whole cerebral arteries and ∼45‐fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT1Rb for pressure‐induced vasoconstriction. Abstract Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT1R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1R – AT1Ra and AT1Rb – and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1Ra knockout animals compared with controls. In patch‐clamp experiments using native cerebral arterial myocytes, membrane stretch‐induced cation currents were blocked by the TRPM4 inhibitor 9‐phenanthrol in both groups. Further, the AT1R blocker losartan (1 μm) diminished myogenic tone and blocked stretch‐induced cation currents in cerebral arteries from both groups. Activation of AT1R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1Rb mRNA was ∼30‐fold greater than AT1Ra in cerebral arteries, and knockdown of AT1Rb selectively diminished myogenic constriction. We conclude that AT1Rb, acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

  June 01, 2017   doi: 10.1113/JP274310   open full text
• Does the intercept of the heat–stress relation provide an accurate estimate of cardiac activation heat?
  Toan Pham, Kenneth Tran, Kimberley M Mellor, Anthony Hickey, Amelia Power, Marie‐Louise Ward, Andrew Taberner, June‐Chiew Han, Denis Loiselle.
  The Journal of Physiology. June 01, 2017
  Key points The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15–20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. Abstract Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+‐ATPase and Ca2+ translocation by the Na+/Ca2+ exchanger coupled to the Na+,K+‐ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l−1), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat–stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat–force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long‐standing disputes in the literature.

  June 01, 2017   doi: 10.1113/JP274174   open full text
• Genotype‐specific pathogenic effects in human dilated cardiomyopathy.
  Ilse A. E. Bollen, Maike Schuldt, Magdalena Harakalova, Aryan Vink, Folkert W. Asselbergs, Jose R. Pinto, Martina Krüger, Diederik W. D. Kuster, Jolanda Velden.
  The Journal of Physiology. June 01, 2017
  Key points Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+‐sensitivity and reduced length‐dependent activation. TNNT2p.K217del caused increased passive tension. A mutation in the gene encoding Lamin A/C (LMNAp.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy. Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. Abstract Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.

  June 01, 2017   doi: 10.1113/JP274145   open full text
• Light adaptation and the evolution of vertebrate photoreceptors.
  Ala Morshedian, Gordon L. Fain.
  The Journal of Physiology. June 01, 2017
  Key Points Lamprey are cyclostomes, a group of vertebrates that diverged from lines leading to jawed vertebrates (including mammals) in the late Cambrian, 500 million years ago. It may therefore be possible to infer properties of photoreceptors in early vertebrate progenitors by comparing lamprey to other vertebrates. We show that lamprey rods and cones respond to light much like rods and cones in amphibians and mammals. They operate over a similar range of light intensities and adapt to backgrounds and bleaches nearly identically. These correspondences are pervasive and detailed; they argue for the presence of rods and cones very early in the evolution of vertebrates with properties much like those of rods and cones in existing vertebrate species. Abstract The earliest vertebrates were agnathans – fish‐like organisms without jaws, which first appeared near the end of the Cambrian radiation. One group of agnathans became cyclostomes, which include lamprey and hagfish. Other agnathans gave rise to jawed vertebrates or gnathostomes, the group including all other existing vertebrate species. Because cyclostomes diverged from other vertebrates 500 million years ago, it may be possible to infer some of the properties of the retina of early vertebrate progenitors by comparing lamprey to other vertebrates. We have previously shown that rods and cones in lamprey respond to light much like photoreceptors in other vertebrates and have a similar sensitivity. We now show that these affinities are even closer. Both rods and cones adapt to background light and to bleaches in a manner almost identical to other vertebrate photoreceptors. The operating range in darkness is nearly the same in lamprey and in amphibian or mammalian rods and cones; moreover background light shifts response–intensity curves downward and to the right over a similar range of ambient intensities. Rods show increment saturation at about the same intensity as mammalian rods, and cones never saturate. Bleaches decrease sensitivity in part by loss of quantum catch and in part by opsin activation of transduction. These correspondences are so numerous and pervasive that they are unlikely to result from convergent evolution but argue instead that early vertebrate progenitors of both cyclostomes and mammals had photoreceptors much like our own.

  June 01, 2017   doi: 10.1113/JP274211   open full text
• Regionalization of the stretch reflex in the human vastus medialis.
  Alessio Gallina, Jean‐Sébastien Blouin, Tanya D. Ivanova, S. Jayne Garland.
  The Journal of Physiology. June 01, 2017
  Key points Regionalization of the stretch reflex, i.e. the notion that the activation of 1a afferents from a muscle region influences only the activation of motor units in the same region, has been demonstrated previously in animals but not in humans. Mechanical stretches applied to regions of vastus medialis as close as 10 mm apart resulted in recruitment of motor units localized topographically with respect to the location of the mechanical stretch. Stretch reflexes are regionalized in the human vastus medialis. The human spinal cord has the neuromuscular circuitry to preferentially activate motoneurones innervating muscle fibres located in different regions of the vastus medialis. Abstract The localization of motor unit territories provides an anatomical basis to suggest that the CNS may have more independence in motor unit recruitment and control strategies than what was previously thought. In this study, we investigated whether the human spinal cord has the neuromuscular circuitry to independently activate motor units located in different regions of the vastus medialis. Mechanical taps were applied to multiple locations in the vastus medialis (VM) in nine healthy individuals. Regional responses within the muscle were observed using a grid of 5 × 13 surface EMG electrodes. The EMG amplitude was quantified for each channel, and a cluster of channels showing the largest activation was identified. The spatial location of the EMG response was quantified as the position of the channels in the cluster. In a subset of three participants, intramuscular recordings were performed simultaneously with the surface EMG recordings. Mechanical taps resulted in localized, discrete responses for each participant. The spatial location of the elicited responses was dependent on the location of the tap (P < 0.001). Recordings with intramuscular electrodes confirmed the regional activation of the VM for different tap locations. Selective stimulation of 1a afferents localized in a region of the VM results in reflex recruitment of motor units in the same region. These findings suggest that the human spinal cord has the neuromuscular circuitry to modulate spatially the motoneuronal output to vastus medialis regions, which is a neuroanatomical prerequisite for regional activation.

  June 01, 2017   doi: 10.1113/JP274458   open full text
• Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.
  Sergej Pirkmajer, Henriette Kirchner, Leonidas S. Lundell, Pavel V. Zelenin, Juleen R. Zierath, Kira S. Makarova, Yuri I. Wolf, Alexander V. Chibalin.
  The Journal of Physiology. May 29, 2017
  Key points Small transmembrane proteins such as FXYDs, which interact with Na+,K+‐ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+‐ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny‐based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Abstract Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1–12), which regulate Na+,K+‐ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence‐based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

  May 29, 2017   doi: 10.1113/JP274254   open full text
• Long‐term plasticity of corticostriatal synapses is modulated by pathway‐specific co‐release of opioids through κ‐opioid receptors.
  Sarah L. Hawes, Armando G. Salinas, David M. Lovinger, Kim T. Blackwell.
  The Journal of Physiology. May 26, 2017
  Key points Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal‐directed actions, and can also modify long‐term plasticity of corticostriatal synapses. Striatal projection neurons of the direct pathway co‐release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ‐opioid receptors. Theta‐burst stimulation of corticostriatal fibres produces long‐term potentiation (LTP) in striatal projection neurons when measured using whole‐cell patch recording. Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release. Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse. Abstract Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction‐related striatal plasticity. Though the role of opioid receptors in long‐term depression in striatum has been characterized, their effect on long‐term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor‐containing; D1R‐) spiny projection neurons (SPNs) co‐release the opioid neuropeptide dynorphin, which acts at presynaptic κ‐opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co‐released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R‐SPNs in brain slice while using whole‐cell patch recording to measure changes in the synaptic response of SPNs following theta‐burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R‐SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R‐SPNs reduces stimulation‐evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R‐SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R‐SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation.

  May 26, 2017   doi: 10.1113/JP274190   open full text
• Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.
  Mitsue Takeya, Hikaru Hashitani, Tokumasa Hayashi, Ryuhei Higashi, Kei‐ichiro Nakamura, Makoto Takano.
  The Journal of Physiology. May 25, 2017
  Key points The mucosa may have neuron‐like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco‐endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco‐endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. Abstract The role of the mucosa in generating the spontaneous activity of guinea‐pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi‐fluorescence Ca2+ imaging, respectively. Mucosa‐intact but not mucosa‐denuded SV preparations generated TTX‐ (1 μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μm). Consistently, SMCs developed mucosa‐dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μm), a sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor. DIDS (300 μm) and niflumic acid (100 μm), blockers for Ca2+‐activated Cl− channels (CACCs), or low Cl− solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L‐type voltage‐dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco‐endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

  May 25, 2017   doi: 10.1113/JP273872   open full text
• α‐Linolenic acid and exercise training independently, and additively, decrease blood pressure and prevent diastolic dysfunction in obese Zucker rats.
  Pierre‐Andre Barbeau, Tanya M. Holloway, Jamie Whitfield, Brittany L. Baechler, Joe Quadrilatero, Luc J. C. Loon, Adrian Chabowski, Graham P. Holloway.
  The Journal of Physiology. May 24, 2017
  Key points α‐linolenic acid (ALA) and exercise training both attenuate hyperlipidaemia‐related cardiovascular derangements, however, there is a paucity of information pertaining to their mechanisms of action when combined. We investigated both the independent and combined effects of exercise training and ALA consumption in obese Zucker rats, aiming to determine the potential for additive improvements in cardiovascular function. ALA and exercise training independently improved cardiac output, end‐diastolic volume, left ventricular fibrosis and mean blood pressure following a 4 week intervention. Combining ALA and endurance exercise yielded greater improvements in these parameters, independent of changes in markers of oxidative stress or endogenous anti‐oxidants. We postulate that divergent mechanisms of action may explain these changes: ALA increases peripheral vasodilation, and exercise training stimulates angiogenesis. Abstract Although α‐linolenic acid (ALA) and endurance exercise training independently attenuate hyperlipidaemia‐related cardiovascular derangements, there is a paucity of information pertaining to their mechanisms of action and efficacy when combined as a preventative therapeutic approach. Therefore, we used obese Zucker rats to investigate the independent and combined effects of these interventions on cardiovascular disease. Specifically, animals were randomly assigned to one of the following groups: control diet‐sedentary, ALA supplemented‐sedentary, control diet‐exercise trained or ALA supplemented‐exercise trained. Following a 4 week intervention, although the independent and combined effects of ALA and exercise reduced (P < 0.05) the serum free/esterified cholesterol ratio, only the ALA supplemented‐exercise trained animals displayed a reduction in the content of both serum free and esterified cholesterol. Moreover, although ALA and endurance training individually increased cardiac output, stroke volume and end‐diastolic volume, as well as reduced left ventricle fibrosis, mean blood pressure and total peripheral resistance, these responses were all greater following the combined intervention (ALA supplemented‐exercise trained). These effects occurred independent of changes in oxidative phosphorylation proteins, markers of oxidative stress or endogenous anti‐oxidant capacity. We propose that the beneficial effects of a combined intervention occur as a result of divergent mechanisms of action elicited by ALA and endurance exercise because only exercise training increased the capillary content in the left ventricle and skeletal muscle, and tended to decrease protein carbonylation in the left ventricle (P = 0.06). Taken together, our data indicate that combining ALA and endurance exercise provides additional improvements in cardiovascular disease risk reduction compared to singular interventions in the obese Zucker rat.

  May 24, 2017   doi: 10.1113/JP274036   open full text
• Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC‐A.
  Sam El‐Ajouz, Elisa Venturi, Katja Witschas, Matthew Beech, Abigail D. Wilson, Chris Lindsay, David Eberhardt, Fiona O'Brien, Tsunaki Iida, Miyuki Nishi, Hiroshi Takeshima, Rebecca Sitsapesan.
  The Journal of Physiology. May 23, 2017
  Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1). We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+. In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice. Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. Abstract The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.

  May 23, 2017   doi: 10.1113/JP273550   open full text
• Increased transient Na+ conductance and action potential output in layer 2/3 prefrontal cortex neurons of the fmr1−/y mouse.
  Brandy N. Routh, Rahul K. Rathour, Michael E. Baumgardner, Brian E. Kalmbach, Daniel Johnston, Darrin H. Brager.
  The Journal of Physiology. May 23, 2017
  Key points Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1−/y mice. In fmr1−/y L2/3 neurons, action potentials are taller, faster and narrower. Outside‐out patch clamp recordings revealed that the maximum Na+ conductance density is higher in fmr1−/y L2/3 neurons. Measurements of three biophysically distinct K+ currents revealed a depolarizing shift in the activation of a rapidly inactivating (A‐type) K+ conductance. Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype. Abstract Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1−/y mouse is significantly altered due to changes in several voltage‐gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole‐cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1−/y mouse fired more action potentials for a given stimulus compared with wild‐type neurons. In addition, action potentials in fmr1−/y neurons were significantly larger, faster and narrower. Voltage clamp of outside‐out patches from L2/3 neurons revealed that the transient Na+ current was significantly larger in fmr1−/y neurons. Furthermore, the activation curve of somatic A‐type K+ current was depolarized. Realistic conductance‐based simulations revealed that these biophysical changes in Na+ and K+ channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1−/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome.

  May 23, 2017   doi: 10.1113/JP274258   open full text
• Exercise training reverses age‐induced diastolic dysfunction and restores coronary microvascular function.
  Kazuki Hotta, Bei Chen, Bradley J. Behnke, Payal Ghosh, John N. Stabley, Jeremy A. Bramy, Jaime L. Sepulveda, Michael D. Delp, Judy M. Muller‐Delp.
  The Journal of Physiology. May 23, 2017
  Key points In a rat model of ageing that is free of atherosclerosis or hypertension, E/A, a diagnostic measure of diastolic filling, decreases, and isovolumic relaxation time increases, indicating that both active and passive ventricular relaxation are impaired with advancing age. Resting coronary blood flow and coronary functional hyperaemia are reduced with age, and endothelium‐dependent vasodilatation declines with age in coronary resistance arterioles. Exercise training reverses age‐induced declines in diastolic and coronary microvascular function. Thus, microvascular dysfunction and inadequate coronary perfusion are likely mechanisms of diastolic dysfunction in aged rats. Exercise training, initiated at an advanced age, reverses age‐related diastolic and microvascular dysfunction; these data suggest that late‐life exercise training can be implemented to improve coronary perfusion and diastolic function in the elderly. Abstract The risk for diastolic dysfunction increases with advancing age. Regular exercise training ameliorates age‐related diastolic dysfunction; however, the underlying mechanisms have not been identified. We investigated whether (1) microvascular dysfunction contributes to the development of age‐related diastolic dysfunction, and (2) initiation of late‐life exercise training reverses age‐related diastolic and microvascular dysfunction. Young and old rats underwent 10 weeks of exercise training or remained as sedentary, cage‐controls. Isovolumic relaxation time (IVRT), early diastolic filling (E/A), myocardial performance index (MPI) and aortic stiffness (pulse wave velocity; PWV) were evaluated before and after exercise training or cage confinement. Coronary blood flow and vasodilatory responses of coronary arterioles were evaluated in all groups at the end of training. In aged sedentary rats, compared to young sedentary rats, a 42% increase in IVRT, a 64% decrease in E/A, and increased aortic stiffness (PWV: 6.36 ± 0.47 vs.4.89 ± 0.41, OSED vs. YSED, P < 0.05) was accompanied by impaired coronary blood flow at rest and during exercise. Endothelium‐dependent vasodilatation was impaired in coronary arterioles from aged rats (maximal relaxation to bradykinin: 56.4 ± 5.1% vs. 75.3 ± 5.2%, OSED vs. YSED, P < 0.05). After exercise training, IVRT, a measure of active ventricular relaxation, did not differ between old and young rats. In old rats, exercise training reversed the reduction in E/A, reduced aortic stiffness, and eliminated impairment of coronary blood flow responses and endothelium‐dependent vasodilatation. Thus, age‐related diastolic and microvascular dysfunction are reversed by late‐life exercise training. The restorative effect of exercise training on coronary microvascular function may result from improved endothelial function.

  May 23, 2017   doi: 10.1113/JP274172   open full text
• Beneficial effects of leptin treatment in a setting of cardiac dysfunction induced by transverse aortic constriction in mouse.
  Nieves Gómez‐Hurtado, Alejandro Domínguez‐Rodríguez, Philippe Mateo, María Fernández‐Velasco, Almudena Val‐Blasco, Rafael Aizpún, Jessica Sabourin, Ana María Gómez, Jean‐Pierre Benitah, Carmen Delgado.
  The Journal of Physiology. May 23, 2017
  Key points Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non‐hypertensive dose of leptin on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters. Abstract Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg–1 day–1) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin‐treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+/calmodulin‐dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.

  May 23, 2017   doi: 10.1113/JP274030   open full text
• An increased extrasynaptic NMDA tone inhibits A‐type K+ current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.
  Meng Zhang, Vinicia C. Biancardi, Javier E. Stern.
  The Journal of Physiology. May 23, 2017
  Key points A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR–IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA, contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats. Abstract We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR–IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch‐clamp electrophysiology and real‐time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1‐NR2A–D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.

  May 23, 2017   doi: 10.1113/JP274327   open full text
• Hippocampal electrical stimulation disrupts associative learning when targeted at dentate spikes.
  Miriam S. Nokia, Irina Gureviciene, Tomi Waselius, Heikki Tanila, Markku Penttonen.
  The Journal of Physiology. May 23, 2017
  Key points Dentate spikes are fast fluctuations of hilar local‐field potentials that take place during rest and are thought to reflect input arriving from the entorhinal cortex to the hippocampus. During dentate spikes, neuronal firing in hippocampal input (dentate gyrus) and output (CA1/CA3) regions is uncoupled. To date, the behavioural significance of dentate spikes is unknown. Here, we provide evidence that disrupting the dentate spike‐related uncoupling of the dentate gyrus and the CA1/CA3 subregions for 1 h after training retards associative learning. We suggest dentate spikes play a significant role in memory consolidation. Abstract Hippocampal electrophysiological oscillations, namely theta and ripples, have been implicated in encoding and consolidation of new memories, respectively. According to existing literature, hippocampal dentate spikes are prominent, short‐duration (<30 ms), large‐amplitude (∼2–4 mV) fluctuations in hilar local‐field potentials that take place during awake immobility and sleep. Interestingly, previous studies indicate that during dentate spikes dentate gyrus granule cells increase their firing while firing of CA1 pyramidal cells are suppressed, thus resulting in momentary uncoupling of the two hippocampal subregions. To date, the behavioural significance of dentate spikes is unknown. Here, to study the possible role of dentate spikes in learning, we trained adult male Sprague–Dawley rats in trace eyeblink classical conditioning. For 1 h immediately following each conditioning session, one group of animals received hippocampal stimulation via the ventral hippocampal commissure (vHC) contingent on dentate spikes to disrupt the uncoupling between the dentate gyrus and the CA1 subregions. A yoked control group was stimulated during immobility, irrespective of brain state, and another control group was not stimulated at all. As a result, learning was impaired only in the group where vHC stimulation was administered contingent on dentate spikes. Our results suggest dentate spikes and/or the associated uncoupling of the dentate gyrus and the CA1 play a significant role in memory consolidation. Dentate spikes could possibly reflect reactivation and refinement of a memory trace within the dentate gyrus triggered by input from the entorhinal cortex.

  May 23, 2017   doi: 10.1113/JP274023   open full text
• Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.
  Abigail L. Mackey, Mélanie Magnan, Bénédicte Chazaud, Michael Kjaer.
  The Journal of Physiology. May 23, 2017
  Key points Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross‐talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell‐culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell–cell cross‐talk during physiological and pathological muscle remodelling. Abstract Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross‐sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co‐cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross‐sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

  May 23, 2017   doi: 10.1113/JP273997   open full text
• Mechanical tuning and amplification within the apex of the guinea pig cochlea.
  Alberto Recio‐Spinoso, John S. Oghalai.
  The Journal of Physiology. May 21, 2017
  Key points A popular conception of mammalian cochlear physiology is that tuned mechanical vibration of the basilar membrane defines the frequency response of the innervating auditory nerve fibres However, the data supporting these concepts come from vibratory measurements at cochlear locations tuned to high frequencies (>7 kHz). Here, we measured the travelling wave in regions of the guinea pig cochlea that respond to low frequencies (<2 kHz) and found that mechanical tuning was broad and did not match auditory nerve tuning characteristics. Non‐linear amplification of the travelling wave functioned over a broad frequency range and did not substantially sharpen frequency tuning. Thus, the neural encoding of low‐frequency sounds, which includes most of the information conveyed by human speech, is not principally determined by basilar membrane mechanics. Abstract The popular notion of mammalian cochlear function is that auditory nerves are tuned to respond best to different sound frequencies because basilar membrane vibration is mechanically tuned to different frequencies along its length. However, this concept has only been demonstrated in regions of the cochlea tuned to frequencies >7 kHz, not in regions sensitive to lower frequencies where human speech is encoded. Here, we overcame historical technical limitations and non‐invasively measured sound‐induced vibrations at four locations distributed over the apical two turns of the guinea pig cochlea. In turn 3, the responses demonstrated low‐pass filter characteristics. In turn 2, the responses were low‐pass‐like, in that they occasionally did have a slight peak near the corner frequency. The corner frequencies of the responses were tonotopically tuned and ranged from 384 to 668 Hz. Non‐linear gain, or amplification of the vibrations in response to low‐intensity stimuli, was found both below and above the corner frequencies. Post mortem, cochlear gain disappeared. The non‐linear gain was typically 10–30 dB and was broad‐band rather than sharply tuned. However, the gain did reach nearly 50 dB in turn 2 for higher stimulus frequencies, nearly the amount of gain found in basal cochlear regions. Thus, our data prove that mechanical responses do not match neural responses and that cochlear amplification does not appreciably sharpen frequency tuning for cochlear regions that respond to frequencies <2 kHz. These data indicate that the non‐linear processing of sound performed by the guinea pig cochlea varies substantially between the cochlear apex and base.

  May 21, 2017   doi: 10.1113/JP273881   open full text
• Compensatory and decompensatory alterations in cardiomyocyte Ca2+ dynamics in hearts with diastolic dysfunction following aortic banding.
  Sara Gattoni, Åsmund Treu Røe, Jan Magnus Aronsen, Ivar Sjaastad, William E. Louch, Nicolas P. Smith, Steven A. Niederer.
  The Journal of Physiology. May 21, 2017
  Key points At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics. Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality. Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic‐banded rats exhibiting diastolic dysfunction. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively. Results show that the ability to dynamically change systolic Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction. Abstract Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic‐banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel (LCC) and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) as the principal regulators of systolic and diastolic Ca2+, respectively. In the aortic banding model, the sensitivity of systolic Ca2+ to LCC density and diastolic Ca2+ to SERCA density decreased by 16‐fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca2+ is moderately increased.

  May 21, 2017   doi: 10.1113/JP273879   open full text
• Maternal nutrient restriction during pregnancy and lactation leads to impaired right ventricular function in young adult baboons.
  Anderson H. Kuo, Cun Li, Hillary F. Huber, Matthias Schwab, Peter W. Nathanielsz, Geoffrey D. Clarke.
  The Journal of Physiology. May 18, 2017
  Key points Maternal nutrient restriction induces intrauterine growth restriction (IUGR) and leads to heightened cardiovascular risks later in life. We report right ventricular (RV) filling and ejection abnormalities in IUGR young adult baboons using cardiac magnetic resonance imaging. Both functional and morphological indicators of poor RV function were seen, many of which were similar to effects of ageing, but also with a few key differences. We observed more pronounced RV changes compared to our previous report of the left ventricle, suggesting there is likely to be a component of isolated RV abnormality in addition to expected haemodynamic sequelae from left ventricular dysfunction. In particular, our findings raise the suspicion of pulmonary hypertension after IUGR. This study establishes that IUGR also leads to impairment of the right ventricle in addition to the left ventricle classically studied. Abstract Maternal nutrient restriction induces intrauterine growth restriction (IUGR), increasing later life chronic disease including cardiovascular dysfunction. Our left ventricular (LV) CMRI studies in IUGR baboons (8 M, 8 F, 5.7 years – human equivalent approximately 25 years), control offspring (8 M, 8 F, 5.6 years), and normal elderly (OLD) baboons (6 M, 6 F, mean 15.9 years) revealed long‐term LV abnormalities in IUGR offspring. Although it is known that right ventricular (RV) function is dependent on LV health, the IUGR right ventricle remains poorly studied. We examined the right ventricle with cardiac magnetic resonance imaging in the same cohorts. We observed decreased ejection fraction (49 ± 2 vs. 33 ± 3%, P < 0.001), cardiac index (2.73 ± 0.27 vs. 1.89 ± 0.20 l min−1 m−2, P < 0.05), early filling rate/body surface area (BSA) (109.2 ± 7.8 vs. 44.6 ± 7.3 ml s−1 m−2, P < 0.001), wall thickening (61 ± 3 vs. 44 ± 5%, P < 0.05), and longitudinal shortening (26 ± 3 vs. 15 ± 2%, P < 0.01) in IUGR animals with increased chamber volumes. Many, but not all, of these changes share similarities to normal older animals. Our findings suggest IUGR‐induced pulmonary hypertension should be further investigated and that atrial volume, pulmonic outflow and interventricular septal motion may provide valuable insights into IUGR cardiovascular physiology. Overall, our findings reaffirm that gestational and neonatal challenges can result in long‐term programming of poor offspring cardiovascular health. To our knowledge, this is the first study reporting IUGR‐induced programmed adult RV dysfunction in an experimental primate model.

  May 18, 2017   doi: 10.1113/JP273928   open full text
• Phosphatidylinositol 4,5‐bisphosphate (PIP2) modulates afterhyperpolarizations in oxytocin neurons of the supraoptic nucleus.
  Matthew K. Kirchner, Robert C. Foehring, Lie Wang, Giri Kumar Chandaka, Joseph C. Callaway, William E. Armstrong.
  The Journal of Physiology. May 15, 2017
  Key points Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2’s control of these AHPs. We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+]i, and whole cell Ca2+ currents. Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3‐triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2. PIP2 depletion reduced spike‐evoked Ca2+ entry and voltage‐gated Ca2+ currents. PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking. Abstract Oxytocin (OT)‐ and vasopressin (VP)‐secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium‐dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5‐bisphosphate (PIP2) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP) of OT, but not VP neurons with high affinity. We further tested this by introducing a water‐soluble PIP2 analogue (diC8‐PIP2) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP. Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐triggered Ca2+‐store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+. Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage‐gated Ca2+ channels opened during spike trains.

  May 15, 2017   doi: 10.1113/JP274219   open full text
• Refuting the myth of non‐response to exercise training: ‘non‐responders’ do respond to higher dose of training.
  David Montero, Carsten Lundby.
  The Journal of Physiology. May 14, 2017
  Key points The prevalence of cardiorespiratory fitness (CRF) non‐response gradually declines in healthy individuals exercising 60, 120, 180, 240 or 300 min per week for 6 weeks. Following a successive identical 6‐week training period but comprising 120 min of additional exercise per week, CRF non‐response is universally abolished. The magnitude of CRF improvement is primarily attributed to changes in haemoglobin mass. The potential for CRF improvement may be present and unveiled with appropriate exercise training stimuli in healthy individuals without exception. Abstract One in five adults following physical activity guidelines are reported to not demonstrate any improvement in cardiorespiratory fitness (CRF). Herein, we sought to establish whether CRF non‐response to exercise training is dose‐dependent, using a between‐ and within‐subject study design. Seventy‐eight healthy adults were divided into five groups (1–5) respectively comprising one, two, three, four and five 60 min exercise sessions per week but otherwise following an identical 6‐week endurance training (ET) programme. Non‐response was defined as any change in CRF, determined by maximal incremental exercise power output (Wmax), within the typical error of measurement (±3.96%). Participants classified as non‐responders after the ET intervention completed a successive 6‐week ET period including two additional exercise sessions per week. Maximal oxygen consumption (V̇O2 max ), haematology and muscle biopsies were assessed prior to and after each ET period. After the first ET period, Wmax increased (P < 0.05) in groups 2, 3, 4 and 5, but not 1. In groups 1, 2, 3, 4 and 5, 69%, 40%, 29%, 0% and 0% of individuals, respectively, were non‐responders. After the second ET period, non‐response was eliminated in all individuals. The change in V̇O2 max with exercise training independently determined Wmax response (partial correlation coefficient, rpartial ≥ 0.74, P < 0.001). In turn, total haemoglobin mass was the strongest independent determinant of V̇O2 max (rpartial = 0.49, P < 0.001). In conclusion, individual CRF non‐response to exercise training is abolished by increasing the dose of exercise and primarily a function of haematological adaptations in oxygen‐carrying capacity.

  May 14, 2017   doi: 10.1113/JP273480   open full text
• When size matters: transient receptor potential vanilloid 4 channel as a volume‐sensor rather than an osmo‐sensor.
  Trine L. Toft‐Bertelsen, David Križaj, Nanna MacAulay.
  The Journal of Physiology. May 14, 2017
  Key points Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume‐sensing ion channel irrespective of the origin of the cell swelling. Abstract Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non‐selective cation channel permeable to Ca2+, it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+‐dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4–AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume‐sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co‐expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4‐mediated transmembrane currents. Similar TRPV4 activation was induced by co‐expression of a cognate channel, AQP1. The level of osmotically‐induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co‐expressed water‐translocating Na+/K+/2Cl− cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume‐dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume‐sensor, rather than as an osmo‐sensor.

  May 14, 2017   doi: 10.1113/JP274135   open full text
• Sensitivity to ischaemia of single sympathetic nerve fibres innervating the dorsum of the human foot.
  W. J. Z'Graggen, R. Solà, N. E. Graf, J. Serra, H. Bostock.
  The Journal of Physiology. May 14, 2017
  Key points Changes in nerve conduction velocity following an impulse (i.e. velocity recovery cycles) reflect after‐potentials, and can provide an indication of altered nerve membrane properties. This study used microneurography to assess the effects of ischaemia on single human sympathetic fibres innervating the dorsum of the foot. It was found that velocity recovery cycles can distinguish whether a sympathetic nerve fibre is depolarized or not. The method may be used to detect membrane depolarization of sympathetic nerve fibres in human patients when autonomic neuropathy is suspected. Abstract The aim of this study was to determine whether velocity recovery cycles (VRCs) could detect the effects of ischaemia on sympathetic nerve fibres. VRCs of human sympathetic nerve fibres of the superficial peroneal nerve innervating the dorsum of the foot were recorded by microneurography in seven healthy volunteers. Sympathetic nerve fibres were identified by studying their response to manoeuvres increasing sympathetic outflow and by measuring activity‐dependent slowing at 2 Hz stimulation. VRCs were assessed at rest, during 30 min of induced limb ischaemia and during 20 min of recovery after ischaemia. From each VRC was measured the relative refractory period (RRP), the supernormality and the time to peak supernormality (SN@). During ischaemia, RRP increased from the baseline value of 37.4 ± 8.7 ms (mean ± SEM) to 67.1 ± 12.1 ms (P < 0.01) and SN@ increased from 68.6 ± 9.8 ms to 133.8 ± 11.0 ms (P < 0.005). The difference between SN@ and RRP separated ischaemic from non‐ischaemic sympathetic nerve fibres. It is concluded that these sympathetic nerve fibres are sensitive to ischaemia, and that VRCs provide a method to study changes of axonal membrane potential of human sympathetic nerve fibres in vivo.

  May 14, 2017   doi: 10.1113/JP274324   open full text
• The impact of age and frailty on ventricular structure and function in C57BL/6J mice.
  H. A. Feridooni, A. E. Kane, O. Ayaz, A. Boroumandi, N. Polidovitch, R. G. Tsushima, R. A. Rose, S. E. Howlett.
  The Journal of Physiology. May 14, 2017
  Key points Heart size increases with age (called hypertrophy), and its ability to contract declines. However, these reflect average changes that may not be present, or present to the same extent, in all older individuals. That aging happens at different rates is well accepted clinically. People who are aging rapidly are frail and frailty is measured with a ‘frailty index’. We quantified frailty with a validated mouse frailty index tool and evaluated the impacts of age and frailty on cardiac hypertrophy and contractile dysfunction. Hypertrophy increased with age, while contractions, calcium currents and calcium transients declined; these changes were graded by frailty scores. Overall health status, quantified as frailty, may promote maladaptive changes associated with cardiac aging and facilitate the development of diseases such as heart failure. To understand age‐related changes in heart structure and function, it is essential to know both chronological age and the health status of the animal. Abstract On average, cardiac hypertrophy and contractile dysfunction increase with age. Still, individuals age at different rates and their health status varies from fit to frail. We investigated the influence of frailty on age‐dependent ventricular remodelling. Frailty was quantified as deficit accumulation in adult (≈7 months) and aged (≈27 months) C57BL/6J mice by adapting a validated frailty index (FI) tool. Hypertrophy and contractile function were evaluated in Langendorff‐perfused hearts; cellular correlates/mechanisms were investigated in ventricular myocytes. FI scores increased with age. Mean cardiac hypertrophy increased with age, but values in the adult and aged groups overlapped. When plotted as a function of frailty, hypertrophy was graded by FI score (r = 0.67–0.55, P < 0.0003). Myocyte area also correlated positively with FI (r = 0.34, P = 0.03). Left ventricular developed pressure (LVDP) plus rates of pressure development (+dP/dt) and decay (−dP/dt) declined with age and this was graded by frailty (r = −0.51, P = 0.0007; r = −0.48, P = 0.002; r = −0.56, P = 0.0002 for LVDP, +dP/dt and −dP/dt). Smaller, slower contractions graded by FI score were also seen in ventricular myocytes. Contractile dysfunction in cardiomyocytes isolated from frail mice was attributable to parallel changes in underlying Ca2+ transients. These changes were not due to reduced sarcoplasmic reticulum stores, but were graded by smaller Ca2+ currents (r = −0.40, P = 0.008), lower gain (r = −0.37, P = 0.02) and reduced expression of Cav1.2 protein (r = −0.68, P = 0.003). These results show that cardiac hypertrophy and contractile dysfunction in naturally aging mice are graded by overall health and suggest that frailty, in addition to chronological age, can help explain heterogeneity in cardiac aging.

  May 14, 2017   doi: 10.1113/JP274134   open full text
• Endogenous nitric oxide formation in cardiac myocytes does not control respiration during β‐adrenergic stimulation.
  Michael Kohlhaas, Alexander G. Nickel, Stefanie Bergem, Barbara Casadei, Ulrich Laufs, Christoph Maack.
  The Journal of Physiology. May 14, 2017
  Key points In the heart, endothelial nitric oxide (NO) controls oxygen consumption in the working heart through paracrine mechanisms. While cardiac myocytes contain several isoforms of NO synthases, it is unclear whether these can control respiration in an intracrine fashion. A long‐standing controversy is whether a NOS exists within mitochondria. By combining fluorescence technologies with electrical field stimulation or the patch‐clamp technique in beating cardiac myocytes, we identified a neuronal NO synthase (nNOS) as the most relevant source of intracellular NO during β‐adrenergic stimulation, while no evidence for a mitochondria‐located NOS was obtained. The amounts of NO produced by non‐mitochondrial nNOS were insufficient to regulate respiration during β‐adrenergic stimulation, arguing against intracrine control of respiration by NO within cardiac myocytes. Abstract Endothelial nitric oxide (NO) controls cardiac oxygen (O2) consumption in a paracrine way by slowing respiration at the mitochondrial electron transport chain. While NO synthases (NOSs) are also expressed in cardiac myocytes, it is unclear whether they control respiration in an intracrine way. Furthermore, the existence of a mitochondrial NOS is controversial. Here, by combining fluorescence imaging with electrical field stimulation, the patch‐clamp method and knock‐out technology, we determined the sources and consequences of intracellular NO formation during workload transitions in isolated murine and guinea pig cardiac myocytes and mitochondria. Using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF) as a fluorescent NO‐sensor that locates to the cytosol and mitochondria, we observed that NO increased by ∼12% within 3 min of β‐adrenergic stimulation in beating cardiac myocytes. This NO stems from neuronal NOS (nNOS), but not endothelial (eNOS). After patch clamp‐mediated dialysis of cytosolic DAF, the remaining NO signals (mostly mitochondrial) were blocked by nNOS deletion, but not by inhibiting the mitochondrial Ca2+ uniporter with Ru360. While in isolated mitochondria exogenous NO inhibited respiration and reduced the NAD(P)H redox state, pyridine nucleotide redox states were unaffected by pharmacological or genetic disruption of endogenous nNOS or eNOS during workload transitions in cardiac myoctyes. We conclude that under physiological conditions, nNOS is the most relevant source for NO in cardiac myocytes, but this nNOS is not located in mitochondria and does not control respiration. Therefore, cardiac O2 consumption is controlled by endothelial NO in a paracrine, but not intracrine, fashion.

  May 14, 2017   doi: 10.1113/JP273750   open full text
• Vasopressin casts light on the suprachiasmatic nucleus.
  Takahiro Tsuji, Andrew J. Allchorne, Meng Zhang, Chiharu Tsuji, Vicky A. Tobin, Rafael Pineda, Androniki Raftogianni, Javier E. Stern, Valery Grinevich, Gareth Leng, Mike Ludwig.
  The Journal of Physiology. May 14, 2017
  Key points A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light‐induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo‐entrainment of biological rhythms. Abstract In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light‐evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c‐fos in the SCN, which is involved in photic entrainment of circadian rhythms.

  May 14, 2017   doi: 10.1113/JP274025   open full text
• Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial‐specific Na+/Ca2+ exchange knockout mice.
  Angelo G. Torrente, Rui Zhang, Heidi Wang, Audrey Zaini, Brian Kim, Xin Yue, Kenneth D. Philipson, Joshua I. Goldhaber.
  The Journal of Physiology. May 13, 2017
  Key points Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+. Abstract Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q‐PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+‐sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial‐specific Na+/Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+‐dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+. We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.

  May 13, 2017   doi: 10.1113/JP274249   open full text
• Calcium‐activated BKCa channels govern dynamic membrane depolarizations of horizontal cells in rodent retina.
  Xiaoping Sun, Arlene A. Hirano, Nicholas C. Brecha, Steven Barnes.
  The Journal of Physiology. May 13, 2017
  Key points Large conductance, Ca2+‐activated K+ (BKCa) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells. BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 mm K+, single channels had a conductance close to 250 pS, within the range of all known BKCa channels. In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells. Abstract Large conductance, calcium‐activated potassium (BKCa) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between −30 and +60 mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole‐cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100 nm), iberiotoxin (10 μm), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30 μm). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS (n = 17; symmetrical 150 mm K+) with open probability being both voltage‐ and Ca2+‐dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells.

  May 13, 2017   doi: 10.1113/JP274132   open full text
• Non‐muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.
  Wenwu Zhang, Susan J. Gunst.
  The Journal of Physiology. May 08, 2017
  Key points Non‐muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non‐phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh‐induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. Abstract The molecular function of non‐muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non‐phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh‐induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross‐bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA‐mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross‐bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

  May 08, 2017   doi: 10.1113/JP273906   open full text
• Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature.
  M. Nocella, G. Cecchi, B. Colombini.
  The Journal of Physiology. May 08, 2017
  Key points Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi] in the myoplasm. High [Pi] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low‐ or no‐force state and show that fibre length changes perturb the Pi‐sensitive force generation of the crossbridge cycle. Abstract Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi] in the myoplasm. Experiments in skinned fibres at low temperature (10–12°C) have shown that [Pi] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.

  May 08, 2017   doi: 10.1113/JP273672   open full text
• Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.
  Erin V. McGillick, Sandra Orgeig, Beth J. Allison, Kirsty L. Brain, Youguo Niu, Nozomi Itani, Katie L. Skeffington, Andrew D. Kane, Emilio A. Herrera, Dino A. Giussani, Janna L. Morrison.
  The Journal of Physiology. May 07, 2017
  Key points Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air‐breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. Abstract Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2) or hypoxia (10% O2) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT‐PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti‐oxidant marker CAT and decreased expression of the pro‐oxidant marker NOX‐4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF‐3α, KDM3A, SLC2A1, EGLN‐3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B‐1, HSD11B‐2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1‐B, ATP1‐A1, ATP1‐B1) and water (AQP‐4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP‐B, SFTP‐D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth.

  May 07, 2017   doi: 10.1113/JP273842   open full text
• Both standing and postural threat decrease Achilles’ tendon reflex inhibition from tendon electrical stimulation.
  Brian C. Horslen, J. Timothy Inglis, Jean‐Sébastien Blouin, Mark G. Carpenter.
  The Journal of Physiology. May 04, 2017
  Key points Golgi tendon organs (GTOs) and associated Ib reflexes contribute to standing balance, but the potential impacts of threats to standing balance on Ib reflexes are unknown. Tendon electrical stimulation to the Achilles’ tendon was used to probe changes in Ib inhibition in medial gastrocnemius with postural orientation (lying prone vs. upright standing; experiment 1) and height‐induced postural threat (standing at low and high surface heights; experiment 2). Ib inhibition was reduced while participants stood upright, compared to lying prone (42.2%); and further reduced when standing in the high, compared to low, threat condition (32.4%). These experiments will impact future research because they demonstrate that tendon electrical stimulation can be used to probe Ib reflexes in muscles engaged in standing balance. These results provide novel evidence that human short‐latency GTO‐Ib reflexes are dependent upon both task, as evidenced by changes with postural orientation, and context, such as height‐induced postural threat during standing. Abstract Golgi tendon organ Ib reflexes are thought to contribute to standing balance control, but it is unknown if they are modulated when people are exposed to a postural threat. We used a novel application of tendon electrical stimulation (TStim) to elicit Ib inhibitory reflexes in the medial gastrocnemius, while actively engaged in upright standing balance, to examine (a) how Ib reflexes to TStim are influenced by upright stance, and (b) the effects of height‐induced postural threat on Ib reflexes during standing. TStim evoked short‐latency (<47 ms) inhibition apparent in trigger‐averaged rectified EMG, which was quantified in terms of area, duration and mean amplitude of inhibition. In order to validate the use of TStim in a standing model, TStim‐Ib inhibition was compared from conditions where participants were lying prone vs. standing upright. TStim evoked Ib inhibition in both conditions; however, significant reductions in Ib inhibition area (42.2%) and duration (32.9%) were observed during stance. Postural threat, manipulated by having participants stand at LOW (0.8 m high, 0.6 m from edge) and HIGH (3.2 m, at edge) elevated surfaces, significantly reduced Ib inhibition area (32.4%), duration (16.4%) and amplitude (24.8%) in the HIGH, compared to LOW, threat condition. These results demonstrate TStim is a viable technique for investigating Ib reflexes in standing, and confirm Ib reflexes are modulated with postural orientation. The novel observation of reduced Ib inhibition with elevated postural threat reveals that human Ib reflexes are context dependent, and the human Ib reflex pathways are modulated by threat or emotional processing centres of the CNS.

  May 04, 2017   doi: 10.1113/JP273935   open full text
• Mechanically sensitive Aδ nociceptors that innervate bone marrow respond to changes in intra‐osseous pressure.
  Sara Nencini, Jason Ivanusic.
  The Journal of Physiology. May 04, 2017
  Key points Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function. Here we use a novel in vivo bone–nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra‐osseous pressure. We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra‐osseous pressure stimulus and express the Piezo2 mechano‐transducer molecule. Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and that the Piezo2 mechano‐transducer may be involved. Abstract Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic–tonic units that appear to code for different intensities of intra‐osseous pressure, or phasic units that code for the rate of change in intra‐osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic–tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak‐to‐peak action potential amplitude. The majority of small‐diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano‐nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and imply that Piezo2 signalling may be involved in mechano‐transduction in these receptors.

  May 04, 2017   doi: 10.1113/JP273877   open full text
• SIRT1 may play a crucial role in overload‐induced hypertrophy of skeletal muscle.
  Erika Koltai, Zoltán Bori, Clovis Chabert, Hervé Dubouchaud, Hisashi Naito, Shuichi Machida, Kelvin JA Davies, Zsolt Murlasits, Andrew C Fry, Istvan Boldogh, Zsolt Radak.
  The Journal of Physiology. April 28, 2017
  Key points Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload‐induced hypertrophy. SIRT1‐mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload‐induced changes in microRNA levels regulate SIRT1 and insulin‐like growth factor 1 signalling. Abstract Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P < 0.001). SIRT1‐regulated Akt, endothelial nitric oxide synthase and GLUT4 levels were also induced in hypertrophied muscles, and SIRT1 levels correlated with muscle mass, paired box protein 7 (Pax7), proliferating cell nuclear antigen (PCNA) and nicotinamide phosphoribosyltransferase (Nampt) levels. Alternatively, decreased forkhead box O 1 (FOXO1) and increased K48 polyubiquitination also suggest that SIRT1 could be involved in the catabolic process of hypertrophy. Furthermore, increased levels of K63 and muscle RING finger 2 (MuRF2) protein could also be important enhancers of muscle mass. We report here that the levels of miR1 and miR133a decrease in hypertrophy and negatively correlate with muscle mass, SIRT1 and Nampt levels. Our results reveal a strong correlation between SIRT1 levels and activity, SIRT1‐regulated pathways and overload‐induced hypertrophy. These findings, along with the well‐known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload‐induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing.

  April 28, 2017   doi: 10.1113/JP273774   open full text
• Impact of perinatal exposure to sucrose or high fructose corn syrup (HFCS‐55) on adiposity and hepatic lipid composition in rat offspring.
  Carla R. Toop, Beverly S. Muhlhausler, Kerin O'Dea, Sheridan Gentili.
  The Journal of Physiology. April 26, 2017
  Perinatal exposure to excess maternal intake of added sugars, including fructose and sucrose, is associated with an increased risk of obesity and type 2 diabetes in adult life. However, it is unknown to what extent the type of sugar and the timing of exposure affect these outcomes. The aim of this study was to determine the impact of exposure to maternal consumption of a 10% w/v beverage containing sucrose or high fructose corn syrup‐55 (HFCS‐55) during the prenatal and/or suckling periods on offspring at 3 and 12 weeks, utilising a cross‐fostering approach in a rodent model. Perinatal sucrose exposure decreased plasma glucose concentrations in offspring at 3 weeks, but did not alter glucose tolerance. Increased adiposity was observed in 3‐week‐old offspring exposed to sucrose or HFCS‐55 during suckling, with increased hepatic fat content in HFCS‐55‐exposed offspring. In terms of specific fatty acids, hepatic monounsaturated (omega‐7 and ‐9) fatty acid content was elevated at weaning, and was most pronounced in sucrose offspring exposed during both the prenatal and suckling periods, and HFCS‐55 offspring exposed during suckling only. By 12 weeks, the effects on adiposity and hepatic lipid composition were largely normalised. However, exposure to either sucrose or HFCS‐55 during the prenatal period only was associated with elevated plasma free fatty acids at weaning, and this effect persisted until 12 weeks. This study suggests that the type of sugar and the timing of exposure (prenatal or suckling periods) are both important for determining the impact on metabolic health outcomes in the offspring. This article is protected by copyright. All rights reserved

  April 26, 2017   doi: 10.1113/JP274066   open full text
• Functional impact of an oculopharyngeal muscular dystrophy mutation in PABPN1.
  Maricela García‐Castañeda, Ana Victoria Vega, Rocío Rodríguez, Maria Guadalupe Montiel‐Jaen, Bulmaro Cisneros, Angel Zarain‐Herzberg, Guillermo Avila.
  The Journal of Physiology. April 25, 2017
  Key points Mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1) result in oculopharyngeal muscular dystrophy (OPMD). This disease is of late‐onset, but the underlying mechanism is unclear. Ca2+ stimulates muscle growth and contraction and, because OPMD courses with muscle atrophy and weakness, we hypothesized that the homeostasis of Ca2+ is altered in this disorder. C2C12 myotubes were transfected with cDNAs encoding either PABPN1 or the PABPN1‐17A OPMD mutation. Subsequently, they were investigated concerning not only excitation–contraction coupling (ECC) and intracellular levels of Ca2+, but also differentiation stage and nuclear structure. PABPN1‐17A gave rise to: inhibition of Ca2+ release during ECC, depletion of sarcoplasmic reticulum Ca2+ content, reduced expression of ryanodine receptors, altered nuclear morphology and incapability to stimulate myoblast fusion. PABPN1‐17A failed to inhibit ECC in adult muscle fibres, suggesting that its effects are primarily related to muscle regeneration. Abstract Oculopharyngeal muscular dystrophy (OPMD) is linked to mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1). OPMD mutations consist of an expansion of a tract that contains 10 alanines (to 12–17). This disease courses with muscle weakness that begins in adulthood, but the underlying mechanism is unclear. In the present study, we investigated the functional effects of PABPN1 and an OPMD mutation (PABPN1‐17A) using myotubes transfected with cDNAs encoding these proteins (GFP‐tagged). PABPN1 stimulated myoblast fusion (100%), whereas PABPN1‐17A failed to mimic this effect. Additionally, the OPMD mutation markedly altered nuclear morphology; specifically, it led to nuclei with a more convoluted and ovoid shape. Although PABPN1 and PABPN1‐17A modified the expression of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase and calsequestrin, the corresponding changes did not have a clear impact on [Ca2+]. Interestingly, neither L‐type Ca2+ channels, nor voltage‐gated sarcoplasmic reticulum (SR) Ca2+ release (VGCR) was altered by PABPN1. However, PABPN1‐17A produced a selective inhibition of VGCR (50%). This effect probably arises from both lower expression of RyR1 and depletion of SR Ca2+. The latter, however, was not related to inhibition of store‐operated Ca2+ entry. Both PABPN1 constructs promoted a moderated decrease in cytosolic [Ca2+], which apparently results from down‐regulation of excitation‐coupled Ca2+ entry. On the other hand, PABPN1‐17A did not alter ECC in muscle fibres, suggesting that adult muscle is less prone to developing deleterious effects. These results demonstrate that PABPN1 proteins regulate essential processes during myotube formation and support the notion that OPMD involves disruption of myogenesis, nuclear structure and homeostasis of Ca2+.

  April 25, 2017   doi: 10.1113/JP273948   open full text
• In vitro characterization of cell‐level neurophysiological diversity in the rostral nucleus reuniens of adult mice.
  Darren A. Walsh, Jonathan T. Brown, Andrew D. Randall.
  The Journal of Physiology. April 25, 2017
  Key points The nucleus reuniens (Re), a nucleus of the midline thalamus, is part of a cognitive network including the hippocampus and the medial prefrontal cortex. To date, very few studies have examined the electrophysiological properties of Re neurons at a cellular level. The majority of Re neurons exhibit spontaneous action potential firing at rest. This is independent of classical amino‐acid mediated synaptic transmission. When driven by various forms of depolarizing current stimulus, Re neurons display considerable diversity in their firing patterns. As a result of the presence of a low threshold Ca2+ channel, spike output functions are strongly modulated by the prestimulus membrane potential. Finally, we describe a novel form of activity‐dependant intrinsic plasticity that eliminates the high‐frequency burst firing present in many Re neurons. These results provide a comprehensive summary of the intrinsic electrophysiological properties of Re neurons allowing us to better consider the role of the Re in cognitive processes. Abstract The nucleus reuniens (Re) is the largest of the midline thalamic nuclei. We have performed a detailed neurophysiological characterization of neurons in the rostral Re of brain slices prepared from adult male mice. At resting potential (−63.7 ± 0.6 mV), ∼90% of Re neurons fired action potentials, typically continuously at ∼8 Hz. Although Re neurons experience a significant spontaneous barrage of fast, amino‐acid‐mediate synaptic transmission, this was not predominantly responsible for spontaneous spiking because firing persisted in the presence of glutamate and GABA receptor antagonists. With resting potential preset to −80 mV, −20 pA current injections revealed a mean input resistance of 615 MΩ and a mean time constant of 38 ms. Following cessation of this stimulus, a significant rebound potential was seen that was sometimes sufficiently large to trigger a short burst of very high frequency (100–300 Hz) firing. In most cells, short (2 ms), strong (2 nA) current injections elicited a single spike followed by a large afterdepolarizing potential which, when suprathreshold, generated high‐frequency spiking. Similarly, in the majority of cells preset at −80 mV, 500 ms depolarizing current injections to cells led to a brief initial burst of very high‐frequency firing, although this was lost when cells were preset at −72 mV. Biophysical and pharmacological experiments indicate a prominent role for T‐type Ca2+ channels in the high‐frequency bursting of Re neurons. Finally, we describe a novel form of activity‐dependent intrinsic plasticity that persistently eliminates the burst firing potential of Re neurons.

  April 25, 2017   doi: 10.1113/JP273915   open full text
• Evidence that 5‐HT stimulates intracellular Ca2+ signalling and activates pannexin‐1 currents in type II cells of the rat carotid body.
  Sindhubarathi Murali, Min Zhang, Colin A. Nurse.
  The Journal of Physiology. April 25, 2017
  Key points 5‐HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH). In the present study, we show that, in addition to type I cells, adjacent glial‐like type II cells express functional, ketanserin‐sensitive 5‐HT2 receptors, and their stimulation increases cytoplasmic Ca2+ derived from intracellular stores. In type II cells, 5‐HT activated a ketanserin‐sensitive inward current (I5‐HT) that was similar to that (IUTP) activated by the P2Y2R agonist, UTP. As previously shown for IUTP, I5‐HT was inhibited by BAPTA‐AM and carbenoxolone (5 μm), a putative blocker of ATP‐permeable pannexin (Panx)‐1 channels; IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide channel blocker, 10Panx peptide. Paracrine stimulation of type II cells by 5‐HT, leading to ATP release via Panx‐1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea). Abstract Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5‐HT and increased autocrine–paracrine 5‐HT2 receptor signalling contributes to sensory long‐term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial‐like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca2+]i) and activating carbenoxolone‐sensitive, ATP‐permeable, pannexin (Panx)‐1‐like channels. In the present study, using dissociated rat CB cultures, we found that 5‐HT induced Δ[Ca2+]i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5‐HT‐induced Ca2+ response in type II cells was dose‐dependent (EC50 ∼183 nm) and largely inhibited by the 5‐HT2A receptor blocker, ketanserin (1 μm), and also arose mainly from intracellular stores. 5‐HT also activated an inward current (I5‐HT) in type II cells (EC50 ∼200 nm) that was reversibly inhibited by ketanserin (1–10 nm), the Ca2+ chelator BAPTA‐AM (5 μm), and low concentrations of carbenoxolone (5 μm), a putative Panx‐1 channel blocker. I5‐HT reversed direction at approximately −11 mV and was indistinguishable from the UTP‐activated current (IUTP). Consistent with a role for Panx‐1 channels, IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide blocker 10Panx (100 μm), although not by its scrambled control peptide (scPanx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5‐HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx‐1 channels.

  April 25, 2017   doi: 10.1113/JP273473   open full text
• Characterisation and functional mapping of surface potentials in the rat dorsal column nuclei.
  Alastair J. Loutit, Ted Maddess, Stephen J. Redmond, John W. Morley, Greg J. Stuart, Jason R. Potas.
  The Journal of Physiology. April 25, 2017
  Key points The brainstem dorsal column nuclei (DCN) process sensory information arising from the body before it reaches the brain and becomes conscious. Despite significant investigations into sensory coding in peripheral nerves and the somatosensory cortex, little is known about how sensory information arising from the periphery is represented in the DCN. Following stimulation of hind‐limb nerves, we mapped and characterised the evoked electrical signatures across the DCN surface. We show that evoked responses recorded from the DCN surface are highly reproducible and are unique to nerves carrying specific sensory information. Abstract The brainstem dorsal column nuclei (DCN) play a role in early processing of somatosensory information arising from a variety of functionally distinct peripheral structures, before being transmitted to the cortex via the thalamus. To improve our understanding of how sensory information is represented by the DCN, we characterised and mapped low‐ (<200 Hz) and high‐frequency (550–3300 Hz) components of nerve‐evoked DCN surface potentials. DCN surface potentials were evoked by electrical stimulation of the left and right nerves innervating cutaneous structures (sural nerve), or a mix of cutaneous and deep structures (peroneal nerve), in 8‐week‐old urethane‐anaesthetised male Wistar rats. Peroneal nerve‐evoked DCN responses demonstrated low‐frequency events with significantly longer durations, more high‐frequency events and larger magnitudes compared to responses evoked from sural nerve stimulation. Hotspots of low‐ and high‐frequency DCN activity were found ipsilateral to stimulated nerves but were not symmetrically organised. In conclusion, we find that sensory inputs from peripheral nerves evoke unique and characteristic DCN activity patterns that are highly reproducible both within and across animals.

  April 25, 2017   doi: 10.1113/JP273759   open full text
• Early structural and functional signature of 3‐day human skeletal muscle disuse using the dry immersion model.
  Rémi Demangel, Loïc Treffel, Guillaume Py, Thomas Brioche, Allan F. Pagano, Marie‐Pierre Bareille, Arnaud Beck, Laurence Pessemesse, Robin Candau, Claude Gharib, Angèle Chopard, Catherine Millet.
  The Journal of Physiology. April 23, 2017
  Key points Our study contributes to the characterization of muscle loss and weakness processes induced by a sedentary life style, chronic hypoactivity, clinical bed rest, immobilization and microgravity. This study, by bringing together integrated and cellular evaluation of muscle structure and function, identifies the early functional markers and biomarkers of muscle deconditioning. Three days of muscle disuse in healthy adult subjects is sufficient to significantly decrease muscle mass, tone and force, and to induce changes in function relating to a weakness in aerobic metabolism and muscle fibre denervation. The outcomes of this study should be considered in the development of an early muscle loss prevention programme and/or the development of pre‐conditioning programmes required before clinical bed rest, immobilization and spaceflight travel. Abstract Microgravity and hypoactivity are associated with skeletal muscle deconditioning. The decrease of muscle mass follows an exponential decay, with major changes in the first days. The purpose of the study was to dissect out the effects of a short‐term 3‐day dry immersion (DI) on human quadriceps muscle function and structure. The DI model, by suppressing all support zones, accurately reproduces the effects of microgravity. Twelve healthy volunteers (32 ± 5 years) completed 3 days of DI. Muscle function was investigated through maximal voluntary contraction (MVC) tests and muscle viscoelasticity. Structural experiments were performed using MRI analysis and invasive experiments on muscle fibres. Our results indicated a significant 9.1% decrease of the normalized MVC constant (P = 0.048). Contraction and relaxation modelization kinetics reported modifications related to torque generation (kACT = −29%; P = 0.014) and to the relaxation phase (kREL = +34%; P = 0.040) after 3 days of DI. Muscle viscoelasticity was also altered. From day one, rectus femoris stiffness and tone decreased by, respectively, 7.3% (P = 0.002) and 10.2% (P = 0.002), and rectus femoris elasticity decreased by 31.5% (P = 0.004) after 3 days of DI. At the cellular level, 3 days of DI translated into a significant atrophy of type I muscle fibres (−10.6 ± 12.1%, P = 0.027) and an increased proportion of hybrid, type I/IIX fibre co‐expression. Finally, we report an increase (6‐fold; P = 0.002) in NCAM+ muscle fibres, showing an early denervation process. This study is the first to report experiments performed in Europe investigating human short‐term DI‐induced muscle adaptations, and contributes to deciphering the early changes and biomarkers of skeletal muscle deconditioning.

  April 23, 2017   doi: 10.1113/JP273895   open full text
• Direct current stimulation boosts synaptic gain and cooperativity in vitro.
  Asif Rahman, Belen Lafon, Lucas C. Parra, Marom Bikson.
  The Journal of Physiology. April 23, 2017
  Key points Direct current stimulation (DCS) polarity specifically modulates synaptic efficacy during a continuous train of presynaptic inputs, despite synaptic depression. DCS polarizes afferent axons and postsynaptic neurons, boosting cooperativity between synaptic inputs. Polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during transcranial DCS (tDCS). A statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous in enhancing connectivity between co‐active brain regions. Abstract Transcranial direct current stimulation (tDCS) produces sustained and diffuse current flow in the brain with effects that are state dependent and outlast stimulation. A mechanistic explanation for tDCS should capture these spatiotemporal features. It remains unclear how sustained DCS affects ongoing synaptic dynamics and how modulation of afferent inputs by diffuse stimulation changes synaptic activity at the target brain region. We tested the effect of acute DCS (10–20 V m−1 for 3–5 s) on synaptic dynamics with constant rate (5–40 Hz) and Poisson‐distributed (4 Hz mean) trains of presynaptic inputs. Across tested frequencies, sustained synaptic activity was modulated by DCS with polarity‐specific effects. Synaptic depression attenuates the sensitivity to DCS from 1.1% per V m−1 to 0.55%. DCS applied during synaptic activity facilitates cumulative neuromodulation, potentially reversing endogenous synaptic depression. We establish these effects are mediated by both postsynaptic membrane polarization and afferent axon fibre polarization, which boosts cooperativity between synaptic inputs. This potentially extends the locus of neuromodulation from the nominal target to afferent brain regions. Based on these results we hypothesized the polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during tDCS. A multiscale model of transcranial electrical stimulation including a finite element model of brain current flow, numerical simulations of neuronal activity, and a statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous. Thus, we propose that specifically because tDCS is diffuse, weak and sustained it can boost connectivity between co‐active brain regions.

  April 23, 2017   doi: 10.1113/JP273005   open full text
• Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high‐intensity exercise training.
  Victoria L. Wyckelsma, Itamar Levinger, Michael J. McKenna, Luke E. Formosa, Michael T. Ryan, Aaron C. Petersen, Mitchell J. Anderson, Robyn M. Murphy.
  The Journal of Physiology. April 23, 2017
  Key points Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high‐intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. Abstract Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High‐intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin‐2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70–100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults.

  April 23, 2017   doi: 10.1113/JP273950   open full text
• mGluR1 enhances efferent inhibition of inner hair cells in the developing rat cochlea.
  Zhanlei Ye, Juan D. Goutman, Sonja J. Pyott, Elisabeth Glowatzki.
  The Journal of Physiology. April 21, 2017
  Key points Spontaneous activity of the sensory inner hair cells shapes maturation of the developing ascending (afferent) auditory system before hearing begins. Just before the onset of hearing, descending (efferent) input from cholinergic neurons originating in the brainstem inhibit inner hair cell spontaneous activity and may further refine maturation. We show that agonist activation of the group I metabotropic glutamate receptor mGluR1 increases the strength of this efferent inhibition by enhancing the presynaptic release of acetylcholine. We further show that the endogenous release of glutamate from the inner hair cells may increase the strength of efferent inhibition via the activation of group I metabotropic glutamate receptors. Thus, before the onset of hearing, metabotropic glutamate signalling establishes a local negative feedback loop that is positioned to regulate inner hair cell excitability and refine maturation of the auditory system. Abstract Just before the onset of hearing, the inner hair cells (IHCs) receive inhibitory efferent input from cholinergic medial olivocochlear (MOC) neurons originating in the brainstem. This input may serve a role in the maturation of the ascending (afferent) auditory system by inhibiting spontaneous activity of the IHCs. To investigate the molecular mechanisms regulating these IHC efferent synapses, we combined electrical stimulation of the efferent fibres with patch clamp recordings from the IHCs to measure efferent synaptic strength. By examining evoked responses, we show that activation of metabotropic glutamate receptors (mGluRs) by general and group I‐specific mGluR agonists enhances IHC efferent inhibition. This enhancement is blocked by application of a group I mGluR1‐specific antagonist, indicating that enhancement of IHC efferent inhibition is mediated by group I mGluRs and specifically by mGluR1s. By comparing spontaneous and evoked responses, we show that group I mGluR agonists act presynaptically to increase neurotransmitter release without affecting postsynaptic responsiveness. Moreover, endogenous glutamate released from the IHCs also enhances IHC efferent inhibition via the activation of group I mGluRs. Finally, immunofluorescence analysis indicates that the efferent terminals are sufficiently close to IHC glutamate release sites to allow activation of mGluRs on the efferent terminals by glutamate spillover. Together, these results suggest that glutamate released from the IHCs activates group I mGluRs (mGluR1s), probably present on the efferent terminals, which, in turn, enhances release of acetylcholine and inhibition of the IHCs. Thus, mGluRs establish a local negative feedback loop positioned to regulate IHC activity and maturation of the ascending auditory system in the developing cochlea.

  April 21, 2017   doi: 10.1113/JP272604   open full text
• Vasopressin V1a receptors mediate the hypertensive effects of [Pyr1]apelin‐13 in the rat rostral ventrolateral medulla.
  Philip R. Griffiths, Stephen J. Lolait, Louise E. Harris, Julian F. R. Paton, Anne‐Marie O'Carroll.
  The Journal of Physiology. April 21, 2017
  Key points Dysfunctions in CNS regulation of arterial blood pressure lead to an increase in sympathetic nerve activity that participates in the pathogenesis of hypertension. The apelin‐apelin receptor system affects arterial blood pressure homeostasis; however, the central mechanisms underlying apelin‐mediated changes in sympathetic nerve activity and blood pressure have not been clarified. We explored the mechanisms involved in the regulation of [Pyr1]apelin‐13‐mediated cardiovascular control within the rostral ventrolateral medulla (RVLM) using selective receptor antagonists. We show that [Pyr1]apelin‐13 acts as a modulating neurotransmitter in the normotensive RVLM to affect vascular tone through interaction with the vasopressin V1a receptor but that [Pyr1]apelin‐13‐induced sympathoexcitation is independent of angiotensin II receptor type 1, oxytocin, ionotropic glutamate and GABAA receptors. Our data confirm a role for the apelin peptide system in cardiovascular regulation at the level of the RVLM and highlight that this system is a possible potential therapeutic target for the treatment of hypertension. Abstract Apelin is a ubiquitous peptide that can elevate arterial blood pressure (ABP) yet understanding of the mechanisms involved remain incomplete. Bilateral microinjection of [Pyr1]apelin‐13 into the rostral ventrolateral medulla (RVLM), a major source of sympathoexcitatory neurones, increases ABP and sympathetic nerve activity. We aimed to investigate the potential involvement of neurotransmitter systems through which the apelin pressor response may occur within the RVLM. Adult male Wistar rats were anaesthetized and ABP was monitored via a femoral arterial catheter. Bilateral RVLM microinjection of [Pyr1]apelin‐13 significantly increased ABP (9 ± 1 mmHg) compared to saline (−1 ± 2mmHg; P < 0.001), which was blocked by pretreatment with the apelin receptor antagonist, F13A (0 ± 1 mmHg; P < 0.01). The rise in ABP was associated with an increase in the low frequency spectra of systolic BP (13.9 ± 4.3% total power; P < 0.001), indicative of sympathetic vasomotor activation. The [Pyr1]apelin‐13‐mediated pressor response and the increased low frequency spectra of systolic BP response were fully maintained despite RVLM pretreatment with the angiotensin II type 1 receptor antagonist losartan, the oxytocin receptor antagonist desGly‐NH2, d(CH2)5[D‐Tyr2,Thr4]OVT, the ionotropic glutamate receptor antagonist kynurenate or the GABAA antagonist bicuculline (P > 0.05). By contrast, the [Pyr1]apelin‐13 induced pressor and sympathoexcitatory effects were abolished by pretreatment of the RVLM with the vasopressin V1a receptor antagonist, SR 49059 (−1 ± 1 mmHg; 1.1 ± 1.1% total power, respectively; P < 0.001). These findings suggest that the pressor action of [Pyr1]apelin‐13 in the RVLM of normotensive rats is not mediated via angiotensin II type 1 receptor, oxytocin, ionotropic glutamate or GABAA receptors but instead involves a close relationship with the neuropeptide modulator vasopressin.

  April 21, 2017   doi: 10.1113/JP274178   open full text
• Angiotensin II activates CaV1.2 Ca2+ channels through β‐arrestin2 and casein kinase 2 in mouse immature cardiomyocytes.
  Toshihide Kashihara, Tsutomu Nakada, Katsuhiko Kojima, Toshikazu Takeshita, Mitsuhiko Yamada.
  The Journal of Physiology. April 20, 2017
  Key points Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca2+ transients of mouse immature but not mature cardiomyocytes by robustly activating CaV1.2 L‐type Ca2+ channels through a novel signalling pathway involving angiotensin type 1 (AT1) receptors, β‐arrestin2 and casein kinase 2. A β‐arrestin‐biased AT1 receptor agonist, TRV027, was as effective as AngII in activating L‐type Ca2+ channels. Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that β‐arrestin‐biased AT1 receptor agonists may be valuable therapeutics for paediatric heart failure. Abstract Angiotensin II (AngII), the main effector peptide of the renin–angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well‐known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca2+ transients by robustly activating L‐type CaV1.2 Ca2+ (CaV1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT1 receptors and β‐arrestin2. A β‐arrestin‐biased AT1 receptor agonist was as effective as AngII in activating CaV1.2 channels. Src‐family tyrosine kinases (SFKs) and casein kinase 2α’β (CK2α’β) were sequentially activated when AngII activated CaV1.2 channels. A cyclin‐dependent kinase inhibitor, p27Kip1 (p27), inhibited CK2α’β, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α’β but not c‐Src directly activated recombinant CaV1.2 channels composed of C‐terminally truncated α1C, the distal C‐terminus of α1C, β2C and α2δ1 subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C‐terminus of CaV1.2α1C subunits. Co‐immunoprecipitation revealed that CaV1.2 channels, CK2α’β and p27 formed a macromolecular complex. Therefore, stimulation of AT1 receptors by AngII activates CaV1.2 channels through β‐arrestin2 and CK2α’β, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that β‐arrestin‐biased AT1 receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.

  April 20, 2017   doi: 10.1113/JP273883   open full text
• Chronic alcohol feeding potentiates hormone‐induced calcium signalling in hepatocytes.
  Paula J. Bartlett, Anil Noronha Antony, Amit Agarwal, Mauricette Hilly, Victoria L. Prince, Laurent Combettes, Jan B. Hoek, Lawrence D. Gaspers.
  The Journal of Physiology. April 18, 2017
  Key points Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+‐mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol‐dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone‐induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone‐evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol‐induced hepatocyte injury. Abstract ‘Adaptive’ responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide‐dependent cytosolic calcium ([Ca2+]i) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose–response for Ca2+‐mobilizing hormones resulting in more sustained and prolonged [Ca2+]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone‐induced calcium increases in control livers, but not after chronic alcohol‐feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone‐induced inositol 1,4,5 trisphosphate (IP3) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol‐fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone‐stimulated PLC activity, indicating calcium‐dependent PLCs are not upregulated by alcohol. We propose that the liver ‘adapts’ to chronic alcohol exposure by increasing hormone‐dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

  April 18, 2017   doi: 10.1113/JP273891   open full text
• Geniculohypothalamic GABAergic projections gate suprachiasmatic nucleus responses to retinal input.
  Lydia Hanna, Lauren Walmsley, Abigail Pienaar, Michael Howarth, Timothy M. Brown.
  The Journal of Physiology. April 11, 2017
  Key points Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt their physiology and behaviour in line with the solar day. In addition to direct retinal projections, the clock receives input from the visual thalamus, although the role of this geniculohypothalamic pathway in circadian photoreception is poorly understood. In the present study, we develop a novel brain slice preparation that preserves the geniculohypothalamic pathway to show that GABAergic thalamic neurons inhibit retinally‐driven activity in the central clock in a circadian time‐dependent manner. We also show that in vivo manipulation of thalamic signalling adjusts specific features of the hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily activated by crossed retinal inputs. Our data provide a mechanism by which geniculohypothalamic signals can adjust the magnitude of circadian and more acute hypothalamic light responses according to time‐of‐day and establish an important new model for future investigations of the circadian visual system. Abstract Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in allowing animals to optimize physiology and behaviour alongside daily changes in the environment. Retinal inputs encoding changes in external illumination provide the principle source of such information. The SCN also receives input from other retinorecipient brain regions, primarily via the geniculohypothalamic tract (GHT), although the contribution of these indirect projections to circadian photoreception is currently poorly understood. To address this deficit, in the present study, we established an in vitro mouse brain slice preparation that retains connectivity across the extended circadian system. Using multi‐electrode recordings, we first confirm that this preparation retains intact optic projections to the SCN, thalamus and pretectum and a functional GHT. We next show that optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, and also that this effect exhibits a pronounced day/night variation and involves a GABAergic mechanism. This inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside pharmacological inactivation or optogenetic excitation to show that GHT signalling actively modulates specific features of the SCN light response, indicating that GHT cells are primarily activated by crossed retinal projections. Taken together, our data establish a new model for studying network communication in the extended circadian system and provide novel insight into the roles of GHT‐signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the SCN according to time of day.

  April 11, 2017   doi: 10.1113/JP273850   open full text
• Baroreflex control of renal sympathetic nerve activity in early heart failure assessed by the sequence method.
  Renata Maria Lataro, Luiz Eduardo Virgilio Silva, Carlos Alberto Aguiar Silva, Helio Cesar Salgado, Rubens Fazan.
  The Journal of Physiology. April 07, 2017
  Key points The integrity of the baroreflex control of sympathetic activity in heart failure (HF) remains under debate. We proposed the use of the sequence method to assess the baroreflex control of renal sympathetic nerve activity (RSNA). The sequence method assesses the spontaneous arterial pressure (AP) fluctuations and their related changes in heart rate (or other efferent responses), providing the sensitivity and the effectiveness of the baroreflex. Effectiveness refers to the fraction of spontaneous AP changes that elicits baroreflex‐mediated variations in the efferent response. Using three different approaches, we showed that the baroreflex sensitivity between AP and RSNA is not altered in early HF rats. However, the sequence method provided evidence that the effectiveness of baroreflex in changing RSNA in response to AP changes is markedly decreased in HF. The results help us better understand the baroreflex control of the sympathetic nerve activity. Abstract In heart failure (HF), the reflex control of the heart rate is known to be markedly impaired; however, the baroreceptor control of the sympathetic drive remains under debate. Applying the sequence method to a series of arterial pressure (AP) and renal sympathetic nerve activity (RSNA), we demonstrated a clear dysfunction in the baroreflex control of sympathetic activity in rats with early HF. We analysed the baroreflex control of the sympathetic drive using three different approaches: AP vs. RSNA curve, cross‐spectral analysis and sequence method between AP and RSNA. The sequence method also provides the baroreflex effectiveness index (BEI), which represents the percentage of AP ramps that actually produce a reflex response. The methods were applied to control rats and rats with HF induced by myocardial infarction. None of the methods employed to assess the sympathetic baroreflex gain were able to detect any differences between the control and the HF group. However, rats with HF exhibited a lower BEI compared to the controls. Moreover, an optimum delay of 1 beat was observed, i.e. 1 beat is required for the RSNA to respond after AP changing, which corroborates with the findings related to the timing between these two variables. For delay 1, the BEI of the controls was 0.45 ± 0.03, whereas the BEI of rats with HF was 0.29 ± 0.09 (P < 0.05). These data demonstrate that while the gain of the baroreflex is not affected in early HF, its effectiveness is markedly decreased. The analysis of the spontaneous changes in AP and RSNA using the sequence method provides novel insights into arterial baroreceptor reflex function.

  April 07, 2017   doi: 10.1113/JP274065   open full text
• A Western‐style obesogenic diet alters maternal metabolic physiology with consequences for fetal nutrient acquisition in mice.
  Barbara Musial, Owen R. Vaughan, Denise S. Fernandez‐Twinn, Peter Voshol, Susan E. Ozanne, Abigail L. Fowden, Amanda N. Sferruzzi‐Perri.
  The Journal of Physiology. April 05, 2017
  Key points In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy, although their effects on the metabolism of the mother, in relation to feto‐placental glucose utilization and growth, are unknown. In the present study, the consumption of an obesogenic HFHS diet compromised maternal glucose tolerance and insulin sensitivity in late pregnancy in association with dysregulated lipid and glucose handling by the dam. These maternal metabolic changes induced by HFHS feeding were related to altered feto‐placental glucose metabolism and growth. A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth. These findings have implications for the health of women and their infants, who consume obesogenic diets during pregnancy. Abstract In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy. However, the impacts of a HFHS diet during pregnancy on maternal insulin sensitivity and signalling in relation to feto‐placental growth and glucose utilization are unknown. The present study examined the effects of a HFHS diet during mouse pregnancy on maternal glucose tolerance and insulin resistance, as well as, on feto‐placental glucose metabolism. Female mice were fed a control or HFHS diet from day (D) 1 of pregnancy (term = D20.5). At D16 or D19, dams were assessed for body composition, metabolite and hormone concentrations, tissue abundance of growth and metabolic signalling pathways, glucose tolerance and utilization and insulin sensitivity. HFHS feeding perturbed maternal insulin sensitivity in late pregnancy; hepatic insulin sensitivity was higher, whereas sensitivity of the skeletal muscle and white adipose tissue was lower in HFHS than control dams. These changes were accompanied by increased adiposity and reduced glucose production and glucose tolerance of HFHS dams. The HFHS diet also disturbed the hormone and metabolite milieu and altered expression of growth and metabolic signalling pathways in maternal tissues. Furthermore, HFHS feeding was associated with impaired feto‐placental glucose metabolism and growth. A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth. These findings have implications for the health of women and their infants, who consume HFHS diets during pregnancy.

  April 05, 2017   doi: 10.1113/JP273684   open full text
• mTOR folate sensing links folate availability to trophoblast cell function.
  Fredrick J. Rosario, Theresa L. Powell, Thomas Jansson.
  The Journal of Physiology. April 04, 2017
  Folate is a water‐soluble B vitamin that is essential for cellular methylation reactions and DNA synthesis and repair. Low maternal folate levels in pregnancy are associated with fetal growth restriction, however the underlying mechanisms are poorly understood. Mechanistic target of rapamycin (mTOR) links nutrient availability to cell growth and function by regulating gene expression and protein translation. Here we show that mTOR functions as a folate sensor in primary human trophoblast (PHT) cells. Folate deficiency in PHT cells caused inhibition of mTOR signalling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton‐coupled folate transporter (PCFT, SLC46A1). The involvement of PCFT in mTOR folate sensing is not dependent on its function as a plasma membrane folate transporter. Increasing levels of homocysteine had no effect on PHT mTOR signalling, suggesting that mTOR senses low folate rather than high homocysteine. In addition, we demonstrate that maternal serum folate is positively correlated to placental mTORC1 and mTORC2 signalling activity in human pregnancy. We have identified a previously unknown molecular link between folate availability and cell function involving PCFT and mTOR signalling. We propose that mTOR folate sensing in trophoblast cells matches placental nutrient transport, and therefore fetal growth, to folate availability. These findings may have implications for our understanding of how altered folate availability causes human diseases such as fetal growth restriction, fetal malformations and cancer. This article is protected by copyright. All rights reserved

  April 04, 2017   doi: 10.1113/JP272424   open full text
• Differential serotonergic modulation across the main and accessory olfactory bulbs.
  Zhenbo Huang, Nicolas Thiebaud, Debra Ann Fadool.
  The Journal of Physiology. March 31, 2017
  Key points There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current‐clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5‐HT2A receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5‐HT2 receptors on GABAergic interneurons and direct inhibition via the 5‐HT1 receptors on MCs. Differential 5‐HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression. Abstract Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5‐HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15–30 day mice. In the MOB, 5‐HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)– first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5‐HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5‐HT2A receptors through a direct activation. In comparison, 5‐HT‐evoked inhibitory responses in the AOB arose due to a polysynaptic, slow‐onset inhibition attributed to 5‐HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5‐HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.

  March 31, 2017   doi: 10.1113/JP273945   open full text
• Computational analysis of the human sinus node action potential: model development and effects of mutations.
  Alan Fabbri, Matteo Fantini, Ronald Wilders, Stefano Severi.
  The Journal of Physiology. March 30, 2017
  Key points We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the ‘funny current’ (If) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart‐rate modulating drugs. Abstract The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (If) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss‐of‐function and gain‐of‐function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

  March 30, 2017   doi: 10.1113/JP273259   open full text
• Uteroplacental insufficiency reduces rat plasma leptin concentrations and alters placental leptin transporters: ameliorated with enhanced milk intake and nutrition.
  Jessica F. Briffa, Rachael O'Dowd, Karen M. Moritz, Tania Romano, Lisa R. Jedwab, Andrew J. McAinch, Deanne H. Hryciw, Mary E. Wlodek.
  The Journal of Physiology. March 29, 2017
  Key points Uteroplacental insufficiency compromises maternal mammary development, milk production and pup organ development; this is ameliorated by cross‐fostering, which improves pup growth and organ development and prevents adult diseases in growth‐restricted (Restricted) offspring by enhancing postnatal nutrition. Leptin is transported to the fetus from the mother by the placenta; we report reduced plasma leptin concentrations in Restricted fetuses associated with sex‐specific alterations in placental leptin transporter expression. Pup plasma leptin concentrations were also reduced during suckling, which may suggest reduced milk leptin transport or leptin reabsorption. Mothers suckled by Restricted pups had impaired mammary development and changes in milk fatty acid composition with no alterations in milk leptin; cross‐fostering restored pup plasma leptin concentrations, which may be correlated to improved milk composition and intake. Increased plasma leptin and altered milk fatty acid composition in Restricted pups suckling mothers with normal lactation may improve postnatal growth and prevent adult diseases. Abstract Uteroplacental insufficiency reduces birth weight and adversely affects fetal organ development, increasing adult disease risk. Cross‐fostering improves postnatal nutrition and restores these deficits. Mothers with growth‐restricted pups have compromised milk production and composition; however, the impact cross‐fostering has on milk production and composition is unknown. Plasma leptin concentrations peak during the completion of organogenesis, which occurs postnatally in rats. Leptin is transferred to the fetus via the placenta and to the pup via the lactating mammary gland. This study investigated the effect of uteroplacental insufficiency on pup plasma leptin concentrations and placental leptin transporters. We additionally examined whether cross‐fostering improves mammary development, milk composition and pup plasma leptin concentrations. Fetal growth restriction was induced by bilateral uterine vessel ligation surgery on gestation day 18 in Wistar Kyoto rats (termed uteroplacental insufficiency surgery mothers). Growth‐restricted (Restricted) fetuses had reduced plasma leptin concentrations, persisting throughout lactation, and sex‐specific alterations in placental leptin transporters. Mothers suckled by Restricted pups had impaired mammary development, altered milk fatty acid composition and increased plasma leptin concentrations, despite no changes in milk leptin. Milk intake was reduced in Restricted pups suckling uteroplacental insufficiency surgery mothers compared to Restricted pups suckling sham‐operated mothers. Cross‐fostering Restricted pups onto a sham‐operated mother improved postnatal growth and restored plasma leptin concentrations compared to Restricted pups suckling uteroplacental insufficiency surgery mothers. Uteroplacental insufficiency alters leptin homeostasis. This is ameliorated with cross‐fostering and enhanced milk fatty acid composition and consumption, which may protect the pups from developing adverse health conditions in adulthood.

  March 29, 2017   doi: 10.1113/JP273825   open full text
• A calcium‐dependent pathway underlies activity‐dependent plasticity of electrical synapses in the thalamic reticular nucleus.
  Jessica Sevetson, Sarah Fittro, Emily Heckman, Julie S. Haas.
  The Journal of Physiology. March 29, 2017
  Recent results have demonstrated modification of electrical synapse strength by varied forms of neuronal activity. However, the mechanisms underlying plasticity induction in central mammalian neurons are unclear. Here we show that the two established inductors of plasticity at electrical synapses in the thalamic reticular nucleus – paired burst spiking in coupled neurons, and mGluR‐dependent tetanization of synaptic input – are separate pathways that converge at a common downstream endpoint. Using occlusion experiments and pharmacology in patched pairs of coupled neurons in vitro, we show that burst‐induced depression depends on calcium entry via voltage‐gated channels, is blocked by BAPTA chelation, and recruits intracellular calcium release on its way to activation of phosphatase activity. In contrast, mGluR‐dependent plasticity is independent of calcium entry or calcium dynamics. Together, these results show that the spiking‐initiated mechanisms underlying electrical synapse plasticity are similar to those that induce plasticity at chemical synapses, and offer the possibility that calcium‐regulated mechanisms may also lead to alternate outcomes, such as potentiation. Because these mechanistic elements are widely found in mature neurons, we expect them to apply broadly to electrical synapses across the brain, acting as the crucial link between neuronal activity and electrical synapse strength. This article is protected by copyright. All rights reserved

  March 29, 2017   doi: 10.1113/JP274049   open full text
• Leg vascular and skeletal muscle mitochondrial adaptations to aerobic high‐intensity exercise training are enhanced in the early postmenopausal phase.
  Michael Nyberg, Jon Egelund, Camilla M. Mandrup, Caroline B. Andersen, Karen M. B. E. Hansen, Ida‐Marie F. Hergel, Nicholai Valbak‐Andersen, Ruth Frikke‐Schmidt, Bente Stallknecht, Jens Bangsbo, Ylva Hellsten.
  The Journal of Physiology. March 29, 2017
  Key points Exercise training effectively improves vascular and skeletal muscle function; however, these effects of training may be blunted in postmenopausal women as a result of the loss of oestrogens. Accordingly, the capacity to deliver oxygen to the active muscles may also be impaired in postmenopausal women. In both premenopausal and recent postmenopausal women, exercise training was shown to improve leg vascular and skeletal muscle mitochondrial function. Interestingly, these effects were more pronounced in postmenopausal women. Skeletal muscle oxygen supply and utilization were similar in the two groups of women. These findings suggest that the early postmenopausal phase is associated with an enhanced capacity of the leg vasculature and skeletal muscle mitochondria to adapt to exercise training and that the ability to deliver oxygen to match the demand of the active muscles is preserved in the early phase following the menopausal transition. Abstract Exercise training leads to favourable adaptations within skeletal muscle; however, this effect of exercise training may be blunted in postmenopausal women as a result of the loss of oestrogens. Furthermore, postmenopausal women may have an impaired vascular response to acute exercise. We examined the haemodynamic response to acute exercise in matched pre‐ and postmenopausal women before and after 12 weeks of aerobic high intensity exercise training. Twenty premenopausal and 16 early postmenopausal (mean ± SEM: 3.1 ± 0.5 years after final menstrual period) women only separated by 4 years of age (mean ± SEM: 50 ± 0 years vs. 54 ± 1 years) were included. Before training, leg blood flow, O2 delivery, O2 uptake and lactate release during knee‐extensor exercise were similar in pre‐ and postmenopausal women. Exercise training reduced (P < 0.05) leg blood flow, O2 delivery, O2 uptake, lactate release, blood pressure and heart rate during the same absolute workloads in postmenopausal women. These effects were not detected in premenopausal women. Quadriceps muscle protein contents of mitochondrial complex II, III and IV; endothelial nitric oxide synthase (eNOS); cyclooxygenase (COX)‐1; COX‐2; and oestrogen‐related receptor α (ERRα) were increased (P < 0.05) with training in postmenopausal women, whereas only the levels of mitochondrial complex V, eNOS and COX‐2 were increased (P < 0.05) in premenopausal women. These findings demonstrate that vascular and skeletal muscle mitochondrial adaptations to aerobic high intensity exercise training are more pronounced in recent post‐ compared to premenopausal women, possibly as an effect of enhanced ERRα signalling. Also, the hyperaemic response to acute exercise appears to be preserved in the early postmenopausal phase.

  March 29, 2017   doi: 10.1113/JP273871   open full text
• The role played by oxidative stress in evoking the exercise pressor reflex in health and simulated peripheral artery disease.
  Jonathan E. Harms, J. Matthew Kuczmarski, Joyce Kim, Gail D. Thomas, Marc P. Kaufman.
  The Journal of Physiology. March 28, 2017
  Contraction of muscle evokes the exercise pressor reflex (EPR), which is expressed partly by increases in heart rate and arterial pressure. Patients with peripheral artery disease (PAD) show an exaggerated EPR, sometimes report pain when walking and are at risk for cardiac arrthymias. Previous research suggested that reactive oxygen species (ROS) mediate the exaggerated EPR associated with PAD. To examine the effects of ROS on the EPR, we infused a superoxide scavenger, tiron, into the superficial epigastric artery of decerebrated rats. In some, we simulated PAD by ligating a femoral artery for 72 h before the experiment. The peak EPR in “ligated” rats during saline infusion averaged 31 ± 4 mmHg, whereas the peak EPR in these rats during tiron infusion averaged 13 ± 2 mmHg (n = 12; P < 0.001); the attenuating effect of tiron on the EPR was partly reversed when saline was reinfused into the superficial epigastric artery (21 ± 2; P < 0.01 vs tiron). The peak EPR in “ligated” rats was also attenuated (n = 7; P < 0.01) by infusion of gp91ds‐tat, a peptide which blocks the activity of NAD(P)H oxidase. Tiron infusion had no effect on the EPR in rats with patent femoral arteries (n = 9). Western blots showed that the triceps surae muscles of “ligated” rats expressed more Nox2 and p67phox, which are components of NADPH oxidase, than did triceps surae muscles of “freely perfused” rats. Tiron added to muscle homogenates reduced ROS production in vitro. Our results provide further evidence that ROS mediates the exaggeration of EPR in rats with simulated PAD. This article is protected by copyright. All rights reserved

  March 28, 2017   doi: 10.1113/JP273816   open full text
• Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.
  Daniel C. Bartos, Stefano Morotti, Kenneth S. Ginsburg, Eleonora Grandi, Donald M. Bers.
  The Journal of Physiology. March 28, 2017
  Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i. When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Abstract The slowly activating delayed rectifier K+ current (IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+]i dependence of IKs in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in IKs as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail IKs, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent IKs activation during normal Ca2+ transients. [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i. When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Membrane topology of a Kv7.1 α‐subunit and regulatory proteins.

  March 28, 2017   doi: 10.1113/JP273676   open full text
• Nutritional status‐dependent endocannabinoid signalling regulates the integration of rat visceral information.
  Abdessattar Khlaifia, Isabelle Matias, Daniela Cota, Fabien Tell.
  The Journal of Physiology. March 27, 2017
  Key points Vagal sensory inputs transmit information from the viscera to brainstem neurones located in the nucleus tractus solitarii to set physiological parameters. These excitatory synapses exhibit a CB1 endocannabinoid‐induced long‐term depression (LTD) triggered by vagal fibre stimulation. We investigated the impact of nutritional status on long‐term changes in this long‐term synaptic plasticity. Food deprivation prevents LTD induction by disrupting CB1 receptor signalling. Short‐term refeeding restores the capacity of vagal synapses to express LTD. Ghrelin and cholecystokinin, respectively released during fasting and refeeding, play a key role in the control of LTD via the activation of energy sensing pathways such as AMPK and the mTOR and ERK pathways. Abstract Communication form the viscera to the brain is essential to set physiological homoeostatic parameters but also to drive more complex behaviours such as mood, memory and emotional states. Here we investigated the impact of the nutritional status on long‐term changes in excitatory synaptic transmission in the nucleus tractus solitarii, a neural hub integrating visceral signals. These excitatory synapses exhibit a CB1 endocannabinoid (eCB)‐induced long‐term depression (LTD) triggered by vagal fibre stimulation. Since eCB signalling is known to be an important component of homoeostatic regulation of the body and is regulated during various stressful conditions, we tested the hypothesis that food deprivation alters eCB signalling in central visceral afferent fibres. Food deprivation prevents eCB‐LTD induction due to the absence of eCB signalling. This loss was reversed by blockade of ghrelin receptors. Activation of the cellular fuel sensor AMP‐activated protein kinase or inhibition of the mechanistic target of rapamycin pathway abolished eCB‐LTD in free‐fed rats. Signals associated with energy surfeit, such as short‐term refeeding, restore eCB‐LTD induction, which in turn requires activation of cholecystokinin receptors and the extracellular signal‐regulated kinase pathway. These data suggest a tight link between eCB‐LTD in the NTS and nutritional status and shed light on the key role of eCB in the integration of visceral information.

  March 27, 2017   doi: 10.1113/JP273484   open full text
• Article update.
  
  The Journal of Physiology. March 26, 2017
  There is no abstract available for this paper.

  March 26, 2017   doi: 10.1113/JP273893   open full text
• Endothelin‐1 mediates natriuresis but not polyuria during vitamin D‐induced acute hypercalcaemia.
  Natsuko Tokonami, Lydie Cheval, Isabelle Monnay, Guillaume Meurice, Johannes Loffing, Eric Feraille, Pascal Houillier.
  The Journal of Physiology. March 23, 2017
  Key points Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism. A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)‐1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia‐induced effects. In the present study, we demonstrate that, during vitamin D‐induced hypercalcaemia, the activation of ET system by increased ET‐1 is responsible for natriuresis but not for polyuria. Vitamin D‐treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited. We have identified an original pathway that specifically mediates the effects of vitamin D‐induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. Abstract Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D‐induced hypercalcaemia increases the renal expression of endothelin (ET)‐1, we hypothesized that ET‐1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8‐week‐old, parathyroid hormone‐supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT‐treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET‐1 and the transcription factors CCAAT‐enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT‐treated mice. To examine the role of the ET system in hypercalcaemia‐induced natriuresis and polyuria, mice were treated with the ET‐1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT‐treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT‐treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D‐induced hypercalcaemia increases the renal production of ET‐1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria.

  March 23, 2017   doi: 10.1113/JP273610   open full text
• Prolactin regulation of oxytocin neurone activity in pregnancy and lactation.
  Rachael A. Augustine, Sharon R. Ladyman, Gregory T. Bouwer, Yousif Alyousif, Tony J. Sapsford, Victoria Scott, Ilona C. Kokay, David R. Grattan, Colin H. Brown.
  The Journal of Physiology. March 23, 2017
  Key points During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin‐secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation. Abstract Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane‐anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s−1, respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s−1 in lactating rats but excited none in virgin or pregnant rats (χ22 = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 μg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s−1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect phosphorylation of extracellular regulated kinase 1 or 2, or of Akt in the supraoptic or paraventricular nuclei of virgin or lactating rats. Hence, prolactin inhibition of oxytocin neurones is lost in lactation, which might allow concurrent elevation of prolactin secretion from the pituitary gland and activation of oxytocin neurones for synthesis and delivery of milk to the newborn.

  March 23, 2017   doi: 10.1113/JP273712   open full text
• Frequency and function in the basal ganglia: the origins of beta and gamma band activity.
  Alexander Blenkinsop, Sean Anderson, Kevin Gurney.
  The Journal of Physiology. March 23, 2017
  Neural oscillations in the basal ganglia are well studied yet remain poorly understood. Behavioural correlates of spectral activity are well described, yet a quantitative hypothesis linking time domain dynamics and spectral properties to basal ganglia function has been lacking. We show, for the first time, that a unified description is possible by interpreting previously ignored structure in data describing GPi responses to cortical stimulation. These data were used to expose a pair of distinctive neuronal responses to the stimulation. This observation formed the basis for a new mathematical model of the BG, quantitatively fitted to the data, which describes the dynamics in the data, and is validated against other stimulus protocol experiments. A key new result is that when the model is run using inputs hypothesised to occur during the performance of a motor task, beta and gamma frequency oscillations emerge naturally during static‐force and movement respectively, consistent with experimental local field potentials. This new model predicts that the pallido‐striatum connection has a key role in the generation of beta band activity, and that the gamma band activity associated with motor task performance has its origins in the pallido‐subthalamic feedback loop. The network's functionality as a selection‐mechanism also occurs as an emergent property, and closer fits to the data gave better selection properties. The model provides a coherent framework for the study of spectral, temporal and functional analyses of the BG and therefore lays the foundation for an integrated approach to study BG pathologies such as Parkinson's disease in silico. This article is protected by copyright. All rights reserved

  March 23, 2017   doi: 10.1113/JP273760   open full text
• Protein kinase A regulates C‐terminally truncated CaV1.2 in Xenopus oocytes: roles of N‐ and C‐termini of the α1C subunit.
  Shimrit Oz, Ines Pankonien, Anouar Belkacemi, Veit Flockerzi, Enno Klussmann, Hannelore Haase, Nathan Dascal.
  The Journal of Physiology. March 23, 2017
  Key points β‐Adrenergic stimulation enhances Ca2+ entry via L‐type CaV1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV1.2 remain controversial despite extensive research. We show that PKA regulation of CaV1.2 can be reconstituted in Xenopus oocytes when the distal C‐terminus (dCT) of the main subunit, α1C, is truncated. The PKA upregulation of CaV1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase‐anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV1.2. The gating element within the initial segment of the N‐terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV1.2 in the heart. Abstract β‐Adrenergic stimulation enhances Ca2+ currents via L‐type, voltage‐gated CaV1.2 channels, strengthening cardiac contraction. The signalling via β‐adrenergic receptors (β‐ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post‐translational proteolytic truncation of the distal C‐terminus (dCT) of the main (α1C) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT‐truncated CaV1.2 in Xenopus oocytes, which previously failed with the non‐truncated α1C. cAMP and the purified catalytic subunit of PKA, PKA‐CS, injected into intact oocytes, enhanced CaV1.2 currents by ∼40% (rabbit α1C) to ∼130% (mouse α1C). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase‐anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaVβ2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long‐N‐terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV1.2 in the heart. We hypothesize that PKA is acting on CaV1.2, in part, by affecting a structural ‘scaffold’ comprising the interacting cytosolic N‐ and C‐termini of α1C.

  March 23, 2017   doi: 10.1113/JP274015   open full text
• T‐type calcium channels contribute to NMDA receptor independent synaptic plasticity in hippocampal regular‐spiking oriens‐alveus interneurons.
  Elizabeth Nicholson, Dimitri M. Kullmann.
  The Journal of Physiology. March 22, 2017
  Key points Regular‐spiking interneurons in the hippocampal stratum oriens exhibit a form of long‐term potentiation of excitatory transmission that is independent of NMDA receptors but requires co‐activation of Ca2+‐permeable AMPA receptors and group I metabotropic glutamate receptors. We show that T‐type Ca2+ channels are present in such interneurons. Blockade of T‐type currents prevents the induction of long‐term potentiation, and also interferes with long‐lasting potentiation induced either by postsynaptic trains of action potentials or by pairing postsynaptic hyperpolarization with activation of group I metabotropic receptors. Several Ca2+ sources thus converge on the induction of NMDA receptor independent synaptic plasticity. Abstract NMDA receptor independent long‐term potentiation (LTP) in hippocampal stratum oriens‐alveus (O/A) interneurons requires co‐activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and Ca2+‐permeable AMPA receptors. The rectification properties of such AMPA receptors contribute to the preferential induction of LTP at hyperpolarized potentials. A persistent increase in excitatory transmission can also be triggered by exogenous activation of group I mGluRs at the same time as the interneuron is hyperpolarized, or by postsynaptic trains of action potentials in the absence of presynaptic stimulation. In the present study, we identify low‐threshold transient (T‐type) channels as a further source of Ca2+ that contributes to synaptic plasticity. T‐type Ca2+ currents were detected in mouse regular‐spiking O/A interneurons. Blocking T‐type currents pharmacologically prevented LTP induced by high‐frequency stimulation of glutamatergic axons, or by application of the group I mGluR agonist dihydroxyphenylglycine, paired with postsynaptic hyperpolarization. T‐type current blockade also prevented synaptic potentiation induced by postsynaptic action potential trains. Several sources of Ca2+ thus converge on NMDA receptor independent LTP induction in O/A interneurons.

  March 22, 2017   doi: 10.1113/JP273695   open full text
• Voltage‐sensitive conductances increase the sensitivity of rod photoresponses following pigment bleaching.
  Johan Pahlberg, Rikard Frederiksen, Gabriel E. Pollock, Kiyoharu J. Miyagishima, Alapakkam P. Sampath, M. Carter Cornwall.
  The Journal of Physiology. March 22, 2017
  Key points Following substantial bleaching of the visual pigment, the desensitization of the rod photovoltage is not as substantial as the desensitization of the rod outer segment photocurrent. The block of cation conductances during the internal dialysis of Cs+ further desensitizes the photovoltage thereby eliminating its difference in desensitization with the rod outer segment photocurrent. Bleached visual pigment produced an acceleration of the rod photovoltage with respect to the outer segment photocurrent, which is eliminated upon internal dialysis of Cs+. Abstract A majority of our visual experience occurs during the day when a substantial fraction of the visual pigment in our photoreceptor cells is bleached. Under these conditions it is widely believed that rods are saturated and do not contribute substantially to downstream signalling. However, behavioural experiments on subjects with only rod function reveals that these individuals unexpectedly retain substantial vision in daylight. We sought to understand this discrepancy by characterizing the sensitivity of rod photoresponses following exposure to bright bleaching light. Measurements of the rod outer segment photocurrent in transgenic mice, which have only rod function, revealed the well‐studied reduction in the sensitivity of rod photoresponses following pigment bleaching. However, membrane voltage measurements showed that the desensitization of the photovoltage was considerably less than that of the outer segment photocurrent following equivalent pigment bleaching. This discrepancy was largely eliminated during the blockade of cation channels due to the internal dialysis of Cs+, which increased the bleach‐induced desensitization of the photovoltage and slowed its temporal characteristics. Thus, sensitization of the photovoltage by rod inner segment conductances appears to extend the operating range of rod phototransduction following pigment bleaching.

  March 22, 2017   doi: 10.1113/JP273398   open full text
• Diuretic‐sensitive electroneutral Na+ movement and temperature effects on central axons.
  Meneka Kanagaratnam, Christopher Pendleton, Danilo Almeida Souza, Joseph Pettit, James Howells, Mark D. Baker.
  The Journal of Physiology. March 22, 2017
  Key points Optic nerve axons get less excitable with warming. F‐fibre latency does not shorten at temperatures above 30°C. Action potential amplitude falls when the Na+‐pump is blocked, an effect speeded by warming. Diuretics reduce the rate of action potential fall in the presence of ouabain. Our data are consistent with electroneutral entry of Na+ occurring in axons and contributing to setting the resting potential. Abstract Raising the temperature of optic nerve from room temperature to near physiological has effects on the threshold, refractoriness and superexcitability of the shortest latency (fast, F) nerve fibres, consistent with hyperpolarization. The temperature dependence of peak impulse latency was weakened at temperatures above 30°C suggesting a temperature‐sensitive process that slows impulse propagation. The amplitude of the supramaximal compound action potential gets larger on warming, whereas in the presence of bumetanide and amiloride (blockers of electroneutral Na+ movement), the action potential amplitude consistently falls. This suggests a warming‐induced hyperpolarization that is reduced by blocking electroneutral Na+ movement. In the presence of ouabain, the action potential collapses. This collapse is speeded by warming, and exposure to bumetanide and amiloride slows the temperature‐dependent amplitude decline, consistent with a warming‐induced increase in electroneutral Na+ entry. Blocking electroneutral Na+ movement is predicted to be useful in the treatment of temperature‐dependent symptoms under conditions with reduced safety factor (Uhthoff's phenomenon) and provide a route to neuroprotection.

  March 22, 2017   doi: 10.1113/JP273963   open full text
• Lysophosphatidic acid‐induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.
  Hiroki Kittaka, Kunitoshi Uchida, Naomi Fukuta, Makoto Tominaga.
  The Journal of Physiology. March 22, 2017
  Key points Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA‐induced itch‐related behaviours are decreased in TRPA1‐knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+‐independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672–673 and KR977–978 (K: lysine, R: arginine). Abstract Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch‐related behaviours but not pain‐related behaviours. The LPA‐induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA‐induced itch is mediated by LPA5, PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

  March 22, 2017   doi: 10.1113/JP273961   open full text
• Requirement of extracellular Ca2+ binding to specific amino acids for heat‐evoked activation of TRPA1.
  Erkin Kurganov, Shigeru Saito, Claire Tanaka Saito, Makoto Tominaga.
  The Journal of Physiology. March 22, 2017
  Key points We found that extracellular Ca2+, but not other divalent cations (Mg2+ and Ba2+) or intracellular Ca2+, is involved in heat‐evoked activation of green anole (ga) TRPA1. Heat‐evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+. Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat‐evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. Abstract Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non‐selective cation‐permeable channel that has six transmembrane domains and cytoplasmic N‐ and C‐termini. The N‐terminus is characterized by an unusually large number of ankyrin repeats. Although the 3‐dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature‐dependent TRPA1 activation is unclear. We previously reported that extracellular Ca2+, but not intracellular Ca2+, plays an important role in heat‐evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+‐dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat‐activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca2+, rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat‐evoked TRPA1 activation in the presence of extracellular Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat‐evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat‐evoked channel activation.

  March 22, 2017   doi: 10.1113/JP274083   open full text
• Cardiac diastolic and autonomic dysfunction are aggravated by central chemoreflex activation in heart failure with preserved ejection fraction rats.
  Camilo Toledo, David C. Andrade, Claudia Lucero, Alexis Arce‐Alvarez, Hugo S. Díaz, Valentín Aliaga, Harold D. Schultz, Noah J. Marcus, Mónica Manríquez, Marcelo Faúndez, Rodrigo Del Rio.
  The Journal of Physiology. March 19, 2017
  Key points Heart failure with preserved ejection fraction (HFpEF) is associated with disordered breathing patterns, and sympatho‐vagal imbalance. Although it is well accepted that altered peripheral chemoreflex control plays a role in the progression of heart failure with reduced ejection fraction (HFrEF), the pathophysiological mechanisms underlying deterioration of cardiac function in HFpEF are poorly understood. We found that central chemoreflex is enhanced in HFpEF and neuronal activation is increased in pre‐sympathetic regions of the brainstem. Our data showed that activation of the central chemoreflex pathway in HFpEF exacerbates diastolic dysfunction, worsens sympatho‐vagal imbalance and markedly increases the incidence of cardiac arrhythmias in rats with HFpEF. Abstract Heart failure (HF) patients with preserved ejection fraction (HFpEF) display irregular breathing, sympatho‐vagal imbalance, arrhythmias and diastolic dysfunction. It has been shown that tonic activation of the central and peripheral chemoreflex pathway plays a pivotal role in the pathophysiology of HF with reduced ejection fraction. In contrast, no studies to date have addressed chemoreflex function or its effect on cardiac function in HFpEF. Therefore, we tested whether peripheral and central chemoreflexes are hyperactive in HFpEF and if chemoreflex activation exacerbates cardiac dysfunction and autonomic imbalance. Sprague‐Dawley rats (n = 32) were subjected to sham or volume overload to induce HFpEF. Resting breathing variability, chemoreflex gain, cardiac function and sympatho‐vagal balance, and arrhythmia incidence were studied. HFpEF rats displayed [mean ± SD; chronic heart failure (CHF) vs. Sham, respectively] a marked increase in the incidence of apnoeas/hypopnoeas (20.2 ± 4.0 vs. 9.7 ± 2.6 events h−1), autonomic imbalance [0.6 ± 0.2 vs. 0.2 ± 0.1 low/high frequency heart rate variability (LF/HFHRV)] and cardiac arrhythmias (196.0 ± 239.9 vs. 19.8 ± 21.7 events h−1). Furthermore, HFpEF rats showed increase central chemoreflex sensitivity but not peripheral chemosensitivity. Accordingly, hypercapnic stimulation in HFpEF rats exacerbated increases in sympathetic outflow to the heart (229.6 ± 43.2% vs. 296.0 ± 43.9% LF/HFHRV, normoxia vs. hypercapnia, respectively), incidence of cardiac arrhythmias (196.0 ± 239.9 vs. 576.7 ± 472.9 events h−1) and diastolic dysfunction (0.008 ± 0.004 vs. 0.027 ± 0.027 mmHg μl−1). Importantly, the cardiovascular consequences of central chemoreflex activation were related to sympathoexcitation since these effects were abolished by propranolol. The present results show that the central chemoreflex is enhanced in HFpEF and that acute activation of central chemoreceptors leads to increases of cardiac sympathetic outflow, cardiac arrhythmogenesis and impairment in cardiac function in rats with HFpEF.

  March 19, 2017   doi: 10.1113/JP273558   open full text
• The influence of adrenergic stimulation on sex differences in left ventricular twist mechanics.
  Alexandra M. Williams, Rob E. Shave, William S. Cheyne, Neil D. Eves.
  The Journal of Physiology. March 19, 2017
  Key points Sex differences in left ventricular (LV) mechanics occur during acute physiological challenges; however, it is unknown whether sex differences in LV mechanics are fundamentally regulated by differences in adrenergic control. Using two‐dimensional echocardiography and speckle tracking analysis, this study compared LV mechanics in males and females matched for LV length during post‐exercise ischaemia (PEI) and β1‐adrenergic receptor blockade. Our data demonstrate that while basal rotation was increased in males, LV twist was not significantly different between the sexes during PEI. In contrast, during β1‐adrenergic receptor blockade, LV apical rotation, twist and untwisting velocity were reduced in males compared to females. Significant relationships were observed between LV twist and LV internal diameter and sphericity index in females, but not males. These findings suggest that LV twist mechanics may be more sensitive to alterations in adrenergic stimulation in males, but more highly influenced by ventricular structure and geometry in females. Abstract Sex differences in left ventricular (LV) mechanics exist at rest and during acute physiological stress. Differences in cardiac autonomic and adrenergic control may contribute to sex differences in LV mechanics and LV haemodynamics. Accordingly, this study aimed to investigate sex differences in LV mechanics with altered adrenergic stimulation achieved through post‐handgrip‐exercise ischaemia (PEI) and β1‐adrenergic receptor (AR) blockade. Twenty males (23 ± 5 years) and 20 females (22 ± 3 years) were specifically matched for LV length (males: 8.5 ± 0.5 cm, females: 8.2 ± 0.6 cm, P = 0.163), and two‐dimensional speckle‐tracking echocardiography was used to assess LV structure and function at baseline, during PEI and following administration of 5 mg bisoprolol (β1‐AR antagonist). During PEI, LV end‐diastolic volume and stroke volume were increased in both groups (P < 0.001), as was end‐systolic wall stress (P < 0.001). LV twist and apical rotation were not altered from baseline or different between the sexes; however, basal rotation increased in males (P = 0.035). During β1‐AR blockade, LV volumes were unchanged but blood pressure and heart rate were reduced in both groups (P < 0.001). LV apical rotation (P = 0.036) and twist (P = 0.029) were reduced in males with β1‐AR blockade but not females, resulting in lower apical rotation (males: 6.8 ± 2.1 deg, females: 8.8 ± 2.3 deg, P = 0.007) and twist (males: 8.6 ± 1.9 deg, females: 10.7 ± 2.8 deg, P = 0.008), and slower untwisting velocity (males: 68.2 ± 22.1 deg s−1, females: 82.0 ± 18.7 deg s−1, P = 0.046) compared to females. LV twist mechanics are reduced in males compared to females during reductions to adrenergic stimulation, providing preliminary evidence that LV twist mechanics may be more sensitive to adrenergic control in males than in females.

  March 19, 2017   doi: 10.1113/JP273368   open full text
• Mechanisms of pruritogen‐induced activation of itch nerves in isolated mouse skin.
  F. Ru, H. Sun, D. Jurcakova, R. A. Herbstsomer, J. Meixong, X. Dong, B. J. Undem.
  The Journal of Physiology. March 19, 2017
  Key points Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G‐protein coupled receptor (GPCR) MrgprA3; it is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that transient receptor potential A1 (TRPA1) gene deletion blocks CQ‐induced scratching. In the present study we used a novel dorsal skin–nerve preparation to evaluate mechanisms underlying CQ‐ and histamine‐induced action potential discharge in itch nerve terminals. We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, but TRPA1 or other TRP channel are not required. Evidence is provided for a role for calcium‐activated chloride channels such as TMEM16a in GPCR‐activation of itch nerve terminals. The mechanism by which TRP channels participate in pruritogen‐induced scratching may involve sites of action other than the primary afferent terminals. Abstract Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin–nerve preparation was used to evaluate chloroquine (CQ)‐ and histamine‐induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C‐fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr‐cluster Δ−/− mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ‐induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild‐type and TRPA1‐deficient mice. A selective inhibitor of the calcium‐activated chloride channel TMEM16A, N‐((4‐methoxy)‐2‐naphthyl)‐5‐nitroanthranilic acid (MONNA), inhibited CQ‐induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role.

  March 19, 2017   doi: 10.1113/JP273795   open full text
• Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short‐term facilitation at mossy fibre to CA3 pyramidal cell synapses.
  Sam A. Booker, Graham R. Campbell, Karolina S. Mysiak, Peter J. Brophy, Peter C. Kind, Don J. Mahad, David J. A. Wyllie.
  The Journal of Physiology. March 15, 2017
  Key points Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission. High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice. Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function. Abstract Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole‐cell patch‐clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV‐deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy‐fibre synapse because the amplitude, input–output relationship and 50 ms paired‐pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short‐term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired‐pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy‐fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short‐term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction.

  March 15, 2017   doi: 10.1113/JP273581   open full text
• The role of dentate nuclei in human oculomotor control: insights from cerebrotendinous xanthomatosis.
  Francesca Rosini, Elena Pretegiani, Andrea Mignarri, Lance M. Optican, Valeria Serchi, Nicola Stefano, Marco Battaglini, Lucia Monti, Maria T. Dotti, Antonio Federico, Alessandra Rufa.
  The Journal of Physiology. March 14, 2017
  Key points A cerebellar dentate nuclei (DN) contribution to volitional oculomotor control has recently been hypothesized but not fully understood. Cerebrotendinous xanthomatosis (CTX) is a rare neurometabolic disease typically characterized by DN damage. In this study, we compared the ocular movement characteristics of two sets of CTX patients, with and without brain MRI evidence of DN involvement, with a set of healthy subjects. Our results suggest that DN participate in voluntary behaviour, such as the execution of antisaccades, and moreover are involved in controlling the precision of the ocular movement. The saccadic abnormalities related to DN involvement were independent of global and regional brain atrophy. Our study confirms the relevant role of DN in voluntary aspects of oculomotion and delineates specific saccadic abnormalities that could be used to detect the involvement of DN in other cerebellar disorders. Abstract It is well known that the medial cerebellum controls saccadic speed and accuracy. In contrast, the role of the lateral cerebellum (cerebellar hemispheres and dentate nuclei, DN) is less well understood. Cerebrotendinous xanthomatosis (CTX) is a lipid storage disorder due to mutations in CYP27A1, typically characterized by DN damage. CTX thus provides a unique opportunity to study DN in human oculomotor control. We analysed horizontal and vertical visually guided saccades and horizontal antisaccades of 19 CTX patients. Results were related to the presence/absence of DN involvement and compared with those of healthy subjects. To evaluate the contribution of other areas, abnormal saccadic parameters were compared with global and regional brain volumes. CTX patients executed normally accurate saccades with normal main sequence relationships, indicating that the brainstem and medial cerebellar structures were functionally spared. Patients with CTX executed more frequent multistep saccades and directional errors during the antisaccade task than controls. CTX patients with DN damage showed less precise saccades with longer latencies, and more frequent directional errors, usually not followed by corrections, than either controls or patients without DN involvement. These saccadic abnormalities related to DN involvement but were independent of global and regional brain atrophy. We hypothesize that two different cerebellar networks contribute to the metrics of a movement: the medial cerebellar structures determine accuracy, whereas the lateral cerebellar structures control precision. The lateral cerebellum (hemispheres and DN) also participates in modulating goal directed gaze behaviour, by prioritizing volitional over reflexive movements.

  March 14, 2017   doi: 10.1113/JP273670   open full text
• Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel.
  Etsuko Minobe, Masayuki X. Mori, Masaki Kameyama.
  The Journal of Physiology. March 13, 2017
  Key points Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent. Abstract Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed.

  March 13, 2017   doi: 10.1113/JP273736   open full text
• Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation.
  Shelley E. Harris, Miles J. Blasio, Melissa A. Davis, Amy C. Kelly, Hailey M. Davenport, F. B. Peter Wooding, Dominique Blache, David Meredith, Miranda Anderson, Abigail L. Fowden, Sean W. Limesand, Alison J. Forhead.
  The Journal of Physiology. March 13, 2017
  Key points Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose‐dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas. Abstract Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long‐term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T3), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic islets isolated from intact fetal sheep, beta cell proliferation in vitro was reduced by T3 in a dose‐dependent manner and increased by insulin at high concentrations only. Leptin induced a bimodal response whereby beta cell proliferation was suppressed at the lowest, and increased at the highest, concentrations. Therefore, proliferation of beta cells isolated from the ovine fetal pancreas is sensitive to physiological concentrations of T3, insulin and leptin. Alterations in these hormones may be responsible for the increased beta cell proliferation and mass observed in the hypothyroid sheep fetus and may have consequences for pancreatic function in later life.

  March 13, 2017   doi: 10.1113/JP273555   open full text
• KATP channel inhibition blunts electromechanical decline during hypoxia in left ventricular working rabbit hearts.
  Kara Garrott, Sarah Kuzmiak‐Glancy, Anastasia Wengrowski, Hanyu Zhang, Jack Rogers, Matthew W. Kay.
  The Journal of Physiology. March 13, 2017
  Key points Heart function is critically dependent upon the balance of energy production and utilization. Sarcolemmal ATP‐sensitive potassium channels (KATP channels) in cardiac myocytes adjust contractile function to compensate for the level of available energy. Understanding the activation of KATP channels in working myocardium during high‐stress situations is crucial to the treatment of cardiovascular disease, especially ischaemic heart disease. Using a new optical mapping approach, we measured action potentials from the surface of excised contracting rabbit hearts to assess when sarcolemmal KATP channels were activated during physiologically relevant workloads and during gradual reductions in myocardial oxygenation. We demonstrate that left ventricular pressure is closely linked to KATP channel activation and that KATP channel inhibition with a low concentration of tolbutamide prevents electromechanical decline when oxygen availability is reduced. As a result, KATP channel inhibition probably exacerbates a mismatch between energy demand and energy production when myocardial oxygenation is low. Abstract Sarcolemmal ATP‐sensitive potassium channel (KATP channel) activation in isolated cells is generally understood, although the relationship between myocardial oxygenation and KATP activation in excised working rabbit hearts remains unknown. We optically mapped action potentials (APs) in excised rabbit hearts to test the hypothesis that hypoxic changes would be more severe in left ventricular (LV) working hearts (LWHs) than Langendorff (LANG) perfused hearts. We further hypothesized that KATP inhibition would prevent those changes. Optical APs were mapped when measuring LV developed pressure (LVDP), coronary flow rate and oxygen consumption in LANG and LWHs. Hearts were paced to increase workload and perfusate was deoxygenated to study the effects of myocardial hypoxia. A subset of hearts was perfused with 1 μm tolbutamide (TOLB) to identify the level of AP duration (APD) shortening attributed to KATP channel activation. During sinus rhythm, APD was shorter in LWHs compared to LANG hearts. APD in both LWHs and LANG hearts dropped steadily during deoxygenation. With TOLB, APDs in LWHs were longer at all workloads and APD reductions during deoxygenation were blunted in both LWHs and LANG hearts. At 50% perfusate oxygenation, APD and LVDP were significantly higher in LWHs perfused with TOLB (199 ± 16 ms; 92 ± 5.3 mmHg) than in LWHs without TOLB (109 ± 14 ms, P = 0.005; 65 ± 6.5 mmHg, P = 0.01). Our results indicate that KATP channels are activated to a greater extent in perfused hearts when the LV performs pressure–volume work. The results of the present study demonstrate the critical role of KATP channels in modulating myocardial function over a wide range of physiological conditions.

  March 13, 2017   doi: 10.1113/JP273873   open full text
• Relationship between cortical state and spiking activity in the lateral geniculate nucleus of marmosets.
  Alexander N.J. Pietersen, Soon Keen Cheong, Brandon Munn, Pulin Gong, Paul R. Martin, Samuel G. Solomon.
  The Journal of Physiology. March 10, 2017
  Key points How parallel are the primate visual pathways? In the present study, we demonstrate that parallel visual pathways in the dorsal lateral geniculate nucleus (LGN) show distinct patterns of interaction with rhythmic activity in the primary visual cortex (V1). In the V1 of anaesthetized marmosets, the EEG frequency spectrum undergoes transient changes that are characterized by fluctuations in delta‐band EEG power. We show that, on multisecond timescales, spiking activity in an evolutionary primitive (koniocellular) LGN pathway is specifically linked to these slow EEG spectrum changes. By contrast, on subsecond (delta frequency) timescales, cortical oscillations can entrain spiking activity throughout the entire LGN. Our results are consistent with the hypothesis that, in waking animals, the koniocellular pathway selectively participates in brain circuits controlling vigilance and attention. Abstract The major afferent cortical pathway in the visual system passes through the dorsal lateral geniculate nucleus (LGN), where nerve signals originating in the eye can first interact with brain circuits regulating visual processing, vigilance and attention. In the present study, we investigated how ongoing and visually driven activity in magnocellular (M), parvocellular (P) and koniocellular (K) layers of the LGN are related to cortical state. We recorded extracellular spiking activity in the LGN simultaneously with local field potentials (LFP) in primary visual cortex, in sufentanil‐anaesthetized marmoset monkeys. We found that asynchronous cortical states (marked by low power in delta‐band LFPs) are linked to high spike rates in K cells (but not P cells or M cells), on multisecond timescales. Cortical asynchrony precedes the increases in K cell spike rates by 1–3 s, implying causality. At subsecond timescales, the spiking activity in many cells of all (M, P and K) classes is phase‐locked to delta waves in the cortical LFP, and more cells are phase‐locked during synchronous cortical states than during asynchronous cortical states. The switch from low‐to‐high spike rates in K cells does not degrade their visual signalling capacity. By contrast, during asynchronous cortical states, the fidelity of visual signals transmitted by K cells is improved, probably because K cell responses become less rectified. Overall, the data show that slow fluctuations in cortical state are selectively linked to K pathway spiking activity, whereas delta‐frequency cortical oscillations entrain spiking activity throughout the entire LGN, in anaesthetized marmosets.

  March 10, 2017   doi: 10.1113/JP273569   open full text
• The TRPM7 channel kinase regulates store‐operated calcium entry.
  Malika Faouzi, Tatiana Kilch, F. David Horgen, Andrea Fleig, Reinhold Penner.
  The Journal of Physiology. March 10, 2017
  Key points Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store‐operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7−/− cells restores SOCE. TRPM7 is not a store‐operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. Abstract The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store‐operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7‐deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild‐type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell‐cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild‐type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store‐operated channel itself. Using kinase‐deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.

  March 10, 2017   doi: 10.1113/JP274006   open full text
• Perinatal nicotine exposure impairs the maturation of glutamatergic inputs in the auditory brainstem.
  Veronika J. Baumann, Ursula Koch.
  The Journal of Physiology. March 10, 2017
  Key points Chronic perinatal nicotine exposure causes abnormal auditory brainstem responses and auditory processing deficits in children and animal models. The effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem was investigated in granule cells in the ventral nucleus of the lateral lemniscus, which receive a single calyx‐like input from the cochlear nucleus. Perinatal nicotine exposure caused a massive reduction in the amplitude of the excitatory input current. This caused a profound decrease in the number and temporal precision of spikes in these neurons. Perinatal nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons. Abstract Maternal smoking causes chronic nicotine exposure during early development and results in auditory processing deficits including delayed speech development and learning difficulties. Using a mouse model of chronic, perinatal nicotine exposure we explored to what extent synaptic inputs to granule cells in the ventral nucleus of the lateral lemniscus are affected by developmental nicotine treatment. These neurons receive one large calyx‐like input from octopus cells in the cochlear nucleus and play a role in sound pattern analysis, including speech sounds. In addition, they exhibit high levels of α7 nicotinic acetylcholine receptors, especially during early development. Our whole‐cell patch‐clamp experiments show that perinatal nicotine exposure causes a profound reduction in synaptic input amplitude. In contrast, the number of inputs innervating each neuron and synaptic release properties of this calyx‐like synapse remained unaltered. Spike number and spiking precision in response to synaptic stimulation were greatly diminished, especially for later stimuli during a stimulus train. Moreover, chronic nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons, indicating a direct action of nicotine in this brain area. This presumably direct effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem might be one of the underlying causes for auditory processing difficulties in children of heavy smoking mothers.

  March 10, 2017   doi: 10.1113/JP274059   open full text
• Exploratory assessment of left ventricular strain–volume loops in severe aortic valve diseases.
  Hugo G. Hulshof, Arie P. Dijk, Keith P. George, Maria T. E. Hopman, Dick H. J. Thijssen, David L. Oxborough.
  The Journal of Physiology. March 09, 2017
  Key points Severe aortic valve diseases are common cardiac abnormalities that are associated with poor long‐term survival. Before any reduction in left ventricular (LV) function, the left ventricle undergoes structural remodelling under the influence of changing haemodynamic conditions. In this study, we combined temporal changes in LV structure (volume) with alterations in LV functional characteristics (strain, ԑ) into a ԑ–volume loop, in order to provide novel insight into the haemodynamic cardiac consequences of aortic valve diseases in those with preserved LV ejection fraction. We showed that our novel ԑ–volume loop and the specific loop characteristics provide additional insight into the functional and mechanical haemodynamic consequences of severe aortic valve diseases (with preserved LV ejection fraction). Finally, we showed that the ԑ–volume loop characteristics provide discriminative capacity compared with conventional measures of LV function. Abstract The purpose of this study was to examine left ventricular (LV) strain (ԑ)–volume loops to provide novel insight into the haemodynamic cardiac consequences of aortic valve stenosis (AS) and aortic valve regurgitation (AR). Twenty‐seven participants were retrospectively recruited: AR (n = 7), AS (n = 10) and control subjects (n = 10). Standard transthoracic echocardiography was used to obtain apical four‐chamber images to construct ԑ–volume relationships, which were assessed using the following parameters: early systolic ԑ (ԑ_ES); slope of ԑ–volume relationship during systole (Sslope); end‐systolic peak ԑ (peak ԑ); and diastolic uncoupling (systolic ԑ–diastolic ԑ at same volume) during early diastole (UNCOUP_ED) and late diastole (UNCOUP_LD). Receiver operating characteristic curves were used to determine the ability to detect impaired LV function. Although LV ejection fraction was comparable between groups, longitudinal peak ԑ was reduced compared with control subjects. In contrast, ԑ_ES and Sslope were lower in both pathologies compared with control subejcts (P < 0.01), but also different between AS and AR (P < 0.05). UNCOUP_ED and UNCOUP_LD were significantly higher in both patient groups compared with control subjects (P < 0.05). Receiver operating characteristic curves revealed that loop characteristics (AUC = 0.99, 1.00 and 1.00; all P < 0.01) were better able then peak ԑ (AUC = 0.75, 0.89 and 0.76; P = 0.06, <0.01 and 0.08, respectively) and LV ejection fraction (AUC = 0.56, 0.69 and 0.69; all P > 0.05) to distinguish AS vs control, AR vs control and AS vs AR groups, respectively. Temporal changes in ԑ–volume characteristics provide novel insight into the haemodynamic cardiac impact of AS and AR. Contrary to traditional measures (i.e. ejection fraction, peak ԑ), these novel measures successfully distinguish between the haemodynamic cardiac impact of AS and AR.

  March 09, 2017   doi: 10.1113/JP273526   open full text
• Low pHo boosts burst firing and catecholamine release by blocking TASK‐1 and BK channels while preserving Cav1 channels in mouse chromaffin cells.
  Laura Guarina, David H. F. Vandael, Valentina Carabelli, Emilio Carbone.
  The Journal of Physiology. March 02, 2017
  Key points Mouse chromaffin cells (MCCs) generate spontaneous burst‐firing that causes large increases of Ca2+‐dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs. With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo) from 7.4 to 6.6 induces cell depolarizations of 10–15 mV that generate bursts of ∼330 ms at 1–2 Hz and a 7.4‐fold increase of cumulative catecholamine‐release. Burst‐firing originates from the inhibition of the pH‐sensitive TASK‐1‐channels and a 60% reduction of BK‐channel conductance at pHo 6.6. Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine. MCCs act as pH‐sensors. At low pHo, they depolarize, undergo burst‐firing and increase catecholamine‐secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue. Abstract Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+‐dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common ‘tonic‐irregular’ to the less frequent ‘burst’ firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1β2‐subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+‐entry and potentiates CA release by ∼3.5‐fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10–15 mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330 ms, occurred at 1–2 Hz and caused an ∼7‐fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH‐sensitive TASK‐1/TASK‐3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK‐1 blocker A1899 (300 nm) and BK blocker paxilline (300 nm) and could be prevented by blocking L‐type channels by adding 3 μm nifedipine. Mixtures of the two blockers raised cumulative CA‐secretion even more than low pHo (∼12‐fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+‐entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA‐secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.

  March 02, 2017   doi: 10.1113/JP273735   open full text
• Calcium‐calmodulin‐dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.
  Marilen Federico, Enrique L. Portiansky, Leandro Sommese, Francisco J. Alvarado, Paula G. Blanco, Carolina N. Zanuzzi, John Dedman, Marcia Kaetzel, Xander H. T. Wehrens, Alicia Mattiazzi, Julieta Palomeque.
  The Journal of Physiology. March 02, 2017
  Key points Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose‐rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD‐WT mice, which was prevented by co‐treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD‐SR‐autocamide inhibitory peptide (AIP) mice, expressing the SR‐targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD‐SR‐AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD‐WT mice but not in FRD‐S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. Abstract The impact of cardiac apoptosis in pre‐diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose‐rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin‐protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre‐diabetes. We generated a pre‐diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control‐diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia‐induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD‐WT mice. Co‐treatment with the reactive oxygen species scavenger Tempol prevented FRD‐induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD‐SR‐AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD‐WT hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre‐diabetic heart. The FRD‐induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.

  March 02, 2017   doi: 10.1113/JP273714   open full text
• Visceral and somatic pain modalities reveal NaV1.7‐independent visceral nociceptive pathways.
  James R. F. Hockley, Rafael González‐Cano, Sheridan McMurray, Miguel A. Tejada‐Giraldez, Cian McGuire, Antonio Torres, Anna L. Wilbrey, Vincent Cibert‐Goton, Francisco R. Nieto, Thomas Pitcher, Charles H. Knowles, José Manuel Baeyens, John N. Wood, Wendy J. Winchester, David C. Bulmer, Cruz Miguel Cendán, Gordon McMurray.
  The Journal of Physiology. March 01, 2017
  Key points Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics. Abstract Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

  March 01, 2017   doi: 10.1113/JP272837   open full text
• Pre‐ischaemic mitochondrial substrate constraint by inhibition of malate‐aspartate shuttle preserves mitochondrial function after ischaemia–reperfusion.
  Nichlas Riise Jespersen, Takashi Yokota, Nicolaj Brejnholt Støttrup, Andreas Bergdahl, Kim Bolther Pælestik, Jonas Agerlund Povlsen, Flemming Dela, Hans Erik Bøtker.
  The Journal of Physiology. February 27, 2017
  Key points Pre‐ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate‐aspartate shuttle (MAS), provides cardioprotection against ischaemia–reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR‐injured heart. Abstract Mitochondrial dysfunction plays a central role in ischaemia–reperfusion (IR) injury. Pre‐ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate‐aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR‐injured heart to the same extent as ischaemic preconditioning (IPC), which is a well‐validated cardioprotective strategy against IR injury. In the present study, we show that pre‐ischaemic administration of AOA preserved mitochondrial complex I‐linked state 3 respiration and fatty acid oxidation during late reperfusion in IR‐injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I‐linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR‐injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)‐cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR‐injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

  February 27, 2017   doi: 10.1113/JP273408   open full text
• Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states.
  Han‐Jun Wang, George J. Rozanski, Irving H. Zucker.
  The Journal of Physiology. February 27, 2017
  Key points Cardiac sympathetic afferents are considered to be essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia. However, a potential contribution of the CSAR control of cardiac dysfunction in both normal and chronic heart failure (CHF) states remains unknown. We found that activation of the CSAR evokes little increase in cardiac contractility with an exaggerated peripheral vasoconstriction in the CHF state. CSAR inhibition by epicardial lidocaine decreased cardiac contractility to a greater extent in CHF rats than sham rats. Furthermore, we also found that epicardial lidocaine paradoxically decreased left ventricular end‐diastolic pressure (LVEDP) and left ventricular end‐diastolic volume (preload) in CHF rats, which was not observed in sham rats. Chronic ablation of the CSAR by epicardial application of the afferent neurotoxin, RTX, selectively lowered diastolic blood pressure CHF rats. The observation suggests that CSAR has a differential effect on cardiac function in normal and CHF states. CSAR activation in normal state causes significant increase in cardiac contractility and cardiac output. Abstract The enhanced ‘cardiac sympathetic afferent reflex’ (CSAR) critically contributes to the exaggerated global sympathetic tone in chronic heart failure (CHF). However, a potential contribution of the cardio‐cardiac reflex control of cardiac function in both normal and CHF states remains unknown. In this study, we evaluated the effects of direct activation or inhibition of the CSAR on cardiac function by pressure–volume (P–V) loop analysis in ∼12‐week sham‐operated and myocardial infarcted (MI) rats. In sham rats, acute CSAR activation by epicardial application of bradykinin (BK) increased heart rate (HR), left ventricular systolic pressure (LVSP), the maximum first derivative of left ventricular pressure (dp/dtmax), and the slope of the end‐systolic P–V relationship (ESPVR), suggesting that acute CSAR activation in the normal state enhances myocardial contractility. CSAR activation also decreased left ventricular (LV) systolic and diastolic volumes with little effect on LV end‐diastolic pressure (LVEDP) or the end‐diastolic P–V relationship (EDPVR) in sham rats. Compared to sham, CHF rats exhibit a reduced increase in the slope of the ESPVR and dp/dtmax in response to BK, indicating a poor contractile response to CSAR activation. Interestingly, BK application in CHF rats increased cardiac systolic and diastolic volumes and further increased the elevated LVEDP, neither of which was seen in sham rats. Following CSAR inhibition by epicardial lidocaine, blood pressure, HR, LVSP, dp/dt, LVEDP and ESPVR decreased in CHF rats whereas lidocaine had little effect in sham rats, indicating that the CSAR is tonically active in CHF and contributes to cardiac dysfunction. Furthermore, we found that epicardial lidocaine paradoxically decreased LV end‐diastolic volume (preload) in CHF rats, which was not observed in sham rats. The decreased preload by lidocaine in CHF rats may be due to a reduction in peripheral vascular resistance since epicardial lidocaine significantly lowered peripheral (renal) sympathetic nerve activity in CHF rats but not in sham rats. Furthermore, chronic ablation of CSAR by epicardial application of a selective afferent neurotoxin, resiniferatoxin, selectively lowered diastolic blood pressure both at daytime and night‐time with less effect on systolic blood pressure in CHF rats. Our data suggest that there is an imbalance between cardiac and peripheral responses to CSAR in CHF animals compared to sham‐operated controls.

  February 27, 2017   doi: 10.1113/JP273764   open full text
• Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin‐3–RXFP3 signalling.
  Alan Kania, Anna Gugula, Agnieszka Grabowiecka, Camila Ávila, Tomasz Blasiak, Zenon Rajfur, Marian H. Lewandowski, Grzegorz Hess, Elena Timofeeva, Andrew L. Gundlach, Anna Blasiak.
  The Journal of Physiology. February 27, 2017
  Key points Relaxin‐3 is a stress‐responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3‐dependent, inhibitory action of relaxin‐3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin‐3. We observed a Gαi/o‐protein‐dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium‐sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin‐3‐immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin‐3‐containing fibres/terminals within the PVN. Abstract The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra‐PVN injections of the neuropeptide relaxin‐3 or selective relaxin‐3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin‐3, acting through its cognate G‐protein‐coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin‐3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3‐A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3‐A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri‐PVN and sparse intra‐PVN relaxin‐3‐immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin‐3 neurons, identify a strong inhibitory influence of relaxin‐3–RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

  February 27, 2017   doi: 10.1113/JP273787   open full text
• Cortical and reticular contributions to human precision and power grip.
  Toshiki Tazoe, Monica A. Perez.
  The Journal of Physiology. February 27, 2017
  Key points The corticospinal tract contributes to the control of finger muscles during precision and power grip. We explored the neural mechanisms contributing to changes in corticospinal excitability during these gripping configurations. Motor evoked potentials (MEPs) elicited by cortical, but not by subcortical, stimulation were more suppressed during power grip compared with precision grip and index finger abduction. Intracortical inhibition was more reduced during power grip compared with the other tasks. An acoustic startle cue, a stimulus that engages the reticular system, suppressed MEP size during power grip to a lesser extent than during the other tasks at a cortical level and this positively correlated with changes in intracortical inhibition. Our findings suggest that changes in corticospinal excitability during gross more than fine finger manipulations are largely cortical in origin and that the reticular system contributed, at least in part, to these effects. Abstract It is well accepted that the corticospinal tract contributes to the control of finger muscles during precision and power grip in humans but the neural mechanisms involved remain poorly understood. Here, we examined motor evoked potentials elicited by cortical and subcortical stimulation of corticospinal axons (MEPs and CMEPs, respectively) and the activity in intracortical circuits (suppression of voluntary electromyography) and spinal motoneurons (F‐waves) in an intrinsic hand muscle during index finger abduction, precision grip and power grip. We found that the size of MEPs, but not CMEPs, was more suppressed during power grip compared with precision grip and index finger abduction, suggesting a cortical origin for these effects. Notably, intracortical inhibition was more reduced during power grip compared with the other tasks. To further examine the origin of changes in intracortical inhibition we assessed the contribution of the reticular system, which projects to cortical neurons, and projects to spinal motoneurons controlling hand muscles. An acoustic startle cue, which engages the reticular system, suppressed MEP size during power grip to a lesser extent than during the other tasks and this positively correlated with changes in intracortical inhibition. A startle cue decreased intracortical inhibition, but not CMEPs, during power grip. F‐waves remained unchanged across conditions. Our novel findings show that changes in corticospinal excitability present during power grip compared with fine finger manipulations are largely cortical in origin and suggest that the reticular system contributed, at least in part, to these effects.

  February 27, 2017   doi: 10.1113/JP273679   open full text
• Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability.
  Vladimir Grubišić, Brian D. Gulbransen.
  The Journal of Physiology. February 22, 2017
  Key points The role of enteric glial cell activity in the acute regulation of epithelial barrier and secretomotor functions of the intestines under physiological conditions is not clear. We used transgenic mice to modify glial activity and found that enteric glia significantly contribute to the neurogenic ion transport while glial activity does not appear to play a major role in the acute regulation of barrier function. The selective activation of glial activity evoked electrogenic ion transport primarily through neural pathways and was sufficient to drive electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine and might provide novel therapeutic avenues for functional diarrheal diseases. Abstract Enteric glial cells are often implicated in the regulation of epithelial barrier and secretomotor functions of the intestines. But whether glial cell activity regulates these functions acutely under physiological conditions is not clear. We addressed this issue by using transgenic animal models to modify the activity of enteric glia, either reducing glial expression of connexin 43 in Sox10::CreERT2+/−/Cx43f/f mice or activating glial calcium responses in GFAP::hM3Dq mice, and tested the effects on colonic barrier function and electrogenic ion transport in Ussing chambers. We assessed neuronal‐dependent and ‐independent contributions by activating or inhibiting neurogenic activity with veratridine and tetrodotoxin, respectively. Our results show that the reduction of glial Cx43 expression in Sox10::CreERT2+/−/Cx43f/f mice significantly reduced neurogenic ion transport. The selective glial activation in tissues from GFAP::hM3Dq mice evoked electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport with veratridine and glial driven responses consisted of both tetrodotoxin‐sensitive and ‐insensitive components. The selective glial stimulation did not affect transmural ion conductance or cell‐impermeant dye flux but the baseline ion conductance was more variable in Sox10::CreERT2+/−/Cx43f/f tissues. Together, our findings show that glial activity contributes to the regulation of electrogenic ion transport in the intestine through effects on neurons and possibly direct effects on epithelial cells. However, glial activity does not appear to play a major role in the acute regulation of barrier function. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine.

  February 22, 2017   doi: 10.1113/JP273492   open full text
• Presynaptic inhibition of transient receptor potential vanilloid type 1 (TRPV1) receptors by noradrenaline in nociceptive neurons.
  Saikat Chakraborty, Vincent Elvezio, Martin Kaczocha, Mario Rebecchi, Michelino Puopolo.
  The Journal of Physiology. February 22, 2017
  Key points The transient receptor potential vanilloid type 1 (TRPV1) receptor is a polymodal molecular integrator in the pain pathway expressed in Aδ‐ and C‐fibre nociceptors and is responsible for the thermal hyperalgesia associated with inflammatory pain. Noradrenaline strongly inhibited the activity of TRPV1 channels in dorsal root ganglia neurons. The effect of noradrenaline was reproduced by clonidine and antagonized by yohimbine, consistent with contribution of α2 adrenergic receptors. The inhibitory effect of noradrenaline on TRPV1 channels was dependent on calcium influx and linked to calcium/calmodulin‐dependent protein kinase II. In spinal cord slices, clonidine reduced the frequency of capsaicin‐induced miniature EPSCs in the presence of tetrodotoxin and ω‐conotoxin‐MVIIC, consistent with inhibition of presynaptic TRPV1 channels by α2 adrenergic receptors. We suggest that modulation of presynaptic TRPV1 channels in nociceptive neurons by descending noradrenergic inputs may constitute a mechanism for noradrenaline to modulate incoming noxious stimuli in the dorsal horn of the spinal cord. Abstract The transient receptor potential vanilloid type 1 (TRPV1) receptor is a well‐known contributor to nociceptor excitability. To address whether noradrenaline can down‐regulate TRPV1 channel activity in nociceptors and reduce their synaptic transmission, the effects of noradrenaline and clonidine were tested on the capsaicin‐activated current recorded from acutely dissociated small diameter (<27 μm) dorsal root ganglia (DRG) neurons and on miniature (m)EPSCs recorded from large lamina I neurons in horizontal spinal cord slices. Noradrenaline or clonidine inhibited the capsaicin‐activated current by ∼60%, and the effect was reversed by yohimbine, confirming that it was mediated by activation of α2 adrenergic receptors. Similarly, clonidine reduced the frequency of capsaicin‐induced mEPSCs by ∼60%. Inhibition of capsaicin‐activated current by noradrenaline was mediated by GTP binding proteins, and was highly dependent on calcium influx. The inhibitory effect of noradrenaline on the capsaicin‐activated current was not affected either by blocking the activity of protein kinase A with H89, or by blocking the activity of protein kinase C with bisindolylmaleimide II. In contrast, when the calcium/calmodulin‐dependent protein kinase II (CaMKII) was blocked with KN‐93, the inhibitory effect of noradrenaline on the capsaicin‐activated current was greatly reduced, suggesting that activation of adrenergic receptors in DRG neurons is preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by noradrenaline in nociceptive neurons is a mechanism whereby noradrenaline may suppress incoming noxious stimuli at the primary synaptic afferents in the dorsal horn of the spinal cord.

  February 22, 2017   doi: 10.1113/JP273455   open full text
• Rapid limb‐specific modulation of vestibular contributions to ankle muscle activity during locomotion.
  Patrick A. Forbes, Mark Vlutters, Christopher J. Dakin, Herman der Kooij, Jean‐Sébastien Blouin, Alfred C. Schouten.
  The Journal of Physiology. February 22, 2017
  Key points The vestibular influence on human walking is phase‐dependent and modulated across both limbs with changes in locomotor velocity and cadence. Using a split‐belt treadmill, we show that vestibular influence on locomotor activity is modulated independently in each limb. The independent vestibular modulation of muscle activity from each limb occurs rapidly at the onset of split‐belt walking, over a shorter time course relative to the characteristic split‐belt error‐correction mechanisms (i.e. muscle activity and kinematics) associated with locomotor adaptation. Together, the present results indicate that the nervous system rapidly modulates the vestibular influence of each limb separately through processes involving ongoing sensory feedback loops. These findings help us understand how vestibular information is used to accommodate the variable and commonplace demands of locomotion, such as turning or navigating irregular terrain. Abstract During walking, the vestibular influence on locomotor activity is phase‐dependent and modulated in both limbs with changes in velocity. It is unclear, however, whether this bilateral modulation is due to a coordinated mechanism between both limbs or instead through limb‐specific processes that remain masked by the symmetric nature of locomotion. Here, human subjects walked on a split‐belt treadmill with one belt moving at 0.4 m s−1 and the other moving at 0.8 m s−1 while exposed to an electrical vestibular stimulus. Muscle activity was recorded bilaterally around the ankles of each limb and used to compare vestibulo‐muscular coupling between velocity‐matched and unmatched tied‐belt walking. In general, response magnitudes decreased by ∼20–50% and occurred ∼13–20% earlier in the stride cycle at the higher belt velocity. This velocity‐dependent modulation of vestibular‐evoked muscle activity was retained during split‐belt walking and was similar, within each limb, to velocity‐matched tied‐belt walking. These results demonstrate that the vestibular influence on ankle muscles during locomotion can be adapted independently to each limb. Furthermore, modulation of vestibular‐evoked muscle responses occurred rapidly (∼13–34 strides) after onset of split‐belt walking. This rapid adaptation contrasted with the prolonged adaptation in step length symmetry (∼128 strides) as well as EMG magnitude and timing (∼40–100 and ∼20–70 strides, respectively). These results suggest that vestibular influence on ankle muscle control is adjusted rapidly in sensorimotor control loops as opposed to longer‐term error correction mechanisms commonly associated with split‐belt adaptation. Rapid limb‐specific sensorimotor feedback adaptation may be advantageous for asymmetric overground locomotion, such as navigating irregular terrain or turning.

  February 22, 2017   doi: 10.1113/JP272614   open full text
• The statistics of the vestibular input experienced during natural self‐motion differ between rodents and primates.
  Jérome Carriot, Mohsen Jamali, Maurice J. Chacron, Kathleen E. Cullen.
  The Journal of Physiology. February 22, 2017
  Key points In order to understand how the brain's coding strategies are adapted to the statistics of the sensory stimuli experienced during everyday life, the use of animal models is essential. Mice and non‐human primates have become common models for furthering our knowledge of the neuronal coding of natural stimuli, but differences in their natural environments and behavioural repertoire may impact optimal coding strategies. Here we investigated the structure and statistics of the vestibular input experienced by mice versus non‐human primates during natural behaviours, and found important differences. Our data establish that the structure and statistics of natural signals in non‐human primates more closely resemble those observed previously in humans, suggesting similar coding strategies for incoming vestibular input. These results help us understand how the effects of active sensing and biomechanics will differentially shape the statistics of vestibular stimuli across species, and have important implications for sensory coding in other systems. Abstract It is widely believed that sensory systems are adapted to the statistical structure of natural stimuli, thereby optimizing coding. Recent evidence suggests that this is also the case for the vestibular system, which senses self‐motion and in turn contributes to essential brain functions ranging from the most automatic reflexes to spatial perception and motor coordination. However, little is known about the statistics of self‐motion stimuli actually experienced by freely moving animals in their natural environments. Accordingly, here we examined the natural self‐motion signals experienced by mice and monkeys: two species commonly used to study vestibular neural coding. First, we found that probability distributions for all six dimensions of motion (three rotations, three translations) in both species deviated from normality due to long tails. Interestingly, the power spectra of natural rotational stimuli displayed similar structure for both species and were not well fitted by power laws. This result contrasts with reports that the natural spectra of other sensory modalities (i.e. vision, auditory and tactile) instead show a power‐law relationship with frequency, which indicates scale invariance. Analysis of natural translational stimuli revealed important species differences as power spectra deviated from scale invariance for monkeys but not for mice. By comparing our results to previously published data for humans, we found the statistical structure of natural self‐motion stimuli in monkeys and humans more closely resemble one another. Our results thus predict that, overall, neural coding strategies used by vestibular pathways to encode natural self‐motion stimuli are fundamentally different in rodents and primates.

  February 22, 2017   doi: 10.1113/JP273734   open full text
• Rapid frequency‐dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.
  Rob C. I. Wüst, Michiel Helmes, Jody L. Martin, Thomas J. T. der Wardt, René J. P. Musters, Jolanda der Velden, Ger J. M. Stienen.
  The Journal of Physiology. February 22, 2017
  Key points Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Abstract Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an acceleration of Ca2+ release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat‐to‐beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

  February 22, 2017   doi: 10.1113/JP273589   open full text
• EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision.
  Esa‐Ville Immonen, Andrew S. French, Päivi H. Torkkeli, Hongxia Liu, Mikko Vähäsöyrinki, Roman V. Frolov.
  The Journal of Physiology. February 22, 2017
  Key points The principles underlying the evolutionary selection of ion channels for expression in sensory neurons are unclear. Photoreceptor depolarization in the diurnal Drosophila melanogaster is predominantly provided by light‐activated transient receptor potential (TRP) channels, whereas repolarization is mediated by sustained voltage‐gated K+ channels of the Shab family. In the present study, we show that phototransduction in the nocturnal cockroach Periplaneta americana is predominantly mediated by TRP‐like channels, whereas membrane repolarization is based on EAG channels. Although bright light stimulates Shab channels in Drosophila, further restricting depolarization and improving membrane bandwidth, it strongly suppresses EAG conductance in Periplaneta. This light‐dependent inhibition (LDI) is caused by calcium and is abolished by chelating intracellular calcium or suppressing eag gene expression. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth. This makes EAG unsuitable for light response conditioning during the day and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects. Abstract The principles underlying evolutionary selection of ion channels for expression in sensory neurons are unclear. Among species possessing microvillar photoreceptors, the major ionic conductances have only been identified in Drosophila melanogaster. In Drosophila, depolarization is provided by light‐activated transient receptor potential (TRP) channels with a minor contribution from TRP‐like (TRPL) channels, whereas repolarization is mediated by sustained voltage‐gated K+ (Kv) channels of the Shab family. Bright light stimulates Shab channels, further restricting depolarization and improving membrane bandwidth. In the present study, data obtained using a combination of electrophysiological, pharmacological and molecular knockdown techniques strongly suggest that in photoreceptors of the nocturnal cockroach Periplaneta americana the major excitatory channel is TRPL, whereas the predominant delayed rectifier is EAG, a ubiquitous but enigmatic Kv channel. By contrast to the diurnal Drosophila, bright light strongly suppresses EAG conductance in Periplaneta. This light‐dependent inhibition (LDI) is caused by calcium entering the cytosol and is amplified following inhibition of calcium extrusion, and it can also be abolished by chelating intracellular calcium or suppressing eag gene expression by RNA interference. LDI increases membrane resistance, augments gain and reduces the signalling bandwidth, impairing information transfer. LDI is also observed in the nocturnal cricket Gryllus integer, whereas, in the diurnal water strider Gerris lacustris, the delayed rectifier is up‐regulated by light. Although LDI is not expected to reduce delayed rectifier current in the normal illumination environment of nocturnal cockroaches and crickets, it makes EAG unsuitable for light response conditioning during the day, and might have resulted in the evolutionary replacement of EAG by other delayed rectifiers in diurnal insects.

  February 22, 2017   doi: 10.1113/JP273612   open full text
• Late gestational intermittent hypoxia induces metabolic and epigenetic changes in male adult offspring mice.
  Abdelnaby Khalyfa, Rene Cortese, Zhuanhong Qiao, Honggang Ye, Riyue Bao, Jorge Andrade, David Gozal.
  The Journal of Physiology. February 22, 2017
  Key points Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia, a hallmark of OSA, could impose significant long‐term effects on somatic growth, energy homeostasis and metabolic function in offspring. Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue. Fetal perturbations by OSA during pregnancy impose long‐term detrimental effects manifesting as metabolic dysfunction in adult male offspring. Abstract Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long‐term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG‐IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time‐pregnant female C57BL/6 mice were exposed to LG‐IH or room air (LG‐RA) during days 13–18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP‐chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG‐IH male but not female offspring. LG‐IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro‐inflammatory macrophages emerged in VWAT along with 1520 DMRs (P < 0.0001), associated with 693 genes. Pathway analyses showed that genes affected by LG‐IH were mainly associated with molecular processes related to metabolic regulation and inflammation. LG‐IH induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in VWAT. Thus, perturbations to fetal environment by OSA during pregnancy can have long‐term detrimental effects on the fetus, and lead to persistent metabolic dysfunction in adulthood.

  February 22, 2017   doi: 10.1113/JP273570   open full text
• Residual force enhancement is regulated by titin in skeletal and cardiac myofibrils.
  Nabil Shalabi, Anabelle Cornachione, Felipe de Souza Leite, Srikar Vengallatore, Dilson E. Rassier.
  The Journal of Physiology. February 19, 2017
  Key points When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement. Residual force enhancement is puzzling because it cannot be directly explained by the classical force–length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field. We used custom‐built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils. Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments. Abstract When a skeletal muscle contracts isometrically, the muscle produces a force that is relative to the final isometric sarcomere length (SL). However, when the same final SL is reached by stretching the muscle while it contracts, the muscle produces a relatively higher force: a phenomenon commonly referred to as residual force enhancement. In this study, we investigated residual force enhancement in rabbit skeletal psoas myofibrils and, for the first time, cardiac papillary myofibrils. A custom‐built atomic force microscope was used in experiments that stretched myofibrils before and after inhibiting myosin and actin interactions to determine whether the different cardiac and skeletal titin isoforms regulate residual force enhancement. At SLs ranging from 2.24 to 3.13 μm, the skeletal myofibrils enhanced the force by an average of 9.0%, and by 29.5% after hindering myosin and actin interactions. At SLs ranging from 1.80 to 2.29 μm, the cardiac myofibrils did not enhance the force before or after hindering myosin and actin interactions. We conclude that residual force enhancement is present only in skeletal muscles and is dependent on the titin isoforms.

  February 19, 2017   doi: 10.1113/JP272983   open full text
• A critical period of corticomuscular and EMG–EMG coherence detection in healthy infants aged 9–25 weeks.
  Anina Ritterband‐Rosenbaum, Anna Herskind, Xi Li, Maria Willerslev‐Olsen, Mikkel Damgaard Olsen, Simon Francis Farmer, Jens Bo Nielsen.
  The Journal of Physiology. February 15, 2017
  Key points The early postnatal development of functional corticospinal connections in human infants is not fully clarified. Corticospinal drive to upper and lower limb muscle shows developmental changes with an increased functional coupling in infants between 9 and 25 weeks in the beta frequency band. The changes in functional coupling coincide with the developmental period where fidgety movements are present in healthy infants. Data support a possible sensitive period where functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization. Abstract The early postnatal development of functional corticospinal connections in human infants is not fully clarified. We used EEG and EMG to investigate the development of corticomuscular and intramuscular coherence as indicators of functional corticospinal connectivity in healthy infants aged 1–66 weeks. EEG was recorded over leg and hand area of motor cortex. EMG recordings were made from right ankle dorsiflexor and right wrist extensor muscles. Quantification of the amount of corticomuscular coherence in the 20–40 Hz frequency band showed a significantly larger coherence for infants aged 9–25 weeks compared to younger and older infants. Coherence between paired EMG recordings from tibialis anterior muscle in the 20–40 Hz frequency band was also significantly larger for the 9–25 week age group. A low‐amplitude, broad‐duration (40–50 ms) central peak of EMG–EMG synchronization was observed for infants younger than 9 weeks, whereas a short‐lasting (10–20 ms) central peak was observed for EMG–EMG synchronization in older infants. This peak was largest for infants aged 9–25 weeks. These data suggest that the corticospinal drive to lower and upper limb muscles shows significant developmental changes with an increase in functional coupling in infants aged 9–25 weeks, a period which coincides partly with the developmental period of normal fidgety movements. We propose that these neurophysiological findings may reflect the existence of a sensitive period where the functional connections between corticospinal tract fibres and spinal motoneurones undergo activity‐dependent reorganization. This may be relevant for the timing of early therapy interventions in infants with pre‐ and perinatal brain injury.

  February 15, 2017   doi: 10.1113/JP273090   open full text
• Bicarbonate sensing in mouse cortical astrocytes during extracellular acid/base disturbances.
  Shefeeq M. Theparambil, Zinnia Naoshin, Sabrina Defren, Jana Schmaelzle, Tobias Weber, Hans‐Peter Schneider, Joachim W. Deitmer.
  The Journal of Physiology. February 15, 2017
  Key points The present study suggests that the electrogenic sodium–bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes. A decrease in extracellular [HCO3−] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3−] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1‐knockout astrocytes. Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1‐ and CAII‐expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes. We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3−]‐dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3−] in brain tissue. Abstract Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium–bicarbonate cotransporter, NBCe1, is a high‐affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in astrocytes from wild‐type (WT) and from NBCe1‐knockout (KO) mice, using ion‐selective dyes, during isocapnic acidosis, hypercapnic acidosis and hypocapnia. We also analysed NBCe1‐mediated membrane currents in Xenopus laevis oocytes under similar conditions. Comparing WT and NBCe1‐KO astrocytes, we could dissect the contribution of NBCe1, of diffusion of CO2 across the cell membrane and, after blocking carbonic anhydrase (CA) activity with ethoxyzolamide, of the role of CA, for the amplitude and rate of acid/base fluxes. Our results suggest that NBCe1 transport activity in astrocytes, supported by CA activity, renders astrocytes bicarbonate sensors in the mouse cortex. NBCe1 carried bicarbonate into and out of the cell by sensing the variations of transmembrane [HCO3−], irrespective of the changes in intra‐ and extracellular pH, and played a major role in setting pHi responses to the extracellular acid/base challenges. We propose that bicarbonate sensing of astrocytes may have potential functional significance during extracellular acid/base alterations in the brain.

  February 15, 2017   doi: 10.1113/JP273394   open full text
• Effect of gravity and microgravity on intracranial pressure.
  Justin S. Lawley, Lonnie G. Petersen, Erin J. Howden, Satyam Sarma, William K. Cornwell, Rong Zhang, Louis A. Whitworth, Michael A. Williams, Benjamin D. Levine.
  The Journal of Physiology. February 14, 2017
  Key Points Astronauts have recently been discovered to have impaired vision, with a presentation that resembles syndromes of elevated intracranial pressure on Earth. Gravity has a profound effect on fluid distribution and pressure within the human circulation. In contrast to prevailing theory, we observed that microgravity reduces central venous and intracranial pressure. This being said, intracranial pressure is not reduced to the levels observed in the 90 deg seated upright posture on Earth. Thus, over 24 h in zero gravity, pressure in the brain is slightly above that observed on Earth, which may explain remodelling of the eye in astronauts. Abstract Astronauts have recently been discovered to have impaired vision, with a presentation that resembles syndromes of elevated intracranial pressure (ICP). This syndrome is considered the most mission‐critical medical problem identified in the past decade of manned spaceflight. We recruited five men and three women who had an Ommaya reservoir inserted for the delivery of prophylactic CNS chemotherapy, but were free of their malignant disease for at least 1 year. ICP was assessed by placing a fluid‐filled 25 gauge butterfly needle into the Ommaya reservoir. Subjects were studied in the upright and supine position, during acute zero gravity (parabolic flight) and prolonged simulated microgravity (6 deg head‐down tilt bedrest). ICP was lower when seated in the 90 deg upright posture compared to lying supine (seated, 4 ± 1 vs. supine, 15 ± 2 mmHg). Whilst lying in the supine posture, central venous pressure (supine, 7 ± 3 vs. microgravity, 4 ± 2 mmHg) and ICP (supine, 17 ± 2 vs. microgravity, 13 ± 2 mmHg) were reduced in acute zero gravity, although not to the levels observed in the 90 deg seated upright posture on Earth. Prolonged periods of simulated microgravity did not cause progressive elevations in ICP (supine, 15 ± 2 vs. 24 h head‐down tilt, 15 ± 4 mmHg). Complete removal of gravity does not pathologically elevate ICP but does prevent the normal lowering of ICP when upright. These findings suggest the human brain is protected by the daily circadian cycles in regional ICPs, without which pathology may occur.

  February 14, 2017   doi: 10.1113/JP273557   open full text
• Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide‐gated channels.
  Almke Bader, Willem Bintig, Daniela Begandt, Anne Klett, Ina G. Siller, Carola Gregor, Frank Schaarschmidt, Babette Weksler, Ignacio Romero, Pierre‐Olivier Couraud, Stefan W. Hell, Anaclet Ngezahayo.
  The Journal of Physiology. February 14, 2017
  Key points Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell‐to‐cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood–brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP‐dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide‐gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood–brain barrier. Abstract The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT‐PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2‐phenylaminoadenosine (2‐PAA) on the gap junction coupling. We found that 2‐PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration‐dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2‐PAA‐related enhancement of gap junction coupling. In contrast, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2‐PAA‐related enhancement of gap junction coupling. Moreover, we observed a 2‐PAA‐dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP‐dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor‐dependent signalling of endothelial cells of the blood–brain barrier.

  February 14, 2017   doi: 10.1113/JP273150   open full text
• ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter.
  C. A. Cobine, E. E. Hannah, M. H. Zhu, H. E. Lyle, J. R. Rock, K. M. Sanders, S. M. Ward, K. D. Keef.
  The Journal of Physiology. February 14, 2017
  Key points The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC‐IM) by activation of Ca2+‐activated Cl− channels (ANO1, encoded by Ano1) and voltage‐dependent L‐type Ca2+ channels (CavL, encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence‐activated cell sorting. ICC‐IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC‐IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. Abstract The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC‐IM) by activation of Ca2+‐activated Cl− channels (encoded by Ano1) and voltage‐dependent L‐type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un‐stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence‐activated cell sorting, Ano1 expression was 26.5‐fold greater in ICC than in SMCs while Cacna1c expression was only 2‐fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC‐IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC‐IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

  February 14, 2017   doi: 10.1113/JP273618   open full text
• Low carbohydrate, high fat diet impairs exercise economy and negates the performance benefit from intensified training in elite race walkers.
  Louise M. Burke, Megan L. Ross, Laura A. Garvican‐Lewis, Marijke Welvaert, Ida A. Heikura, Sara G. Forbes, Joanne G. Mirtschin, Louise E. Cato, Nicki Strobel, Avish P. Sharma, John A. Hawley.
  The Journal of Physiology. February 14, 2017
  Key points Three weeks of intensified training and mild energy deficit in elite race walkers increases peak aerobic capacity independent of dietary support. Adaptation to a ketogenic low carbohydrate, high fat (LCHF) diet markedly increases rates of whole‐body fat oxidation during exercise in race walkers over a range of exercise intensities. The increased rates of fat oxidation result in reduced economy (increased oxygen demand for a given speed) at velocities that translate to real‐life race performance in elite race walkers. In contrast to training with diets providing chronic or periodised high carbohydrate availability, adaptation to an LCHF diet impairs performance in elite endurance athletes despite a significant improvement in peak aerobic capacity. Abstract We investigated the effects of adaptation to a ketogenic low carbohydrate (CHO), high fat diet (LCHF) during 3 weeks of intensified training on metabolism and performance of world‐class endurance athletes. We controlled three isoenergetic diets in elite race walkers: high CHO availability (g kg−1 day−1: 8.6 CHO, 2.1 protein, 1.2 fat) consumed before, during and after training (HCHO, n = 9); identical macronutrient intake, periodised within or between days to alternate between low and high CHO availability (PCHO, n = 10); LCHF (< 50 g day−1 CHO; 78% energy as fat; 2.1 g kg−1 day−1 protein; LCHF, n = 10). Post‐intervention, V̇O2 peak during race walking increased in all groups (P < 0.001, 90% CI: 2.55, 5.20%). LCHF was associated with markedly increased rates of whole‐body fat oxidation, attaining peak rates of 1.57 ± 0.32 g min−1 during 2 h of walking at ∼80% V̇O2 peak . However, LCHF also increased the oxygen (O2) cost of race walking at velocities relevant to real‐life race performance: O2 uptake (expressed as a percentage of new V̇O2 peak ) at a speed approximating 20 km race pace was reduced in HCHO and PCHO (90% CI: −7.047, −2.55 and −5.18, −0.86, respectively), but was maintained at pre‐intervention levels in LCHF. HCHO and PCHO groups improved times for 10 km race walk: 6.6% (90% CI: 4.1, 9.1%) and 5.3% (3.4, 7.2%), with no improvement (−1.6% (−8.5, 5.3%)) for the LCHF group. In contrast to training with diets providing chronic or periodised high‐CHO availability, and despite a significant improvement in V̇O2 peak , adaptation to the topical LCHF diet negated performance benefits in elite endurance athletes, in part due to reduced exercise economy.

  February 14, 2017   doi: 10.1113/JP273230   open full text
• Vagal denervation inhibits the increase in pulmonary blood flow during partial lung aeration at birth.
  Justin A. R. Lang, James T. Pearson, Corinna Binder‐Heschl, Megan J. Wallace, Melissa L. Siew, Marcus J. Kitchen, Arjan B. te Pas, Robert A. Lewis, Graeme R. Polglase, Mikiyasu Shirai, Stuart B. Hooper.
  The Journal of Physiology. February 14, 2017
  Key points Lung aeration at birth significantly increases pulmonary blood flow, which is unrelated to increased oxygenation or other spatial relationships that match ventilation to perfusion. Using simultaneous X‐ray imaging and angiography in near‐term rabbits, we investigated the relative contributions of the vagus nerve and oxygenation to the increase in pulmonary blood flow at birth. Vagal denervation inhibited the global increase in pulmonary blood flow induced by partial lung aeration, although high inspired oxygen concentrations can partially mitigate this effect. The results of the present study indicate that a vagal reflex may mediate a rapid global increase in pulmonary blood flow in response to partial lung aeration. Abstract Air entry into the lungs at birth triggers major cardiovascular changes, including a large increase in pulmonary blood flow (PBF) that is not spatially related to regional lung aeration. To investigate the possible underlying role of a vagally‐mediated stimulus, we used simultaneous phase‐contrast X‐ray imaging and angiography in near‐term (30 days of gestation) vagotomized (n = 15) or sham‐operated (n = 15) rabbit kittens. Rabbits were imaged before ventilation, when one lung was ventilated (unilateral) with 100% nitrogen (N2), air or 100% oxygen (O2), and after all kittens were switched to unilateral ventilation in air and then ventilation of both lungs using air. Compared to control kittens, vagotomized kittens had little or no increase in PBF in both lungs following unilateral ventilation when ventilation occurred with 100% N2 or with air. However, relative PBF did increase in vagotomized animals ventilated with 100% O2, indicating the independent stimulatory effects of local oxygen concentration and autonomic innervation on the changes in PBF at birth. These findings demonstrate that vagal denervation inhibits the previously observed increase in PBF with partial lung aeration, although high inspired oxygen concentrations can partially mitigate this effect.

  February 14, 2017   doi: 10.1113/JP273682   open full text
• Bicarbonate‐rich fluid secretion predicted by a computational model of guinea‐pig pancreatic duct epithelium.
  Makoto Yamaguchi, Martin C. Steward, Kieran Smallbone, Yoshiro Sohma, Akiko Yamamoto, Shigeru B. H. Ko, Takaharu Kondo, Hiroshi Ishiguro.
  The Journal of Physiology. February 08, 2017
  Key points The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3− concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea‐pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3−. Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3− concentrations is to minimize the secretion of Cl− ions. These findings help to clarify the mechanism responsible for pancreatic HCO3− secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. Abstract A computational model of guinea‐pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3− ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least‐squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP‐stimulated secretion were well replicated by increasing the activities of the basolateral Na+‐HCO3− cotransporter (NBC1) and apical Cl−/HCO3− exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl− and HCO3− permeabilities (CFTR), and reducing the activity of the basolateral Cl−/HCO3− exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3− at a rate of ∼3 nl min−1 mm−2, which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl−/HCO3− exchange via SLC26A6 at the apical membrane were able to support a HCO3−‐rich secretion. Raising the HCO3−/Cl− permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3− concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl− concentration following cAMP stimulation and thereby maximizing the secreted HCO3− concentration. The addition of a basolateral Na+‐K+‐2Cl− cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl− and resulted in a lower secreted HCO3− concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl− secretion is the main requirement for secreting 140 mm HCO3−.

  February 08, 2017   doi: 10.1113/JP273306   open full text
• Western diet induces colonic nitrergic myenteric neuropathy and dysmotility in mice via saturated fatty acid‐ and lipopolysaccharide‐induced TLR4 signalling.
  François Reichardt, Benoit Chassaing, Behtash Ghazi Nezami, Ge Li, Sahar Tabatabavakili, Simon Mwangi, Karan Uppal, Bill Liang, Matam Vijay‐Kumar, Dean Jones, Andrew T. Gewirtz, Shanthi Srinivasan.
  The Journal of Physiology. February 08, 2017
  Key points A high‐fat diet (60% kcal from fat) is associated with motility disorders inducing constipation and loss of nitrergic myenteric neurons in the proximal colon. Gut microbiota dysbiosis, which occurs in response to HFD, contributes to endotoxaemia. High levels of lipopolysaccharide lead to apoptosis in cultured myenteric neurons that express Toll‐like receptor 4 (TLR4). Consumption of a Western diet (WD) (35% kcal from fat) for 6 weeks leads to gut microbiota dysbiosis associated with altered bacterial metabolites and increased levels of plasma free fatty acids. These disorders precede the nitrergic myenteric cell loss observed in the proximal colon. Mice lacking TLR4 did not exhibit WD‐induced myenteric cell loss and dysmotility. Lipopolysaccharide‐induced in vitro enteric neurodegeneration requires the presence of palmitate and may be a result of enhanced NO production. The present study highlights the critical role of plasma saturated free fatty acids that are abundant in the WD with respect to driving enteric neuropathy and colonic dysmotility. Abstract The consumption of a high‐fat diet (HFD) is associated with myenteric neurodegeneration, which in turn is associated with delayed colonic transit and constipation. We examined the hypothesis that an inherent increase in plasma free fatty acids (FFA) in the HFD together with an HFD‐induced alteration in gut microbiota contributes to the pathophysiology of these disorders. C57BL/6 mice were fed a Western diet (WD) (35% kcal from fat enriched in palmitate) or a purified regular diet (16.9% kcal from fat) for 3, 6, 9 and 12 weeks. Gut microbiota dysbiosis was investigated by fecal lipopolysaccharide (LPS) measurement and metabolomics (linear trap quadrupole‐Fourier transform mass spectrometer) analysis. Plasma FFA and LPS levels were assessed, in addition to colonic and ileal nitrergic myenteric neuron quantifications and motility. Compared to regular diet‐fed control mice, WD‐fed mice gained significantly more weight without blood glucose alteration. Dysbiosis was exhibited after 6 weeks of feeding, as reflected by increased fecal LPS and bacterial metabolites and concomitant higher plasma FFA. The numbers of nitrergic myenteric neurons were reduced in the proximal colon after 9 and 12 weeks of WD and this was also associated with delayed colonic transit. WD‐fed Toll‐like receptor 4 (TLR4)−/− mice did not exhibit myenteric cell loss or dysmotility. Finally, LPS (0.5–2 ng·ml–1) and palmitate (20 and 30 μm) acted synergistically to induce neuronal cell death in vitro, which was prevented by the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester. In conclusion, WD‐feeding results in increased levels of FFA and microbiota that, even in absence of hyperglycaemia or overt endotoxaemia, synergistically induce TLR4‐mediated neurodegeneration and dysmotility.

  February 08, 2017   doi: 10.1113/JP273269   open full text
• Effect of reproductive ageing on pregnant mouse uterus and cervix.
  Rima Patel, James D. Moffatt, Evangelia Mourmoura, Luc Demaison, Paul T. Seed, Lucilla Poston, Rachel M. Tribe.
  The Journal of Physiology. February 08, 2017
  Key points Older pregnant women have a greater risk of operative delivery, still birth and post‐term induction. This suggests that maternal age can influence the timing of birth and processes of parturition. We have found that increasing maternal age in C57BL/6J mice is associated with prolongation of gestation and length of labour. Older pregnant mice also had delayed progesterone withdrawal and impaired myometrial function. Uterine ageing and labour dysfunction should be investigated further in older primigravid women. Abstract Advanced maternal age (≥35 years) is associated with increased rates of operative delivery, stillbirth and post‐term labour induction. The physiological causes remain uncertain, although impaired myometrial function has been implicated. To investigate the hypothesis that maternal age directly influences successful parturition, we assessed the timing of birth and fetal outcome in pregnant C57BL/6J mice at 3 months (young) and 5 months (intermediate) vs. 8 months (older) of age using infrared video recording. Serum progesterone profiles, myometrium and cervix function, and mitochondrial electron transport chain complex enzymatic activities were also examined. Older pregnant mice had a longer mean gestation and labour duration (P < 0.001), as well as reduced litter size (P < 0.01) vs. 3‐month‐old mice. Older mice did not exhibit the same decline in serum progesterone concentrations as younger mice. Cervical tissues from older mice were more distensible than younger mice (P < 0.05). Oxytocin receptor and connexin‐43 mRNA expression were reduced in the myometrium from 8‐month‐old vs. 3‐month‐old mice (P < 0.05 and P < 0.01 respectively) in tandem with more frequent but shorter duration spontaneous myometrial contractions (P < 0.05) and an attenuated contractile response to oxytocin. Myometrial mitochondrial copy number was reduced in older mice, although there were no age‐induced changes to the enzymatic activities of the mitochondrial electron transport chain complexes. In conclusion, 8‐month‐old mice provide a useful model of reproductive ageing. The present study has identified potential causes of labour dysfunction amenable to investigation in older primigravid women.

  February 08, 2017   doi: 10.1113/JP273350   open full text
• N‐Cadherin, a novel and rapidly remodelling site involved in vasoregulation of small cerebral arteries.
  Zhe Sun, Min Li, Zhaohui Li, Michael A. Hill, Gerald A. Meininger.
  The Journal of Physiology. February 07, 2017
  Key points N‐cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N‐cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10−5 m) and ACh (10−5 m). N‐cadherin‐coated beads were able to induce clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10−5 m) or sodium nitroprusside (10−5 m) induced a significant increase or decrease in the N‐cadherin‐EGFP clustering, respectively. Application of pulling force (∼1 nN) to the N‐cadherin‐coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. Abstract N‐cadherin is the major cell–cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N‐cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N‐cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90 mmHg, both the density and the average size of N‐cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10–5 m) induced a significant increase of N‐cadherin AJ density at 50 mmHg, whereas vasodilatation induced by ACh (10–5 m) was accompanied by a significant decrease in density and size of N‐cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano‐responsive properties of N‐cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N‐cadherin‐coated microbead (5 μm) induced a progressive clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1 nN) to the N‐cadherin‐coated‐beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10–5 m) or sodium nitroprusside (10–5 m) induced a significant increase or decrease of the N‐cadherin‐EGFP clustering, respectively. These observations provide compelling evidence that N‐cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N‐cadherin AJs in vasomotor regulation.

  February 07, 2017   doi: 10.1113/JP272995   open full text
• The quantal catecholamine release from mouse chromaffin cells challenged with repeated ACh pulses is regulated by the mitochondrial Na+/Ca2+ exchanger.
  Angela López‐Gil, Carmen Nanclares, Iago Méndez‐López, Carmen Martínez‐Ramírez, Cristóbal los Rios, J. Fernando Padín‐Nogueira, Mayte Montero, Luis Gandía, Antonio G. García.
  The Journal of Physiology. February 07, 2017
  Key points Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady‐state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release‐vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh‐sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+/Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long‐term stimulation of chromaffin cells by endogenously released ACh. Abstract Using caged‐Ca2+ photorelease or paired depolarising pulses in voltage‐clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve‐CC synapse, is unknown. We have explored here whether an ACh‐sensitive ready‐release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K+ pulse given at the end (75 mm K+). After the first 2–3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady‐state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage‐clamped cells elicited non‐desensitising nicotinic currents. Also, the [Ca2+]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+/Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.

  February 07, 2017   doi: 10.1113/JP273339   open full text
• Diltiazem prevents stress‐induced contractile deficits in cardiomyocytes, but does not reverse the cardiomyopathy phenotype in Mybpc3‐knock‐in mice.
  Frederik Flenner, Birgit Geertz, Silke Reischmann‐Düsener, Florian Weinberger, Thomas Eschenhagen, Lucie Carrier, Felix W. Friedrich.
  The Journal of Physiology. February 07, 2017
  Key points Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non‐dihydropyridine Ca2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3‐targeted knock‐in), which is based on a mutation commonly found in patients. Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM. Our study shows that diltiazem has beneficial effects in HCM, but long‐term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment. Abstract Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β‐adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre‐clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3‐targeted knock‐in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild‐type (WT) cardiomyocytes in basal conditions (1‐Hz pacing) and under stress conditions (30 nm isoprenaline, 5‐Hz pacing) with or without pre‐treatment with 1 μm diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than WT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre‐treatment of cardiomyocytes with 1 μm diltiazem reduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Six months of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. In conclusion, diltiazem blunted the response to isoprenaline in WT and KI cardiomyocytes and improved diastolic relaxation under stress conditions in KI cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in KI mice.

  February 07, 2017   doi: 10.1113/JP273769   open full text
• Angiotensin II reduces the surface abundance of KV1.5 channels in arterial myocytes to stimulate vasoconstriction.
  Michael W. Kidd, Simon Bulley, Jonathan H. Jaggar.
  The Journal of Physiology. February 05, 2017
  Key points Several different voltage‐dependent K+ (KV) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit KV currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of KV1.5, but not KV2.1, channels through a protein kinase C‐ and lysosome‐dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II‐induced decrease in cell surface KV1.5 channels reduces whole‐cell KV1.5 currents and attenuates KV1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C‐dependent KV1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Abstract Smooth muscle cells (myocytes) of resistance‐size arteries express several different voltage‐dependent K+ (KV) channels, including KV1.5 and KV2.1, which regulate contractility. Myocyte KV currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits KV currents by reducing the plasma membrane abundance of KV channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total KV1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface KV2.1, or KV1.5 or KV2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II‐induced decrease in total and surface KV1.5. Immunofluorescence also suggested that Ang II reduced surface KV1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch‐clamp electrophysiology. Angiotensin II reduced both whole‐cell KV currents and currents inhibited by Psora‐4, a KV1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora‐4 or 4‐aminopyridine, another KV channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates KV1.5 channel degradation, leading to a decrease in surface KV1.5, a reduction in whole‐cell KV1.5 currents and a loss of functional KV1.5 channels in myocytes of pressurized arteries.

  February 05, 2017   doi: 10.1113/JP272893   open full text
• Four‐week cold acclimation in adult humans shifts uncoupling thermogenesis from skeletal muscles to brown adipose tissue.
  Denis P. Blondin, Amani Daoud, Taryn Taylor, Hans C. Tingelstad, Véronic Bézaire, Denis Richard, André C. Carpentier, Albert W. Taylor, Mary‐Ellen Harper, Céline Aguer, François Haman.
  The Journal of Physiology. February 05, 2017
  Key points Muscle‐derived thermogenesis during acute cold exposure in humans consists of a combination of cold‐induced increases in skeletal muscle proton leak and shivering. Daily cold exposure results in an increase in brown adipose tissue oxidative capacity coupled with a decrease in the cold‐induced skeletal muscle proton leak and shivering intensity. Improved coupling between electromyography‐determined muscle activity and whole‐body heat production following cold acclimation suggests a maintenance of ATPase‐dependent thermogenesis and decrease in skeletal muscle ATPase independent thermogenesis. Although daily cold exposure did not change the fibre composition of the vastus lateralis, the fibre composition was a strong predictor of the shivering pattern evoked during acute cold exposure. Abstract We previously showed that 4 weeks of daily cold exposure in humans can increase brown adipose tissue (BAT) volume by 45% and oxidative metabolism by 182%. Surprisingly, we did not find a reciprocal reduction in shivering intensity when exposed to a mild cold (18°C). The present study aimed to determine whether changes in skeletal muscle oxidative metabolism or shivering activity could account for these unexpected findings. Nine men participated in a 4 week cold acclimation intervention (10°C water circulating in liquid‐conditioned suit, 2 h day–1, 5 days week–1). Shivering intensity and pattern were measured continuously during controlled cold exposure (150 min at 4 °C) before and after the acclimation. Muscle biopsies from the m. vastus lateralis were obtained to measure oxygen consumption rate and proton leak of permeabilized muscle fibres. Cold acclimation elicited a modest 21% (P < 0.05) decrease in whole‐body and m. vastus lateralis shivering intensity. Furthermore, cold acclimation abolished the acute cold‐induced increase in proton leak. Although daily cold exposure did not change the fibre composition of the m. vastus lateralis, fibre composition was a strong predictor of the shivering pattern evoked during acute cold. We conclude that muscle‐derived thermogenesis during acute cold exposure in humans is not only limited to shivering, but also includes cold‐induced increases in proton leak. The efficiency of muscle oxidative phosphorylation improves with cold acclimation, suggesting that reduced muscle thermogenesis occurs through decreased proton leak, in addition to decreased shivering intensity as BAT capacity and activity increase. These changes occur with no net difference in whole‐body thermogenesis.

  February 05, 2017   doi: 10.1113/JP273395   open full text
• Differential regulation of blood flow‐induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.
  Oliver A. Stone, James G. Carter, P. Charles Lin, Ewa Paleolog, Maria J. C. Machado, David O. Bates.
  The Journal of Physiology. February 02, 2017
  Key points Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis. This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. Abstract Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains‐2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin‐1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)‐mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over‐expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms‐like tyrosine kinase receptor‐1 (sFlt1). We found that NO‐mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin‐1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide‐mediated vasodilatation and angiopoietin signalling (NO–Tie‐mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

  February 02, 2017   doi: 10.1113/JP273430   open full text
• Non‐uniform phase sensitivity in spatial frequency maps of the human visual cortex.
  Reza Farivar, Simon Clavagnier, Bruce C. Hansen, Ben Thompson, Robert F. Hess.
  The Journal of Physiology. February 02, 2017
  Key points Just as a portrait painting can come from a collection of coarse and fine details, natural vision can be decomposed into coarse and fine components. Previous studies have shown that the early visual areas in the brain represent these components in a map‐like fashion. Other studies have shown that these same visual areas can be sensitive to how coarse and fine features line up in space. We found that the brain actually jointly represents both the scale of the feature (fine, medium, or coarse) and the alignment of these features in space. The results suggest that the visual cortex has an optimized representation particularly for the alignment of fine details, which are crucial in understanding the visual scene. Abstract Complex natural scenes can be decomposed into their oriented spatial frequency (SF) and phase relationships, both of which are represented locally at the earliest stages of cortical visual processing. The SF preference map in the human cortex, obtained using synthetic stimuli, is orderly and correlates strongly with eccentricity. In addition, early visual areas show sensitivity to the phase information that describes the relationship between SFs and thereby dictates the structure of the image. Taken together, two possibilities arise for the joint representation of SF and phase: either the entirety of the cortical SF map is uniformly sensitive to phase, or a particular set of SFs is selectively phase sensitive – for example, greater phase sensitivity for higher SFs that define fine‐scale edges in a complex scene. To test between these two possibilities, we constructed a novel continuous natural scene video whereby phase information was maintained in one SF band but scrambled elsewhere. By shifting the central frequency of the phase‐aligned band in time, we mapped the phase‐sensitive SF preference of the visual cortex. Using functional magnetic resonance imaging, we found that phase sensitivity in early visual areas is biased toward higher SFs. Compared to a SF map of the same scene obtained using linear‐filtered stimuli, a much larger patch of areas V1 and V2 is sensitive to the phase alignment of higher SFs. The results of early areas cannot be explained by attention. Our results suggest non‐uniform sensitivity to phase alignment in population‐level SF representations, with phase alignment being particularly important for fine‐scale edge representations of natural scenes.

  February 02, 2017   doi: 10.1113/JP273206   open full text
• High‐fat diet induces protein kinase A and G‐protein receptor kinase phosphorylation of β2‐adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.
  Qin Fu, Yuting Hu, Qingtong Wang, Yongming Liu, Ning Li, Bing Xu, Sungjin Kim, Nipavan Chiamvimonvat, Yang K. Xiang.
  The Journal of Physiology. February 02, 2017
  Key points Patients with diabetes show a blunted cardiac inotropic response to β‐adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β‐adrenergically induced contractile function in isolated cardiomyocytes and Langendorff‐perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high‐fat diet (HFD) feeding on the cardiac β2‐adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β‐adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2‐adrenergic receptor phosphorylation at protein kinase A and G‐protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Abstract Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high‐fat diet (HFD) on the insulin–adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD‐fed mice displayed a significant elevation of phosphorylation of the β2‐adrenergic receptor (β2AR) at both the protein kinase A site serine 261/262 and the G‐protein‐coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD‐fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the β2AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD‐fed mice. Together, these data indicate that HFD promotes phosphorylation of the β2AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin–adrenergic signalling network might be effective in prevention of cardiac complications in diabetes.

  February 02, 2017   doi: 10.1113/JP273314   open full text
• The internal representation of head orientation differs for conscious perception and balance control.
  Brian H. Dalton, Brandon G. Rasman, J. Timothy Inglis, Jean‐Sébastien Blouin.
  The Journal of Physiology. February 01, 2017
  Key points We tested perceived head‐on‐feet orientation and the direction of vestibular‐evoked balance responses in passively and actively held head‐turned postures. The direction of vestibular‐evoked balance responses was not aligned with perceived head‐on‐feet orientation while maintaining prolonged passively held head‐turned postures. Furthermore, static visual cues of head‐on‐feet orientation did not update the estimate of head posture for the balance controller. A prolonged actively held head‐turned posture did not elicit a rotation in the direction of the vestibular‐evoked balance response despite a significant rotation in perceived angular head posture. It is proposed that conscious perception of head posture and the transformation of vestibular signals for standing balance relying on this head posture are not dependent on the same internal representation. Rather, the balance system may operate under its own sensorimotor principles, which are partly independent from perception. Abstract Vestibular signals used for balance control must be integrated with other sensorimotor cues to allow transformation of descending signals according to an internal representation of body configuration. We explored two alternative models of sensorimotor integration that propose (1) a single internal representation of head‐on‐feet orientation is responsible for perceived postural orientation and standing balance or (2) conscious perception and balance control are driven by separate internal representations. During three experiments, participants stood quietly while passively or actively maintaining a prolonged head‐turned posture (>10 min). Throughout the trials, participants intermittently reported their perceived head angular position, and subsequently electrical vestibular stimuli were delivered to elicit whole‐body balance responses. Visual recalibration of head‐on‐feet posture was used to determine whether static visual cues are used to update the internal representation of body configuration for perceived orientation and standing balance. All three experiments involved situations in which the vestibular‐evoked balance response was not orthogonal to perceived head‐on‐feet orientation, regardless of the visual information provided. For prolonged head‐turned postures, balance responses consistent with actual head‐on‐feet posture occurred only during the active condition. Our results indicate that conscious perception of head‐on‐feet posture and vestibular control of balance do not rely on the same internal representation, but instead treat sensorimotor cues in parallel and may arrive at different conclusions regarding head‐on‐feet posture. The balance system appears to bypass static visual cues of postural orientation and mainly use other sensorimotor signals of head‐on‐feet position to transform vestibular signals of head motion, a mechanism appropriate for most daily activities.

  February 01, 2017   doi: 10.1113/JP272998   open full text
• Dynamics of volume‐averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage‐clamp depolarizations.
  Kun‐Han Lin, Holger Taschenberger, Erwin Neher.
  The Journal of Physiology. February 01, 2017
  Key points The intracellular concentration of free calcium ions ([Ca2+]i) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro‐ or nanodomain of highly elevated [Ca2+]i in the vicinity of open voltage‐gated Ca2+ channels, whereas short‐term synaptic plasticity is often controlled by global changes in residual [Ca2+]i, averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+]i in the calyx of Held – a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short‐term plasticity of glutamatergic synapses. Abstract Many aspects of short‐term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+]i) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity‐dependent dynamic changes in global [Ca2+]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch‐clamp and microfluorimetry. We use low concentrations of a low‐affinity Ca2+ indicator dye (100 μm Fura‐6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage‐clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500 μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD = 543 ± 51 nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38 ± 0.20 s−1), which we need to postulate in order to model the measured presynaptic [Ca2+]i transients.

  February 01, 2017   doi: 10.1113/JP272773   open full text
• Degeneracy in the regulation of short‐term plasticity and synaptic filtering by presynaptic mechanisms.
  Chinmayee L. Mukunda, Rishikesh Narayanan.
  The Journal of Physiology. February 01, 2017
  Key points We develop a new biophysically rooted, physiologically constrained conductance‐based synaptic model to mechanistically account for short‐term facilitation and depression, respectively through residual calcium and transmitter depletion kinetics. We address the specific question of how presynaptic components (including voltage‐gated ion channels, pumps, buffers and release‐handling mechanisms) and interactions among them define synaptic filtering and short‐term plasticity profiles. Employing global sensitivity analyses (GSAs), we show that near‐identical synaptic filters and short‐term plasticity profiles could emerge from disparate presynaptic parametric combinations with weak pairwise correlations. Using virtual knockout models, a technique to address the question of channel‐specific contributions within the GSA framework, we unveil the differential and variable impact of each ion channel on synaptic physiology. Our conclusions strengthen the argument that parametric and interactional complexity in biological systems should not be viewed from the limited curse‐of‐dimensionality standpoint, but from the evolutionarily advantageous perspective of providing functional robustness through degeneracy. Abstract Information processing in neurons is known to emerge as a gestalt of pre‐ and post‐synaptic filtering. However, the impact of presynaptic mechanisms on synaptic filters has not been quantitatively assessed. Here, we developed a biophysically rooted, conductance‐based model synapse that was endowed with six different voltage‐gated ion channels, calcium pumps, calcium buffer and neurotransmitter‐replenishment mechanisms in the presynaptic terminal. We tuned our model to match the short‐term plasticity profile and band‐pass structure of Schaffer collateral synapses, and performed sensitivity analyses to demonstrate that presynaptic voltage‐gated ion channels regulated synaptic filters through changes in excitability and associated calcium influx. These sensitivity analyses also revealed that calcium‐ and release‐control mechanisms were effective regulators of synaptic filters, but accomplished this without changes in terminal excitability or calcium influx. Next, to perform global sensitivity analysis, we generated 7000 randomized models spanning 15 presynaptic parameters, and computed eight different physiological measurements in each of these models. We validated these models by applying experimentally obtained bounds on their measurements, and found 104 (∼1.5%) models to match the validation criteria for all eight measurements. Analysing these valid models, we demonstrate that analogous synaptic filters emerge from disparate combinations of presynaptic parameters exhibiting weak pairwise correlations. Finally, using virtual knockout models, we establish the variable and differential impact of different presynaptic channels on synaptic filters, underlining the critical importance of interactions among different presynaptic components in defining synaptic physiology. Our results have significant implications for protein‐localization strategies required for physiological robustness and for degeneracy in long‐term synaptic plasticity profiles.

  February 01, 2017   doi: 10.1113/JP273482   open full text
• Non‐chemosensitive parafacial neurons simultaneously regulate active expiration and airway patency under hypercapnia in rats.
  Alan A. Britto, Davi J. A. Moraes.
  The Journal of Physiology. February 01, 2017
  Key points Hypercapnia or parafacial respiratory group (pFRG) disinhibition at normocapnia evokes active expiration in rats by recruitment of pFRG late‐expiratory (late‐E) neurons. We show that hypercapnia simultaneously evoked active expiration and exaggerated glottal dilatation by late‐E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic expiratory activity in previously silent pFRG late‐E neurons, which did not express the marker of ventral medullary CO2‐sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. Hypercapnia‐evoked active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. Hypercapnia produces disinhibition of non‐chemosensitive pFRG late‐E neurons to evoke active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Abstract Hypercapnia produces active expiration in rats and the recruitment of late‐expiratory (late‐E) neurons located in the parafacial respiratory group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by disinhibition of pFRG late‐E neurons, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration, exaggerated late‐E discharges in cranial respiratory motor outflows, and glottal dilatation via late‐E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic late‐E activity in previously silent pFRG neurons, which did not express the marker of ventral medullary CO2‐sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. In addition, hypercapnia‐evoked AbN active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. On the other hand, pFRG inhibition did not affect either hypercapnia‐induced inspiratory increases in respiratory motor outflows or CO2 sensitivity of the more medial Phox2b‐positive neurons in the retrotrapezoid nucleus (RTN). Our data suggest that neither RTN Phox2b‐positive nor other CO2‐sensitive brainstem neurons activate Phox2b‐negative pFRG late‐E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia produces disinhibition of non‐chemosensitive pFRG late‐E neurons in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.

  February 01, 2017   doi: 10.1113/JP273335   open full text
• A novel mechanism of tandem activation of ryanodine receptors by cytosolic and SR luminal Ca2+ during excitation–contraction coupling in atrial myocytes.
  Joshua T. Maxwell, Lothar A. Blatter.
  The Journal of Physiology. February 01, 2017
  Key points In atrial myocytes excitation–contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+‐induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store. The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra‐SR calcium revealed a transient elevation of store Ca2+ that we termed ‘Ca2+ sensitization signal’. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a ‘fire–diffuse–uptake–fire’ (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold. Abstract In atrial myocytes Ca2+ release during excitation–contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j‐SR) and the much more abundant central non‐junctional (nj‐SR) SR. Action potential (AP)‐induced Ca2+ entry activates Ca2+‐induced Ca2+ release (CICR) from j‐SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+]i initiates CICR from nj‐SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+]i; with the fluorescent Ca2+ indicator rhod‐2) and intra‐SR ([Ca2+]SR; fluo‐5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j‐SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj‐SR; however, the degree of depletion of j‐SR [Ca2+]SR was smaller than nj‐SR [Ca2+]SR. Similarly, Ca2+ signals from individual release sites of the j‐SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj‐SR. During AP‐induced Ca2+ release the rise of [Ca2+]i detected at individual release sites of the nj‐SR preceded the depletion of [Ca2+]SR, and during this latency period a transient elevation of [Ca2+]SR occurred. We propose that Ca2+ release from nj‐SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel ‘fire—diffuse–uptake–fire’ (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) at the propagation front elevates local [Ca2+]SR, leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

  February 01, 2017   doi: 10.1113/JP273611   open full text
• Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III.
  Esther Udina, Charles T. Putman, Luke R. Harris, Neil Tyreman, Victoria E. Cook, Tessa Gordon.
  The Journal of Physiology. January 25, 2017
  Key points Smn+/− transgenic mouse is a model of the mildest form of spinal muscular atrophy. Although there is a loss of spinal motoneurons in 11‐month‐old animals, muscular force is maintained. This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons. The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity. We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die‐back. Abstract Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn+/− transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die‐back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die‐back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast‐twitch and one slow‐twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn+/− transgenic mouse increases their susceptibility to cell death demonstrated that all the motoneurons survived and they sustained their capacity to regenerate their nerve fibres. It is concluded the systematic die‐back of motoneurons that innervate both fast‐ and slow‐twitch muscle fibres is not related to immaturity of the neuromuscular system in SMA.

  January 25, 2017   doi: 10.1113/JP273404   open full text
• Loop G in the GABAA receptor α1 subunit influences gating efficacy.
  Daniel T. Baptista‐Hon, Simona Gulbinaite, Tim G. Hales.
  The Journal of Physiology. January 25, 2017
  Key points The functional importance of residues in loop G of the GABAA receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA. While the T47C substitution had no significant effect, non‐conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol potentiated maximal GABA‐evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non‐stationary variance analysis revealed a reduction in maximal GABA‐evoked Popen, suggesting impaired agonist efficacy. Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol) relative to GABA was impaired. GABA‐, THIP‐ and propofol‐evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors, indicating that the mutation impairs agonist‐evoked gating. Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation. Abstract The modification of cysteine residues (substituted for D43 and T47) by 2‐aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G is known to impair activation of α1β2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non‐conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub‐maximal but not maximal GABA‐evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol) was used to activate WT or α1(T47R)β2γ2 receptors. Propofol‐evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist‐dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline.

  January 25, 2017   doi: 10.1113/JP273752   open full text
• The age‐dependent association between aortic pulse wave velocity and telomere length.
  Barry J. McDonnell, Yasmin, Lee Butcher, John R. Cockcroft, Ian B. Wilkinson, Jorge D. Erusalimsky, Carmel M. McEniery.
  The Journal of Physiology. January 24, 2017
  Key points Age significantly modifies the relationship between aortic pulse wave velocity and telomere length. The differential relationships observed between aortic pulse wave velocity and telomere length in younger and older individuals suggest that the links between cellular and vascular ageing reflect a complex interaction between genetic and environmental factors acting over the life‐course. Abstract Ageing is associated with marked large artery stiffening. Telomere shortening, a marker of cellular ageing, is linked with arterial stiffening. However, the results of existing studies are inconsistent, possibly because of the confounding influence of variable exposure to cardiovascular risk factors. Therefore, we investigated the relationship between telomere length (TL) and aortic stiffness in well‐characterized, younger and older healthy adults, who were pre‐selected on the basis of having either low or high aortic pulse wave velocity (aPWV), a robust measure of aortic stiffness. Demographic, haemodynamic and biochemical data were drawn from participants in the Anglo‐Cardiff Collaborative Trial. Two age groups with an equal sex ratio were examined: those aged <30 years (younger) or >50 years (older). Separately for each age group and sex, DNA samples representing the highest (n = 125) and lowest (n = 125) extremes of aPWV (adjusted for blood pressure) were selected for analysis of leukocyte TL. Ultimately, this yielded complete phenotypic data on 904 individuals. In younger subjects, TL was significantly shorter in those with high aPWV vs. those with low aPWV (P = 0.017). By contrast, in older subjects, TL was significantly longer in those with high aPWV (P = 0.001). Age significantly modified the relationship between aPWV and TL (P < 0.001). Differential relationships are observed between aPWV and TL, with an inverse association in younger individuals and a positive association in older individuals. The links between cellular and vascular ageing reflect a complex interaction between genetic and environmental factors acting over the life‐course.

  January 24, 2017   doi: 10.1113/JP273689   open full text
• Parasympathetic withdrawal increases heart rate after 2 weeks at 3454 m altitude.
  Christoph Siebenmann, Peter Rasmussen, Mike Hug, Stefanie Keiser, Daniela Flück, James P. Fisher, Matthias P. Hilty, Marco Maggiorini, Carsten Lundby.
  The Journal of Physiology. January 24, 2017
  Key points Heart rate is increased in chronic hypoxia and we tested whether this is the result of increased sympathetic nervous activity, reduced parasympathetic nervous activity, or a non‐autonomic mechanism. In seven lowlanders, heart rate was measured at sea level and after 2 weeks at high altitude after individual and combined pharmacological inhibition of sympathetic and/or parasympathetic control of the heart. Inhibition of parasympathetic control of the heart alone or in combination with inhibition of sympathetic control abolished the high altitude‐induced increase in heart rate. Inhibition of sympathetic control of the heart alone did not prevent the high altitude‐induced increase in heart rate. These results indicate that a reduced parasympathetic nervous activity is the main mechanism underlying the elevated heart rate in chronic hypoxia. Abstract Chronic hypoxia increases resting heart rate (HR), but the underlying mechanism remains incompletely understood. We investigated the relative contributions of the sympathetic and parasympathetic nervous systems, along with potential non‐autonomic mechanisms, by individual and combined pharmacological inhibition of muscarinic and/or β‐adrenergic receptors. In seven healthy lowlanders, resting HR was determined at sea level (SL) and after 15–18 days of exposure to 3454 m high altitude (HA) without drug intervention (control, CONT) as well as after intravenous administration of either propranolol (PROP), or glycopyrrolate (GLYC), or PROP and GLYC in combination (PROP+GLYC). Circulating noradrenaline concentration increased from 0.9 ± 0.4 nmol l−1 at SL to 2.7 ± 1.5 nmol l−1 at HA (P = 0.03). The effect of HA on HR depended on the type of autonomic inhibition (P = 0.006). Specifically, HR was increased at HA from 64 ± 10 to 74 ± 12 beats min−1 during the CONT treatment (P = 0.007) and from 52 ± 4 to 59 ± 5 beats min−1 during the PROP treatment (P < 0.001). In contrast, HR was similar between SL and HA during the GLYC treatment (110 ± 7 and 112 ± 5 beats min−1, P = 0.28) and PROP+GLYC treatment (83 ± 5 and 85 ± 5 beats min−1, P = 0.25). Our results identify a reduction in cardiac parasympathetic activity as the primary mechanism underlying the elevated HR associated with 2 weeks of exposure to hypoxia. Unexpectedly, the sympathoactivation at HA that was evidenced by increased circulating noradrenaline concentration had little effect on HR, potentially reflecting down‐regulation of cardiac β‐adrenergic receptor function in chronic hypoxia. These effects of chronic hypoxia on autonomic control of the heart may concern not only HA dwellers, but also patients with disorders that are associated with hypoxaemia.

  January 24, 2017   doi: 10.1113/JP273726   open full text
• The Ca2+ sensitizer CK‐2066260 increases myofibrillar Ca2+ sensitivity and submaximal force selectively in fast skeletal muscle.
  Darren T. Hwee, Arthur J. Cheng, James J. Hartman, Aaron C. Hinken, Ken Lee, Nickie Durham, Alan J. Russell, Fady I. Malik, Håkan Westerblad, Jeffrey R. Jasper.
  The Journal of Physiology. January 24, 2017
  Key points We report that the small molecule CK‐2066260 selectively slows the off‐rate of Ca2+ from fast skeletal muscle troponin, leading to increased myofibrillar Ca2+ sensitivity in fast skeletal muscle. Rodents dosed with CK‐2066260 show increased hindlimb muscle force and power in response to submaximal rates of nerve stimulation in situ. CK‐2066260 has no effect on free cytosolic [Ca2+] during contractions of isolated muscle fibres. We conclude that fast skeletal muscle troponin sensitizers constitute a potential therapy to address an unmet need of improving muscle function in conditions of weakness and premature muscle fatigue. Abstract Skeletal muscle dysfunction occurs in many diseases and can lead to muscle weakness and premature muscle fatigue. Here we show that the fast skeletal troponin activator, CK‐2066260, counteracts muscle weakness by increasing troponin Ca2+ affinity, thereby increasing myofibrillar Ca2+ sensitivity. Exposure to CK‐2066260 resulted in a concentration‐dependent increase in the Ca2+ sensitivity of ATPase activity in isolated myofibrils and reconstituted hybrid sarcomeres containing fast skeletal muscle troponin C. Stopped‐flow experiments revealed a ∼2.7‐fold decrease in the Ca2+ off‐rate of isolated troponin complexes in the presence of CK‐2066260 (6 vs. 17 s−1 under control conditions). Isolated mouse flexor digitorum brevis fibres showed a rapidly developing, reversible and concentration‐dependent force increase at submaximal stimulation frequencies. This force increase was not accompanied by any changes in the free cytosolic [Ca2+] or its kinetics. CK‐2066260 induced a slowing of relaxation, which was markedly larger at 26°C than at 31°C and could be linked to the decreased Ca2+ off‐rate of troponin C. Rats dosed with CK‐2066260 showed increased hindlimb isometric and isokinetic force in response to submaximal rates of nerve stimulation in situ producing significantly higher absolute forces at low isokinetic velocities, whereas there was no difference in force at the highest velocities. Overall muscle power was increased and the findings are consistent with a lack of effect on crossbridge kinetics. In conclusion, CK‐2066260 acts as a fast skeletal troponin activator that may be used to increase muscle force and power in conditions of muscle weakness.

  January 24, 2017   doi: 10.1113/JP273248   open full text
• Resveratrol supplementation of high‐fat diet‐fed pregnant mice promotes brown and beige adipocyte development and prevents obesity in male offspring.
  Tiande Zou, Daiwen Chen, Qiyuan Yang, Bo Wang, Mei‐Jun Zhu, Peter W. Nathanielsz, Min Du.
  The Journal of Physiology. January 24, 2017
  Key points Maternal high‐fat diet impairs brown adipocyte function and correlates with obesity in offspring. Maternal resveratrol administration recovers metabolic activity of offspring brown adipose tissue. Maternal resveratrol promotes beige adipocyte development in offspring white adipose tissue. Maternal resveratrol intervention protects offspring against high‐fat diet‐induced obesity. Abstract Promoting beige/brite adipogenesis and thermogenic activity is considered as a promising therapeutic approach to reduce obesity and metabolic syndrome. Maternal obesity impairs offspring brown adipocyte function and correlates with obesity in offspring. We previously found that dietary resveratrol (RES) induces beige adipocyte formation in adult mice. Here, we evaluated further the effect of resveratrol supplementation of pregnant mice on offspring thermogenesis and energy expenditure. Female C57BL/6 J mice were fed a control diet (CON) or a high‐fat diet (HFD) with or without 0.2% (w/w) RES during pregnancy and lactation. Male offspring were weaned onto a HFD and maintained on this diet for 11 weeks. The offspring thermogenesis and related regulatory factors in adipose tissue were evaluated. At weaning, HFD offspring had lower thermogenesis in brown and white adipose tissues compared with CON offspring, which was recovered by maternal RES supplementation, along with the appearance of multilocular brown/beige adipocytes and elevated thermogenic gene expression. Adult offspring of RES‐treated mothers showed increased energy expenditure and insulin sensitivity when on an obesogenic diet compared with HFD offspring. The elevated metabolic activity was correlated with enhanced brown adipose function and white adipose tissue browning in HFD+RES compared with HFD offspring. In conclusion, RES supplementation of HFD‐fed dams during pregnancy and lactation promoted white adipose browning and thermogenesis in offspring at weaning accompanied by persistent beneficial effects in protecting against HFD‐induced obesity and metabolic disorders.

  January 24, 2017   doi: 10.1113/JP273478   open full text
• Dynamical effects of calcium‐sensitive potassium currents on voltage and calcium alternans.
  Matthew Kennedy, Donald M. Bers, Nipavan Chiamvimonvat, Daisuke Sato.
  The Journal of Physiology. January 24, 2017
  Key points A mathematical model of a small conductance Ca2+‐activated potassium (SK) channel was developed and incorporated into a physiologically detailed ventricular myocyte model. Ca2+‐sensitive K+ currents promote negative intracellular Ca2+ to membrane voltage (CAi2+→ Vm) coupling. Increase of Ca2+‐sensitive K+ currents can be responsible for electromechanically discordant alternans and quasiperiodic oscillations at the cellular level. At the tissue level, Turing‐type instability can occur when Ca2+‐sensitive K+ currents are increased. Abstract Cardiac alternans is a precursor to life‐threatening arrhythmias. Alternans can be caused by instability of the membrane voltage (Vm), instability of the intracellular Ca2+ ( Ca i2+) cycling, or both. Vm dynamics and Ca i2+ dynamics are coupled via Ca2+‐sensitive currents. In cardiac myocytes, there are several Ca2+‐sensitive potassium (K+) currents such as the slowly activating delayed rectifier current (IKs) and the small conductance Ca2+‐activated potassium (SK) current (ISK). However, the role of these currents in the development of arrhythmias is not well understood. In this study, we investigated how these currents affect voltage and Ca2+ alternans using a physiologically detailed computational model of the ventricular myocyte and mathematical analysis. We define the coupling between Vm and Ca i2+ cycling dynamics ( Ca i2+→Vm coupling) as positive (negative) when a larger Ca2+ transient at a given beat prolongs (shortens) the action potential duration (APD) of that beat. While positive coupling predominates at baseline, increasing IKs and ISK promote negative Ca i2+→Vm coupling at the cellular level. Specifically, when alternans is Ca2+‐driven, electromechanically (APD–Ca2+) concordant alternans becomes electromechanically discordant alternans as IKs or ISK increase. These cellular level dynamics lead to different types of spatially discordant alternans in tissue. These findings help to shed light on the underlying mechanisms of cardiac alternans especially when the relative strength of these currents becomes larger under pathological conditions or drug administrations.

  January 24, 2017   doi: 10.1113/JP273626   open full text
• Evidence that an internal schema adapts swallowing to upper airway requirements.
  Seng Mun Wong, Rickie J. Domangue, Sidney Fels, Christy L. Ludlow.
  The Journal of Physiology. January 18, 2017
  Key points To swallow food and liquid safely, airway protection is essential. Upward and forward movements of the hyoid and larynx in the neck during swallowing vary in magnitude between individuals. In healthy human adults, hyoid and laryngeal movements during swallowing were scaled by differences in initial upper airway area before swallowing. Individuals increased laryngeal elevation during swallowing in response to increased airway opening before swallowing. We show that when upper airway protection requirements change, individuals use an internal sensorimotor scaling system to adapt movements to maintain swallow safety. Abstract Hyoid and laryngeal movements contribute to laryngeal vestibule closure and upper oesophageal sphincter opening during swallowing. Evidence of an internal sensorimotor scaling system allowing individuals to achieve these functional goals is lacking. In speech, speakers adjust their articulatory movement magnitude according to the movement distance required to reach an articulatory target for intelligible speech. We investigated if swallowing is similar in that movement amplitude may be scaled by the functional goal for airway protection during swallowing, rather than by head and neck size. We hypothesized that healthy individuals adapt to their own anatomy by adjusting hyo‐laryngeal movements to achieve closure of the upper airway. We also investigated if individuals would automatically compensate for changes in their initial hyo‐laryngeal positions and area when head position was changed prior to swallowing. Videofluoroscopy was performed in 31 healthy adults. Using frame‐by‐frame motion analysis, anterior and superior hyoid and laryngeal displacement, and hyo‐laryngeal area were measured prior to and during swallowing. Kinematic measurements during swallowing were examined for relationships with pharyngeal neck length, and initial hyo‐laryngeal positions, length and area before swallowing. During swallowing, individuals altered laryngeal elevation magnitude to exceed hyoid elevation based on hyo‐laryngeal length before swallowing. Anterior laryngeal displacement was related to initial larynx distance from the spine, while hyoid elevation was predicted by pharyngeal neck length and initial hyoid distance from the mandible prior to the swallow. In conclusion, individuals automatically adapt hyo‐laryngeal movement during swallowing based on targets required for closing the hyo‐laryngeal area for safe swallowing.

  January 18, 2017   doi: 10.1113/JP272368   open full text
• Membrane‐associated guanylate kinase dynamics reveal regional and developmental specificity of synapse stability.
  Jonathan M. Levy, Roger A. Nicoll.
  The Journal of Physiology. January 18, 2017
  Key points The membrane‐associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins anchor glutamate receptors at CNS synapses. MAGUK removal via RNAi‐mediated knockdown in the CA1 hippocampal region in immature animals causes rapid and lasting reductions in glutamatergic transmission. In mature animals, the same manipulation has little acute effect. The hippocampal dentate gyrus, a region with ongoing adult neurogenesis, is sensitive to MAGUK loss in mature animals, behaving like an immature CA1. Over long time courses, removal of MAGUKs in CA1 causes reductions in glutamatergic transmission, indicating that synapses in mature animals require MAGUKs for anchoring glutamate receptors, but are much more stable. These results demonstrate regional and developmental control of synapse stability and suggest the existence of a sensitive period of heightened hippocampal plasticity in CA1 of pre‐adolescent rodents, and in dentate gyrus throughout maturity. Abstract Fast excitatory transmission in the brain requires localization of glutamate receptors to synapses. The membrane‐associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins is critical for localization of glutamate receptors to synapses. Although the MAGUKs are well‐studied in reduced preparations and young animals, few data exist on their role in adult animals. Here, we present a detailed developmental study of the role of the MAGUKs during rat development. We first confirmed by knockdown experiments that MAGUKs are essential for glutamatergic transmission in young animals and cultured slices, and an increase in postsynaptic density protein 95 (PSD‐95) by overexpression caused correlated increases in glutamatergic transmission. We found that CA1 synapses in adults, in contrast, were largely unaffected by overexpression of MAGUKs, and although adult CA1 synapses required MAGUKs to the same degree as synapses in young animals, this was only apparent over long time scales of knockdown. We additionally showed that overexpression of MAGUKs is likely to function to accelerate the developmental strengthening of excitatory transmission. Finally, we showed that adult dentate gyrus appears similar to immature CA1, demonstrating regional and developmental control of MAGUK dynamics. Together, these results demonstrate a period of juvenile instability at CA1 synapses, followed by a period of adult stability in which synapses are acutely unaffected by changes in MAGUK abundance.

  January 18, 2017   doi: 10.1113/JP273147   open full text
• Does trans‐spinal and local DC polarization affect presynaptic inhibition and post‐activation depression?
  D. Kaczmarek, J. Ristikankare, E. Jankowska.
  The Journal of Physiology. January 17, 2017
  Key points Trans‐spinal polarization was recently introduced as a means to improve deficient spinal functions. However, only a few attempts have been made to examine the mechanisms underlying DC actions. We have now examined the effects of DC on two spinal modulatory systems, presynaptic inhibition and post‐activation depression, considering whether they might weaken exaggerated spinal reflexes and enhance excessively weakened ones. Direct current effects were evoked by using local intraspinal DC application (0.3–0.4 μA) in deeply anaesthetized rats and were compared with the effects of trans‐spinal polarization (0.8–1.0 mA). Effects of local intraspinal DC were found to be polarity dependent, as locally applied cathodal polarization enhanced presynaptic inhibition and post‐activation depression, whereas anodal polarization weakened them. In contrast, both cathodal and anodal trans‐spinal polarization facilitated them. The results suggest some common DC‐sensitive mechanisms of presynaptic inhibition and post‐activation depression, because both were facilitated or depressed by DC in parallel. Abstract Direct current (DC) polarization has been demonstrated to alleviate the effects of various deficits in the operation of the central nervous system. However, the effects of trans‐spinal DC stimulation (tsDCS) have been investigated less extensively than the effects of transcranial DC stimulation, and their cellular mechanisms have not been elucidated. The main objectives of this study were, therefore, to extend our previous analysis of DC effects on the excitability of primary afferents and synaptic transmission by examining the effects of DC on two spinal modulatory feedback systems, presynaptic inhibition and post‐activation depression, in an anaesthetized rat preparation. Other objectives were to compare the effects of locally and trans‐spinally applied DC (locDC and tsDCS). Local polarization at the sites of terminal branching of afferent fibres was found to induce polarity‐dependent actions on presynaptic inhibition and post‐activation depression, as cathodal locDC enhanced them and anodal locDC depressed them. In contrast, tsDCS modulated presynaptic inhibition and post‐activation depression in a polarity‐independent fashion because both cathodal and anodal tsDCS facilitated them. The results show that the local presynaptic actions of DC might counteract both excessively strong and excessively weak monosynaptic actions of group Ia and cutaneous afferents. However, they indicate that trans‐spinally applied DC might counteract the exaggerated spinal reflexes but have an adverse effect on pathologically weakened spinal activity by additional presynaptic weakening. The results are also relevant for the analysis of the basic properties of presynaptic inhibition and post‐activation depression because they indicate that some common DC‐sensitive mechanisms contribute to them.

  January 17, 2017   doi: 10.1113/JP272902   open full text
• Gaze‐evoked nystagmus induced by alcohol intoxication.
  Fausto Romano, Alexander A. Tarnutzer, Dominik Straumann, Stefano Ramat, Giovanni Bertolini.
  The Journal of Physiology. January 17, 2017
  Key points The cerebellum is the core structure controlling gaze stability. Chronic cerebellar diseases and acute alcohol intoxication affect cerebellar function, inducing, among others, gaze instability as gaze‐evoked nystagmus. Gaze‐evoked nystagmus is characterized by increased centripetal eye‐drift. It is used as an important diagnostic sign for patients with cerebellar degeneration and to assess the ‘driving while intoxicated’ condition. We quantified the effect of alcohol on gaze‐holding using an approach allowing, for the first time, the comparison of deficits induced by alcohol intoxication and cerebellar degeneration. Our results showed that alcohol intoxication induces a two‐fold increase of centripetal eye‐drift. We establish analysis techniques for using controlled alcohol intake as a model to support the study of cerebellar deficits. The observed similarity between the effect of alcohol and the clinical signs observed in cerebellar patients suggests a possible pathomechanism for gaze‐holding deficits. Abstract Gaze‐evoked nystagmus (GEN) is an ocular‐motor finding commonly observed in cerebellar disease, characterized by increased centripetal eye‐drift with centrifugal correcting saccades at eccentric gaze. With cerebellar degeneration being a rare and clinically heterogeneous disease, data from patients are limited. We hypothesized that a transient inhibition of cerebellar function by defined amounts of alcohol may provide a suitable model to study gaze‐holding deficits in cerebellar disease. We recorded gaze‐holding at varying horizontal eye positions in 15 healthy participants before and 30 min after alcohol intake required to reach 0.6‰ blood alcohol content (BAC). Changes in ocular‐motor behaviour were quantified measuring eye‐drift velocity as a continuous function of gaze eccentricity over a large range (±40 deg) of horizontal gaze angles and characterized using a two‐parameter tangent model. The effect of alcohol on gaze stability was assessed analysing: (1) overall effects on the gaze‐holding system, (2) specific effects on each eye and (3) differences between gaze angles in the temporal and nasal hemifields. For all subjects, alcohol consumption induced gaze instability, causing a two‐fold increase [2.21 (0.55), median (median absolute deviation); P = 0.002] of eye‐drift velocity at all eccentricities. Results were confirmed analysing each eye and hemifield independently. The alcohol‐induced transient global deficit in gaze‐holding matched the pattern previously described in patients with late‐onset cerebellar degeneration. Controlled intake of alcohol seems a suitable disease model to study cerebellar GEN. With alcohol resulting in global cerebellar hypofunction, we hypothesize that patients matching the gaze‐holding behaviour observed here suffered from diffuse deficits in the gaze‐holding system as well.

  January 17, 2017   doi: 10.1113/JP273204   open full text
• Neurovascular mechanisms underlying augmented cold‐induced reflex cutaneous vasoconstriction in human hypertension.
  Jody L. Greaney, W. Larry Kenney, Lacy M. Alexander.
  The Journal of Physiology. January 16, 2017
  Key points In hypertensive adults (HTN), cardiovascular risk increases disproportionately during environmental cold exposure. Despite ample evidence of dysregulated sympathetic control of the peripheral vasculature in hypertension, no studies have examined integrated neurovascular function during cold stress in HTN. The findings of the present study show that whole‐body cold stress elicits greater increases in sympathetic outflow directed to the cutaneous vasculature and, correspondingly, greater reductions in skin blood flow in HTN. We further demonstrate an important role for non‐adrenergic sympathetic co‐transmitters in mediating the vasoconstrictor response to cold stress in hypertension. In the context of thermoregulation and the maintenance of core temperature, sympathetically‐mediated control of the cutaneous vasculature is not only preserved, but also exaggerated in hypertension. Given the increasing prevalence of hypertension, clarifying the mechanistic underpinnings of hypertension‐induced alterations in neurovascular function during cold exposure is clinically relevant. Abstract Despite ample evidence of dysregulated sympathetic control of the peripheral vasculature in hypertension, no studies have examined integrated neurovascular function during cold stress in hypertensive adults (HTN). We hypothesized that (i) whole‐body cooling would elicit greater cutaneous vasoconstriction and greater increases in skin sympathetic nervous system activity (SSNA) in HTN (n = 14; 56 ± 2 years) compared to age‐matched normotensive adults (NTN; n = 14; 55 ± 2 years) and (ii) augmented reflex vasoconstriction in HTN would be mediated by an increase in cutaneous vascular adrenergic sensitivity and a greater contribution of non‐adrenergic sympathetic co‐transmitters. SSNA (peroneal microneurography) and red cell flux (laser Doppler flowmetry; dorsum of foot) were measured during whole‐body cooling (water‐perfused suit). Sympathetic adrenergic‐ and non‐adrenergic‐dependent contributions to reflex cutaneous vasoconstriction and vascular adrenergic sensitivity were assessed pharmacologically using intradermal microdialysis. Cooling elicited greater increases in SSNA (NTN: +64 ± 13%baseline vs. HTN: +194 ± 26%baseline; P < 0.01) and greater reductions in skin blood flow (NTN: −16 ± 2%baseline vs. HTN: −28 ± 3%baseline; P < 0.01) in HTN compared to NTN, reflecting an increased response range for sympathetic reflex control of cutaneous vasoconstriction in HTN. Norepinephrine dose–response curves showed no HTN‐related difference in cutaneous adrenergic sensitivity (logEC50; NTN: −7.4 ± 0.3 log M vs. HTN: −7.5 ± 0.3 log M; P = 0.84); however, non‐adrenergic sympathetic co‐transmitters mediated a significant portion of the vasoconstrictor response to cold stress in HTN. Collectively, these findings indicate that hypertension increases the peripheral cutaneous vasoconstrictor response to cold via greater increases in skin sympathetic outflow coupled with an increased reliance on non‐adrenergic neurotransmitters.

  January 16, 2017   doi: 10.1113/JP273487   open full text
• Post‐translational cleavage of Hv1 in human sperm tunes pH‐ and voltage‐dependent gating.
  Thomas K. Berger, David M. Fußhöller, Normann Goodwin, Wolfgang Bönigk, Astrid Müller, Nasim Dokani Khesroshahi, Christoph Brenker, Dagmar Wachten, Eberhard Krause, U. Benjamin Kaupp, Timo Strünker.
  The Journal of Physiology. January 15, 2017
  Key points In human sperm, proton flux across the membrane is controlled by the voltage‐gated proton channel Hv1. We show that sperm harbour both Hv1 and an N‐terminally cleaved isoform termed Hv1Sper. The pH‐control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. Abstract In human sperm, the voltage‐gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N‐terminus by post‐translational proteolytic cleavage. The pH‐dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance–voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N‐terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.

  January 15, 2017   doi: 10.1113/JP273189   open full text
• Causal relationships between neurons of the nucleus incertus and the hippocampal theta activity in the rat.
  Sergio Martínez‐Bellver, Ana Cervera‐Ferri, Aina Luque‐García, Joana Martínez‐Ricós, Alfonso Valverde‐Navarro, Manuel Bataller, Juan Guerrero, Vicent Teruel‐Marti.
  The Journal of Physiology. January 10, 2017
  Key points The nucleus incertus is a key node of the brainstem circuitry involved in hippocampal theta rhythmicity. Synchronisation exists between the nucleus incertus and hippocampal activities during theta periods. By the Granger causality analysis, we demonstrated a directional information flow between theta rhythmical neurons in the nucleus incertus and the hippocampus in theta‐on states. The electrical stimulation of the nucleus incertus is also able to evoke a phase reset of the hippocampal theta wave. Our data suggest that the nucleus incertus is a key node of theta generation and the modulation network. Abstract In recent years, a body of evidence has shown that the nucleus incertus (NI), in the dorsal tegmental pons, is a key node of the brainstem circuitry involved in hippocampal theta rhythmicity. Ascending reticular brainstem system activation evokes hippocampal theta rhythm with coupled neuronal activity in the NI. In a recent paper, we showed three populations of neurons in the NI with differential firing during hippocampal theta activation. The objective of this work was to better evaluate the causal relationship between the activity of NI neurons and the hippocampus during theta activation in order to further understand the role of the NI in the theta network. A Granger causality analysis was run to determine whether hippocampal theta activity with sensory‐evoked theta depends on the neuronal activity of the NI, or vice versa. The analysis showed causal interdependence between the NI and the hippocampus during theta activity, whose directional flow depended on the different neuronal assemblies of the NI. Whereas type I and II NI neurons mainly acted as receptors of hippocampal information, type III neuronal activity was the predominant source of flow between the NI and the hippocampus in theta states. We further determined that the electrical activation of the NI was able to reset hippocampal waves with enhanced theta‐band power, depending on the septal area. Collectively, these data suggest that hippocampal theta oscillations after sensory activation show dependence on NI neuron activity, which could play a key role in establishing optimal conditions for memory encoding.

  January 10, 2017   doi: 10.1113/JP272841   open full text
• Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics.
  Nipavan Chiamvimonvat, Ye Chen‐Izu, Colleen E. Clancy, Isabelle Deschenes, Dobromir Dobrev, Jordi Heijman, Leighton Izu, Zhilin Qu, Crystal M. Ripplinger, Jamie I. Vandenberg, James N. Weiss, Gideon Koren, Tamas Banyasz, Eleonora Grandi, Michael C. Sanguinetti, Donald M. Bers, Jeanne M. Nerbonne.
  The Journal of Physiology. January 05, 2017
  Abstract This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation–Contraction Coupling and Arrhythmias (3–4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was ‘K+ channels and regulation’, and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen‐Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state‐of‐the‐art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

  January 05, 2017   doi: 10.1113/JP272883   open full text
• Influence of menstrual phase and arid vs. humid heat stress on autonomic and behavioural thermoregulation during exercise in trained but unacclimated women.
  Tze‐Huan Lei, Stephen R. Stannard, Blake G. Perry, Zachary J. Schlader, James D. Cotter, Toby Mündel.
  The Journal of Physiology. January 04, 2017
  Key points Despite an attenuated fluctuation in ovarian hormone concentrations in well‐trained women, one in two of such women believe their menstrual cycle negatively impacts training and performance. Forthcoming large international events will expose female athletes to hot environments, and studies evaluating aerobic exercise performance in such environments across the menstrual cycle are sparse, with mixed findings. We have identified that autonomic heat loss responses at rest and during fixed‐intensity exercise in well‐trained women are not affected by menstrual cycle phase, but differ between dry and humid heat. Furthermore, exercise performance is not different across the menstrual cycle, yet is lower in humid heat, in conjunction with reduced evaporative cooling. Menstrual cycle phase does not appear to affect exercise performance in the heat in well‐trained women, but humidity impairs performance, probably due to reduced evaporative power. Abstract We studied thermoregulatory responses of ten well‐trained [V̇O2 max , 57 (7) ml min−1 kg−1] eumenorrheic women exercising in dry and humid heat, across their menstrual cycle. They completed four trials, each of resting and cycling at fixed intensities (125 and 150 W), to assess autonomic regulation, then self‐paced intensity (30 min work trial), to assess behavioural regulation. Trials were in early‐follicular (EF) and mid‐luteal (ML) phases in dry (DRY) and humid (HUM) heat matched for wet bulb globe temperature (WBGT, 27°C). During rest and fixed‐intensity exercise, rectal temperature was ∼0.2°C higher in ML than EF (P < 0.01) independent of environment (P = 0.66). Mean skin temperature did not differ between menstrual phases (P ≥ 0.13) but was higher in DRY than HUM (P < 0.01). Local sweat rate and/or forearm blood flow differed as a function of menstrual phase and environment (interaction: P ≤ 0.01). Exercise performance did not differ between phases [EF: 257 (37), ML: 255 (43) kJ, P = 0.62], but was 7 (9)% higher in DRY than HUM [263 (39), 248 (40) kJ; P < 0.01] in conjunction with equivalent autonomic regulation and thermal strain but higher evaporative cooling [16 (6) W m2; P < 0.01]. In well‐trained women exercising in the heat: (1) menstrual phase did not affect performance, (2) humidity impaired performance due to reduced evaporative cooling despite matched WBGT and (3) behavioural responses nullified thermodynamic and autonomic differences associated with menstrual phase and dry vs. humid heat.

  January 04, 2017   doi: 10.1113/JP273176   open full text
• Extracellular protons enable activation of the calcium‐dependent chloride channel TMEM16A.
  Silvia Cruz‐Rangel, José J. Jesús‐Pérez, Iván A. Aréchiga‐Figueroa, Aldo A. Rodríguez‐Menchaca, Patricia Pérez‐Cornejo, H. Criss Hartzell, Jorge Arreola.
  The Journal of Physiology. January 03, 2017
  Key points The calcium‐activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage‐dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un‐protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. Abstract Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore‐forming subunit of a Ca2+‐dependent Cl− channel (CaCC), is activated by direct, voltage‐dependent, binding of intracellular Ca2+. Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+]o) on mouse TMEM16A expressed in HEK‐293 cells using whole‐cell and inside‐out patch‐clamp recordings. We found that increasing the [H+]o from 10−10 to 10−5.5 m caused a progressive increase in the chloride current (ICl) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage‐independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10−5.5 and 10−9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10−5.5 m, but 10−9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non‐saturating [Ca2+]i.

  January 03, 2017   doi: 10.1113/JP273111   open full text
• Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl− channel.
  Jeng‐Haur Chen, Weiyi Xu, David N. Sheppard.
  The Journal of Physiology. January 03, 2017
  Key points The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating.

  January 03, 2017   doi: 10.1113/JP273205   open full text
• The effect of α1‐adrenergic blockade on post‐exercise brachial artery flow‐mediated dilatation at sea level and high altitude.
  Michael M. Tymko, Joshua C. Tremblay, Alex B. Hansen, Connor A. Howe, Chris K. Willie, Mike Stembridge, Daniel J. Green, Ryan L. Hoiland, Prajan Subedi, James D. Anholm, Philip N. Ainslie.
  The Journal of Physiology. December 29, 2016
  Key points Our objective was to quantify endothelial function (via brachial artery flow‐mediated dilatation) at sea level (344 m) and high altitude (3800 m) at rest and following both maximal exercise and 30 min of moderate‐intensity cycling exercise with and without administration of an α1‐adrenergic blockade. Brachial endothelial function did not differ between sea level and high altitude at rest, nor following maximal exercise. At sea level, endothelial function decreased following 30 min of moderate‐intensity exercise, and this decrease was abolished with α1‐adrenergic blockade. At high altitude, endothelial function did not decrease immediately after 30 min of moderate‐intensity exercise, and administration of α1‐adrenergic blockade resulted in an increase in flow‐mediated dilatation. Our data indicate that post‐exercise endothelial function is modified at high altitude (i.e. prolonged hypoxaemia). The current study helps to elucidate the physiological mechanisms associated with high‐altitude acclimatization, and provides insight into the relationship between sympathetic nervous activity and vascular endothelial function. Abstract We examined the hypotheses that (1) at rest, endothelial function would be impaired at high altitude compared to sea level, (2) endothelial function would be reduced to a greater extent at sea level compared to high altitude after maximal exercise, and (3) reductions in endothelial function following moderate‐intensity exercise at both sea level and high altitude are mediated via an α1‐adrenergic pathway. In a double‐blinded, counterbalanced, randomized and placebo‐controlled design, nine healthy participants performed a maximal‐exercise test, and two 30 min sessions of semi‐recumbent cycling exercise at 50% peak output following either placebo or α1‐adrenergic blockade (prazosin; 0.05 mg kg −1). These experiments were completed at both sea‐level (344 m) and high altitude (3800 m). Blood pressure (finger photoplethysmography), heart rate (electrocardiogram), oxygen saturation (pulse oximetry), and brachial artery blood flow and shear rate (ultrasound) were recorded before, during and following exercise. Endothelial function assessed by brachial artery flow‐mediated dilatation (FMD) was measured before, immediately following and 60 min after exercise. Our findings were: (1) at rest, FMD remained unchanged between sea level and high altitude (placebo P = 0.287; prazosin: P = 0.110); (2) FMD remained unchanged after maximal exercise at sea level and high altitude (P = 0.244); and (3) the 2.9 ± 0.8% (P = 0.043) reduction in FMD immediately after moderate‐intensity exercise at sea level was abolished via α1‐adrenergic blockade. Conversely, at high altitude, FMD was unaltered following moderate‐intensity exercise, and administration of α1‐adrenergic blockade elevated FMD (P = 0.032). Our results suggest endothelial function is differentially affected by exercise when exposed to hypobaric hypoxia. These findings have implications for understanding the chronic impacts of hypoxaemia on exercise, and the interactions between the α1‐adrenergic pathway and endothelial function.

  December 29, 2016   doi: 10.1113/JP273183   open full text
• Differences in TRPC3 and TRPC6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.
  Inés Álvarez‐Miguel, Pilar Cidad, M. Teresa Pérez‐García, José Ramón López‐López.
  The Journal of Physiology. December 29, 2016
  Key points Canonical transient receptor potential (TRPC)3 and TRPC6 channels of vascular smooth muscle cells (VSMCs) mediate stretch‐ or agonist‐induced cationic fluxes, contributing to membrane potential and vascular tone. Native TRPC3/C6 channels can form homo‐ or heterotetrameric complexes, which can hinder individual TRPC channel properties. The possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. Functional characterization of heterologously expressed channels showed that TRPC6‐containing complexes exhibited Pyr3/Pyr10‐sensitive currents, whereas TRPC3‐mediated currents were blocked by anti‐TRPC3 antibodies. VSMCs from hypertensive (blood pressure high; BPH) mice have larger cationic basal currents insensitive to Pyr10 and sensitive to anti‐TRPC3 antibodies. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN (blood pressure normal) mesenteric arteries. We conclude that the increased TRPC3 channel expression in BPH VSMCs leads to changes in TRPC3/C6 heteromultimeric assembly, with a higher TRPC3 channel contribution favouring depolarization of hypertensive VSMCs. Abstract Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L‐type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol‐activated, non‐selective cationic channels contributing to stretch‐ or agonist‐induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3‐TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non‐selective cationic currents through homo‐ and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti‐TRPC3 antibody selectively blocked TRPC3‐mediated currents. In mesenteric VSMCs, basal and agonist‐induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10‐induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel therapeutical opportunities.

  December 29, 2016   doi: 10.1113/JP273327   open full text
• Differential roles of two delayed rectifier potassium currents in regulation of ventricular action potential duration and arrhythmia susceptibility.
  Ryan A. Devenyi, Francis A. Ortega, Willemijn Groenendaal, Trine Krogh‐Madsen, David J. Christini, Eric A. Sobie.
  The Journal of Physiology. December 28, 2016
  Key points Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood. We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated. A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model. The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias. Abstract Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information‐rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re‐parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K+ current and a drastic decrease in the slow delayed rectifier K+ current, and this prediction was validated experimentally. Subsequent simulations with the adjusted model generated the clinically relevant prediction that the slow delayed rectifier is better able to stabilize the action potential and suppress pro‐arrhythmic events than the rapid delayed rectifier. In summary, iterative coupling of simulations and experiments enabled novel insight into how the balance between cardiac K+ currents influences ventricular arrhythmia susceptibility.

  December 28, 2016   doi: 10.1113/JP273191   open full text
• Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP.
  Saša Jovanovic, Tamara Radulovic, Claudio Coddou, Beatrice Dietz, Jana Nerlich, Stanko S. Stojilkovic, Rudolf Rübsamen, Ivan Milenkovic.
  The Journal of Physiology. December 28, 2016
  Key points Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell‐specific and tonotopically‐determined manner. Developmental down‐regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high‐to‐low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP‐AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors. Abstract Synaptic refinement and strengthening are activity‐dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell‐specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high‐frequency regions, ATP responsiveness diminished before hearing onset. In low‐to‐mid frequency regions, ATP modulation persisted after hearing onset in a subset of low‐frequency bushy cells (characteristic frequency< 10 kHz). Down‐regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high‐to‐low frequency maturation pattern. Facilitated AP generation, measured as higher firing frequency, shorter EPSP‐AP delay in vivo, and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R.

  December 28, 2016   doi: 10.1113/JP273272   open full text
• Inwardly rectifying K+ channels are major contributors to flow‐induced vasodilatation in resistance arteries.
  Sang Joon Ahn, Ibra S. Fancher, Jing‐Tan Bian, Chong Xu Zhang, Sarah Schwab, Robert Gaffin, Shane A. Phillips, Irena Levitan.
  The Journal of Physiology. December 26, 2016
  Key points Endothelial inwardly rectifying K+ (Kir2.1) channels regulate flow‐induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries. Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance. Flow‐induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K+ channels. This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow. Abstract Inwardly rectifying K+ (Kir) channels are known to be sensitive to flow, but their role in flow‐induced endothelial responses is not known. The goal of this study is to establish the role of Kir channels in flow‐induced vasodilatation and to provide first insights into the mechanisms responsible for Kir signalling in this process. First, we establish that primary endothelial cells isolated from murine mesenteric arteries express functional Kir2.1 channels sensitive to shear stress. Then, using the Kir2.1+/− heterozygous mouse model, we establish that downregulation of Kir2.1 results in significant decrease in shear‐activated Kir currents and inhibition of endothelium‐dependent flow‐induced vasodilatation (FIV) assayed in pressurized mesenteric arteries pre‐constricted with endothelin‐1. Deficiency in Kir2.1 also results in the loss of flow‐induced phosphorylation of eNOS and Akt, as well as inhibition of NO generation. All the effects are fully rescued by endothelial cell (EC)‐specific overexpression of Kir2.1. A component of FIV that is Kir independent is abrogated by blocking Ca2+‐sensitive K+ channels. Kir2.1 has no effect on endothelium‐independent and K+‐induced vasodilatation in denuded arteries. Kir2.1+/− mice also show increased mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a tail‐cuff. Importantly, blocking Kir channels also inhibits flow‐induced vasodilatation in human subcutaneous adipose microvessels. Endothelial Kir channels contribute to FIV of mouse mesenteric arteries via an NO‐dependent mechanism, whereas Ca2+‐sensitive K+ channels mediate FIV via an NO‐independent pathway. Kir2 channels also regulate vascular resistance and blood pressure. Finally, Kir channels also contribute to FIV in human subcutaneous microvessels.

  December 26, 2016   doi: 10.1113/JP273255   open full text
• Evidence of viscerally‐mediated cold‐defence thermoeffector responses in man.
  Nathan B. Morris, Davide Filingeri, Mark Halaki, Ollie Jay.
  The Journal of Physiology. December 26, 2016
  Key points Visceral thermoreceptors that modify thermoregulatory responses are widely accepted in animal but not human thermoregulation models. Recently, we have provided evidence of viscerally‐mediated sweating alterations in humans during exercise brought about by warm and cool fluid ingestion. In the present study, we characterize the modification of shivering and whole‐body thermal sensation during cold stress following the administration of a graded thermal stimuli delivered to the stomach via fluid ingestion at 52, 37, 22 and 7°C. Despite no differences in core and skin temperature, fluid ingestion at 52°C rapidly decreased shivering and sensations of cold compared to 37°C, whereas fluid ingestion at 22 and 7°C led to equivalent increases in these responses. Warm and cold fluid ingestion independently modifies cold defence thermoeffector responses, supporting the presence of visceral thermoreceptors in humans. However, the cold‐defence thermoeffector response patterns differed from previously identified hot‐defence thermoeffectors. Abstract Sudomotor activity is modified by both warm and cold fluid ingestion during heat stress, independently of differences in core and skin temperatures, suggesting independent viscerally‐mediated modification of thermoeffectors. The present study aimed to determine whether visceral thermoreceptors modify shivering responses to cold stress. Ten males (mean ± SD: age 27 ± 5 years; height 1.73 ± 0.06 m, weight 78.4 ± 10.7 kg) underwent whole‐body cooling via a water perfusion suit at 5°C, on four occasions, to induce a steady‐state shivering response, at which point two aliquots of 1.5 ml kg–1 (SML) and 3.0 ml kg–1 (LRG), separated by 20 min, of water at 7, 22, 37 or 52°C were ingested. Rectal, mean skin and mean body temperature (Tb), electromyographic activity (EMG), metabolic rate (M) and whole‐body thermal sensation on a visual analogue scale (WBTS) ranging from 0 mm (very cold) to 200 mm (very hot) were all measured throughout. Tb was not different between all fluid temperatures following SML fluid ingestion (7°C: 35.7 ± 0.5°C; 22°C: 35.6 ± 0.5°C; 37°C: 35.5 ± 0.4°C; 52°C: 35.5 ± 0.4°C; P = 0.27) or LRG fluid ingestion (7°C: 35.3 ± 0.6°C; 22°C: 35.3 ± 0.5°C; 37°C: 35.2 ± 0.5°C; 52°C: 35.3 ± 0.5°C; P = 0.99). With SML fluid ingestion, greater metabolic rates and cooler thermal sensations were observed with ingestion at 7°C (M: 179 ± 55 W, WBTS: 29 ± 21 mm) compared to 52°C (M: 164 ± 34 W, WBTS: 51 ± 28 mm; all P < 0.05). With LRG ingestion, compared to shivering and thermal sensations with ingestion at 37°C (M: 215 ± 47 W, EMG: 3.9 ± 2.5% MVC, WBTS: 33 ± 2 mm), values were different (all P < 0.05) following ingestion at 7°C (M: 269 ± 77 W, EMG: 5.5 ± 0.9% MVC, WBTS: 14 ± 12 mm), 22°C (M: 270 ± 86 W, EMG: 5.6 ± 1.0% MVC, WBTS: 18 ± 19 mm) and 52°C (M: 179 ± 34 W, EMG: 3.3 ± 2.1% MVC, WBTS: 53 ± 28 mm). In conclusion, fluid ingestion at 52°C decreased shivering and the sensation of coolness, whereas fluid ingestion at 22 and 7°C increased shivering and sensations of coolness to similar levels, independently of core and skin temperature.

  December 26, 2016   doi: 10.1113/JP273052   open full text
• Cardiac remodelling in a baboon model of intrauterine growth restriction mimics accelerated ageing.
  Anderson H. Kuo, Cun Li, Jinqi Li, Hillary F. Huber, Peter W. Nathanielsz, Geoffrey D. Clarke.
  The Journal of Physiology. December 17, 2016
  Key points Rodent models of intrauterine growth restriction (IUGR) successfully identify mechanisms that can lead to short‐term and long‐term detrimental cardiomyopathies but differences between rodent and human cardiac physiology and placental‐fetal development indicate a need for models in precocial species for translation to human development. We developed a baboon model for IUGR studies using a moderate 30% global calorie restriction of pregnant mothers and used cardiac magnetic resonance imaging to evaluate offspring heart function in early adulthood. Impaired diastolic and systolic cardiac function was observed in IUGR offspring with differences between male and female subjects, compared to their respective controls. Aspects of cardiac impairment found in the IUGR offspring were similar to those found in normal controls in a geriatric cohort. Understanding early cardiac biomarkers of IUGR using non‐invasive imaging in this susceptible population, especially taking into account sexual dimorphisms, will aid recognition of the clinical presentation, development of biomarkers suitable for use in humans and management of treatment strategies. Abstract Extensive rodent studies have shown that reduced perinatal nutrition programmes chronic cardiovascular disease. To enable translation to humans, we developed baboon offspring cohorts from mothers fed ad libitum (control) or 70% of the control ad libitum diet in pregnancy and lactation, which were growth restricted at birth. We hypothesized that intrauterine growth restriction (IUGR) offspring hearts would show impaired function and a premature ageing phenotype. We studied IUGR baboons (8 male, 8 female, 5.7 years), control offspring (8 male, 8 female, 5.6 years – human equivalent approximately 25 years), and normal elderly (OLD) baboons (6 male, 6 female, mean 15.9 years). Left ventricular (LV) morphology and systolic and diastolic function were evaluated with cardiac MRI and normalized to body surface area. Two‐way ANOVA by group and sex (with P < 0.05) indicated ejection fraction, 3D sphericity indices, cardiac index, normalized systolic volume, normalized LV wall thickness, and average filling rate differed by group. Group and sex differences were found for normalized LV wall thickening and normalized myocardial mass, without interactions. Normalized peak LV filling rate and diastolic sphericity index were not correlated in control but strongly correlated in OLD and IUGR baboons. IUGR programming in baboons produces myocardial remodelling, reduces systolic and diastolic function, and results in the emergence of a premature ageing phenotype in the heart. To our knowledge, this is the first demonstration of the specific characteristics of cardiac programming and early life functional decline with ageing in an IUGR non‐human primate model. Further studies across the life span will determine progression of cardiac dysfunction.

  December 17, 2016   doi: 10.1113/JP272908   open full text
• Human motoneurone excitability is depressed by activation of serotonin 1A receptors with buspirone.
  Jessica M. D'Amico, Annie A. Butler, Martin E. Héroux, Florence Cotel, Jean‐François M. Perrier, Jane E. Butler, Simon C. Gandevia, Janet L. Taylor.
  The Journal of Physiology. December 17, 2016
  Key points In the adult turtle spinal cord, action potential generation in motoneurones is inhibited by spillover of serotonin to extrasynaptic serotonin 1A (5‐HT1A) receptors at the axon initial segment. We explored whether ingestion of the 5‐HT1A receptor partial agonist, buspirone, decreases motoneurone excitability in humans. Following ingestion of buspirone, two tests of motoneurone excitability showed decreases. F‐wave areas and persistence in an intrinsic muscle of the hand were reduced, as was the area of cervicomedullary motor evoked potentials in biceps brachii. Our findings suggest that activation of 5‐HT1A receptors depresses human motoneurone excitability. Such a depression could contribute to decreased motoneurone output during fatiguing exercise if there is high serotonergic drive to the motoneurones. Abstract Intense serotonergic drive in the turtle spinal cord results in serotonin spillover to the axon initial segment of the motoneurones where it activates serotonin 1A (5‐HT1A) receptors and inhibits generation of action potentials. We examined whether activation of 5‐HT1A receptors decreases motoneurone excitability in humans by determining the effects of a 5‐HT1A receptor partial agonist, buspirone, on F waves and cervicomedullary motor evoked potentials (CMEPs). In a placebo‐controlled double‐blind study, 10 participants were tested on two occasions where either placebo or 20 mg of buspirone was administered orally. The ulnar nerve was stimulated supramaximally to evoke F waves in abductor digiti minimi (ADM). CMEPs and the maximal M wave were elicited in biceps brachii by cervicomedullary stimulation and brachial plexus stimulation, respectively. Following buspirone intake, F‐wave area and persistence, as well as CMEP area, were significantly decreased. The mean post‐pill difference in normalized F‐wave areas and persistence between buspirone and placebo days was –27% (–42, –12; 95% confidence interval) and –9% (–16, –2), respectively. The mean post‐pill difference in normalized CMEP area between buspirone and placebo days showed greater variation and was –31% (–60, –2). In conclusion, buspirone reduces motoneurone excitability in humans probably via activation of 5‐HT1A receptors at the axon initial segment. This has implications for motor output during high drive to the motoneurones when serotonin may spill over to these inhibitory receptors and consequently inhibit motoneurone output. Such a mechanism could potentially contribute to fatigue with exercise.

  December 17, 2016   doi: 10.1113/JP273200   open full text
• A unifying hypothesis for M1 muscarinic receptor signalling in pyramidal neurons.
  Sameera Dasari, Corey Hill, Allan T. Gulledge.
  The Journal of Physiology. December 17, 2016
  Key points Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes. Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer‐lasting excitation. Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted. We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling. We propose a ‘unifying hypothesis’ for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space. Abstract Gq‐coupled M1‐type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium‐dependent small conductance potassium (‘SK’) channels, and longer‐lasting and voltage‐dependent excitation involving non‐specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the ‘unifying hypothesis’ that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR‐driven excitation acts to refill those stores by promoting voltage‐dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10 mV from RMPs) initiated with variable delay (up to 500 ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time‐locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage‐sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage‐dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK‐mediated inhibition in response to phasic release of ACh.

  December 17, 2016   doi: 10.1113/JP273627   open full text
• Consequences of maternal omega‐3 polyunsaturated fatty acid supplementation on respiratory function in rat pups.
  Luana Tenorio‐Lopes, Cécile Baldy, Alexandra Jochmans‐Lemoine, Océane Mercier, Olivier Pothier‐Piccinin, Tommy Seaborn, Vincent Joseph, Isabelle Marc, Richard Kinkead.
  The Journal of Physiology. December 16, 2016
  Key points Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre‐term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli. Based on evidence suggesting that omega‐3 polyunsaturated fatty acids (n‐3 PUFA) improves brain development, we determined whether n‐3 PUFA supplementation (via the maternal diet) improves respiratory function in 10–11‐day‐old rat pups. n‐3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n‐3 PUFA treatment also reduced microglia cell density in the brainstem. Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n‐3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex. Abstract Most pre‐term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega‐3 polyunsaturated fatty acids (n‐3 PUFA) benefit brain development, we hypothesized that n‐3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n‐3 PUFA supplementation from an enriched diet (13 g kg−1 of n‐3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10–11). Controls received a standard diet (0.3 g kg−1 of n‐3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO2 = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n‐3 PUFA supplementation effectively raised n‐3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n‐3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n‐3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n‐3 PUFA shortened the duration of LCR‐induced apnoeas by 32%. The relative pulmonary surface area of n‐3 PUFA supplemented pups was 12% higher than controls. Although n‐3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

  December 16, 2016   doi: 10.1113/JP273471   open full text
• High resolution three‐dimensional reconstruction of fibrotic skeletal muscle extracellular matrix.
  Allison R. Gillies, Mark A. Chapman, Eric A. Bushong, Thomas J. Deerinck, Mark H. Ellisman, Richard L. Lieber.
  The Journal of Physiology. December 14, 2016
  Key points Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function. It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis. Quantitative and qualitative high‐magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen‐producing cells. Fibrotic muscles are stiffer and have a higher concentration of collagen‐producing cells. These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy. Abstract Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild‐type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function.

  December 14, 2016   doi: 10.1113/JP273376   open full text
• Brainstem sources of cardiac vagal tone and respiratory sinus arrhythmia.
  David G.S. Farmer, Mathias Dutschmann, Julian F.R. Paton, Anthony E. Pickering, Robin M. McAllen.
  The Journal of Physiology. December 14, 2016
  Key points Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. Respiratory‐linked fluctuations in cardiac vagal tone give rise to respiratory sinus arryhthmia (RSA), with maximum tone in the post‐inspiratory phase of respiration. In the present study, we investigated whether respiratory modulation of cardiac vagal tone is intrinsically linked to post‐inspiratory respiratory control using the unanaesthetized working heart‐brainstem preparation of the rat. Abolition of post‐inspiration, achieved by inhibition of the pontine Kolliker‐Fuse nucleus, removed post‐inspiratory peaks in efferent cardiac vagal activity and suppressed RSA, whereas substantial cardiac vagal tone persisted. After transection of the caudal pons, part of the remaining tone was removed by inhibition of nucleus of the solitary tract. We conclude that cardiac vagal tone depends upon at least 3 sites of the pontomedullary brainstem and that a significant proportion arises independently of RSA. Abstract Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. The rat working heart‐brainstem preparation shows strong cardiac vagal tone and pronounced respiratory sinus arrhythmia. In this preparation, recordings from the cut left cardiac vagal branch showed efferent activity that peaked in post‐inspiration, ∼0.5 s before the cyclic minimum in heart rate (HR). We hypothesized that respiratory modulation of cardiac vagal tone and HR is intrinsically linked to the generation of post‐inspiration. Neurons in the pontine Kölliker‐Fuse nucleus (KF) were inhibited with bilateral microinjections of isoguvacine (50–70 nl, 10 mm) to remove the post‐inspiratory phase of respiration. This also abolished the post‐inspiratory peak of cardiac vagal discharge (and cyclical HR modulation), although a substantial level of activity remained. In separate preparations with intact cardiac vagal branches but sympathetically denervated by thoracic spinal pithing, cardiac chronotropic vagal tone was quantified by HR compared to its final level after systemic atropine (0.5 μm). Bilateral KF inhibition removed 88% of the cyclical fluctuation in HR but, on average, only 52% of the chronotropic vagal tone. Substantial chronotropic vagal tone also remained after transection of the brainstem through the caudal pons. Subsequent bilateral isoguvacine injections into the nucleus of the solitary tract further reduced vagal tone: remaining sources were untraced. We conclude that cardiac vagal tone depends on neurons in at least three sites of the pontomedullary brainstem, and much of it arises independently of respiratory sinus arrhythmia.

  December 14, 2016   doi: 10.1113/JP273164   open full text
• Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation.
  Calum Wilson, Matthew D. Lee, John G. McCarron.
  The Journal of Physiology. December 14, 2016
  Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli. The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown. In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling. Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. Abstract Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction.

  December 14, 2016   doi: 10.1113/JP272927   open full text
• Nitric oxide synthase and cyclooxygenase modulate β‐adrenergic cutaneous vasodilatation and sweating in young men.
  Naoto Fujii, Brendan D. McNeely, Glen P. Kenny.
  The Journal of Physiology. December 12, 2016
  Key points β‐Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in β‐adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol. We show that while NOS contributes to β‐adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation. We also show that combined inhibition of NOS and COX augments β‐adrenergic sweating These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of β‐adrenergic receptors in the skin. Abstract β‐Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to β‐adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to β‐adrenergic sweating. In 10 young males (25 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10 mm Nω‐nitro‐l‐arginine (l‐NNA), a non‐specific NOS inhibitor, (3) 10 mm ketorolac, a non‐specific COX inhibitor, or (4) a combination of l‐NNA and ketorolac. All sites were co‐administered with a high dose of isoproterenol (100 μm) for 3 min to maximally induce β‐adrenergic sweating (β‐adrenergic sweating is significantly blunted by subsequent activations). Approximately 60 min after the washout period, three incremental doses of isoproterenol were co‐administered (1, 10 and 100 μm each for 25 min). Increases in CVC induced by the first and second 100 μm isoproterenol were attenuated by l‐NNA alone, and those in response to all doses of isoproterenol were reduced by l‐NNA with co‐infusion of ketorolac (all P ≤ 0.05). Ketorolac alone augmented increases in CVC induced by 10 μm and by the second 100 μm isoproterenol (both P ≤ 0.05). While isoproterenol‐induced sweating was not affected by the separate administration of l‐NNA or ketorolac (all P > 0.05), their combined administration augmented sweating elicited by the first 3 min of 100 μm isoproterenol (P = 0.05). We show that while NOS contributes to β‐adrenergic cutaneous vasodilatation, COX restrains the vasodilatation. Finally, combined inhibition of NOS and COX augments β‐adrenergic sweating.

  December 12, 2016   doi: 10.1113/JP273502   open full text
• FoxO‐dependent atrogenes vary among catabolic conditions and play a key role in muscle atrophy induced by hindlimb suspension.
  Lorenza Brocca, Luana Toniolo, Carlo Reggiani, Roberto Bottinelli, Marco Sandri, Maria Antonietta Pellegrino.
  The Journal of Physiology. December 12, 2016
  Key points Muscle atrophy is a debilitating condition that affects a high percentage of the population with a negative impact on quality of life. Dissecting the molecular level of the atrophy process, and the similarities/dissimilarities among different catabolic conditions, is a necessary step for designing specific countermeasures to attenuate/prevent muscle loss. The FoxO family transcription factors represent one of the most important regulators of atrophy programme stimulating the expression of many atrophy‐related genes. The findings of the present study clearly indicate that the signalling network controlling the atrophy programme is specific for each catabolic condition. Abstract Muscle atrophy is a complex process that is in common with many different catabolic diseases including disuse/inactivity and ageing. The signalling pathways that control the atrophy programme in the different disuse/inactivity conditions have not yet been completely dissected. The inhibition of FoxO is considered to only partially spare muscle mass after denervation. The present study aimed: (i) to determine the involvement of FoxOs in hindlimb suspension disuse model; (ii) to define whether the molecular events of protein breakdown are shared among different unloaded muscles; and finally (iii) to compare the data obtained in this model with another model of inactivity such as denervation. Both wild‐type and muscle‐specific FoxO1,3,4 knockout (FoxO1,3,4−/−) mice were unloaded for 3 and 14 days and muscles were characterized by functional, morphological, biochemical and molecular assays. The data obtained show that FoxOs are required for muscle loss and force drop during unloading. Moreover, we found that FoxO‐dependent atrogenes vary in different unloaded muscles and that they diverge from denervation. The findings of the present study clearly indicate that the signalling network that controls the atrophy programme is specific for each catabolic condition.

  December 12, 2016   doi: 10.1113/JP273097   open full text
• Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.
  Ahmed M. Hashad, Neil Mazumdar, Monica Romero, Anders Nygren, Kamran Bigdely‐Shamloo, Osama F. Harraz, Jose L. Puglisi, Edward J. Vigmond, Sean M. Wilson, Donald G. Welsh.
  The Journal of Physiology. December 12, 2016
  Key points Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+/Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. Abstract Ca2+ sparks are generated in a voltage‐dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (−40 mV) but not depolarized (−20 mV) voltages. In contrast, nifedipine, a CaV1.2 inhibitor, markedly suppressed STOC frequency at −20 mV but not −40 mV. These findings aligned with the voltage‐dependent profiles of L‐ and T‐type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV1.2 and CaV3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+/Ca2+ exchanger blockade. In summary, our work highlights that the voltage‐dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

  December 12, 2016   doi: 10.1113/JP273329   open full text
• Minimum number of myosin motors accounting for shortening velocity under zero load in skeletal muscle.
  Luca Fusi, Valentina Percario, Elisabetta Brunello, Marco Caremani, Pasquale Bianco, Joseph D. Powers, Massimo Reconditi, Vincenzo Lombardi, Gabriella Piazzesi.
  The Journal of Physiology. December 12, 2016
  Key points Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load. Accordingly, the unloaded shortening velocity (V0) is already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state. Here the number of actin‐attached motors per half‐myosin filament (n) during V0 shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half‐sarcomere compliance and force during the force redevelopment after shortening. The value of n decreases progressively with shortening and, during V0 shortening starting at the end of LR, is 1–4. Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening. Abstract The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V0) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca2+‐induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin‐attached motors per half‐thick filament during V0 shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half‐sarcomere (hs) compliance and force during the force development following V0 shortening. When V0 shortening is superimposed on the maximum isometric force T0, n decreases progressively with the increase of shortening (range 30–80 nm per hs) and, when V0 shortening is imposed at the end of LR, n can be as low as 1–4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V0 shortening.

  December 12, 2016   doi: 10.1113/JP273299   open full text
• Molecular recognition at cholinergic synapses: acetylcholine versus choline.
  Iva Bruhova, Anthony Auerbach.
  The Journal of Physiology. December 12, 2016
  Key points Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H‐bonds position ACh and choline differently in the aromatic cage to generate the different affinities. Abstract Acetylcholine (ACh) released at the vertebrate nerve‐muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single‐channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult‐type mouse AChRs having a mutation(s) at the transmitter‐binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist‐independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H‐bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H‐bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.

  December 12, 2016   doi: 10.1113/JP273291   open full text
• Oscillatory dynamics and functional connectivity during gating of primary somatosensory responses.
  Alex I. Wiesman, Elizabeth Heinrichs‐Graham, Nathan M. Coolidge, James E. Gehringer, Max J. Kurz, Tony W. Wilson.
  The Journal of Physiology. December 12, 2016
  Key Points Sensory gating is important for preventing excessive environmental stimulation from overloading neural resources. Gating in the human somatosensory cortices is a critically understudied topic, particularly in the lower extremities. We utilize the unique capabilities of magnetoencephalographic neuroimaging to quantify the normative neural population responses and dynamic functional connectivity of somatosensory gating in the lower extremities of healthy human participants. We show that somatosensory processing is subserved by a robust gating effect in the oscillatory domain, as well as a dynamic effect on interhemispheric functional connectivity between primary sensory cortices. These results provide novel insight into the dynamic neural mechanisms that underlie the processing of somatosensory information in the human brain, and will be vital in better understanding the neural responses that are aberrant in gait‐related neurological disorders (e.g. cerebral palsy). Abstract Sensory gating (SG) is a phenomenon in which neuronal responses to subsequent similar stimuli are weaker, and is considered to be an important mechanism for preventing excessive environmental stimulation from overloading shared neural resources. Although gating has been demonstrated in multiple sensory systems, the neural dynamics and developmental trajectory underlying SG remain poorly understood. In the present study, we adopt a data‐driven approach to map the spectrotemporal amplitude and functional connectivity (FC) dynamics that support gating in the somatosensory system (somato‐SG) in healthy children and adolescents using magnetoencephalography (MEG). These data underwent time‐frequency decomposition and the significant signal changes were imaged using a beamformer. Voxel time series were then extracted from the peak voxels and these signals were examined in the time and time‐frequency domains, and then subjected to dynamic FC analysis. The results obtained indicate a significant decrease in the amplitude of the neural response following the second stimulation relative to the first in the primary somatosensory cortex (SI). A significant decrease in response latency was also found between stimulations, and each stimulation induced a sharp decrease in FC between somatosensory cortical areas. Furthermore, there were no significant correlations between somato‐SG metrics and age. We conclude that somato‐SG can be observed in SI in both the time and oscillatory domains, with rich dynamics and alterations in inter‐hemispheric FC, and that this phenomenon has already matured by early childhood. A better understanding of these dynamics may provide insight to the numerous psychiatric and neurologic conditions that have been associated with aberrant SG across multiple modalities.

  December 12, 2016   doi: 10.1113/JP273192   open full text
• Cholinergic modulation of the parafacial respiratory group.
  Rozlyn C. T. Boutin, Zaki Alsahafi, Silvia Pagliardini.
  The Journal of Physiology. December 11, 2016
  Key points This study investigates the effects of cholinergic transmission on the expiratory oscillator, the parafacial respiratory group (pFRG) in urethane anaesthetized adult rats. Local inhibition of the acetyl cholinesterase enzyme induced activation of expiratory abdominal muscles and active expiration. Local application of the cholinomimetic carbachol elicited recruitment of late expiratory neurons, expiratory abdominal muscle activity and active expiration. This effect was antagonized by local application of the muscarinic antagonists scopolamine, J104129 and 4DAMP. We observed distinct physiological responses between the more medial chemosensitive region of the retrotrapezoid nucleus and the more lateral region of pFRG. These results support the hypothesis that pFRG is under cholinergic neuromodulation and the region surrounding the facial nucleus contains a group of neurons with distinct physiological roles. Abstract Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem: inspiration driven by a neuronal network located in the preBötzinger complex (preBötC) and expiration driven by a neuronal network located in the parafacial respiratory group (pFRG). While continuous activity of the preBötC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in the presence of increased respiratory drive (e.g. hypoxia, hypercapnia, exercise and through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state‐dependent fashion, frequently occurring during periods of REM sleep. We hypothesized that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre‐application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow.

  December 11, 2016   doi: 10.1113/JP273012   open full text
• Sildenafil therapy for fetal cardiovascular dysfunction during hypoxic development: studies in the chick embryo.
  Nozomi Itani, Katie L. Skeffington, Christian Beck, Dino A. Giussani.
  The Journal of Physiology. December 11, 2016
  Key points Common complications of pregnancy, such as chronic fetal hypoxia, trigger a fetal origin of cardiovascular dysfunction and programme cardiovascular disease in later life. Sildenafil treatment protects placental perfusion and fetal growth, but whether the effects of sildenafil transcend the placenta to affect the fetus is unknown. Using the chick embryo model, here we show that sildenafil treatment directly protects the fetal cardiovascular system in hypoxic development, and that the mechanisms of sildenafil protection include reduced oxidative stress and increased nitric oxide bioavailability; Sildenafil does not protect against fetal growth restriction in the chick embryo, supporting the idea that the protective effect of sildenafil on fetal growth reported in mammalian studies, including humans, is secondary to improved placental perfusion. Therefore, sildenafil may be a good candidate for human translational antioxidant therapy to protect the chronically hypoxic fetus in adverse pregnancy. Abstract There is a need for developing clinically translatable therapy for preventing fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia. Evidence shows that sildenafil protects placental perfusion and fetal growth. However, whether beneficial effects of sildenafil transcend onto the fetal heart and circulation in complicated development is unknown. We isolated the direct effects of sildenafil on the fetus using the chick embryo and hypothesised that sildenafil also protects fetal cardiovascular function in hypoxic development. Chick embryos (n = 11 per group) were incubated in normoxia or hypoxia (14% O2) from day 1 and treated with sildenafil (4 mg kg−1 day−1) from day 13 of the 21‐day incubation. Hypoxic incubation increased oxidative stress (4‐hydroxynonenal, 141.1 ± 17.6% of normoxic control), reduced superoxide dismutase (60.7 ± 6.3%), increased phosphodiesterase type 5 expression (167 ± 13.7%) and decreased nitric oxide bioavailability (54.7 ± 6.1%) in the fetal heart, and promoted peripheral endothelial dysfunction (70.9 ± 5.6% AUC of normoxic control; all P < 0.05). Sildenafil treatment after onset of chronic hypoxia prevented the increase in phosphodiesterase expression (72.5 ± 22.4%), protected against oxidative stress (94.7 ± 6.2%) and normalised nitric oxide bioavailability (115.6 ± 22.3%) in the fetal heart, and restored endothelial function in the peripheral circulation (89.8 ± 2.9%). Sildenafil protects the fetal heart and circulation directly in hypoxic development via mechanisms including decreased oxidative stress and enhanced nitric oxide bioavailability. Sildenafil may be a good translational candidate for human antioxidant therapy to prevent fetal origins of cardiovascular dysfunction in adverse pregnancy.

  December 11, 2016   doi: 10.1113/JP273393   open full text
• Cerebral haemodynamic response to somatosensory stimulation in near‐term fetal sheep.
  S. Nakamura, D. W. Walker, F. Y. Wong.
  The Journal of Physiology. December 11, 2016
  Key points Cerebral haemodynamic response to neural stimulation has been extensively investigated in animal and clinical studies, in both adult and paediatric populations, but little is known about cerebral haemodynamic functional response in the fetal brain. The present study describes the cerebral haemodynamic response measured by near‐infrared spectroscopy to somatosensory stimulation in fetal sheep. The cerebral haemodynamic response in the fetal sheep brain changes from a positive (increase in oxyhaemoglobin (oxyHb)) response pattern to a negative or biphasic response pattern when the duration of somatosensory stimulation is increased, probably due to cerebral vasoconstriction with prolonged stimulations. In contrast to adult studies, we have found that changes in fetal cerebral blood flow and oxyHb are positively increased in response to somatosensory stimulation during hypercapnia. We propose this is related to reduced vascular resistance and recruitment of cerebral vasculature in the fetal brain during hypercapnia. Abstract Functional hyperaemia induced by a localised increase in neuronal activity has been suggested to occur in the fetal brain owing to a positive blood oxygen level‐dependent (BOLD) signal recorded by functional magnetic resonance imaging following acoustic stimulation. To study the effect of somatosensory input on local cerebral perfusion we used near‐infrared spectroscopy (NIRS) in anaesthetised, partially exteriorised fetal sheep where the median nerve was stimulated with trains of pulses (2 ms, 3.3 Hz) for durations of 1.8, 4.8 and 7.8 s. Signal averaging of cerebral NIRS responses to 20 stimulus trains repeated every 60 s revealed that a short duration of stimulation (1.8 s) increased oxyhaemoglobin in the contralateral cortex consistent with a positive functional response, whereas longer durations of stimulation (4.8, 7.8 s) produced more variable oxyhaemoglobin responses including positive, negative and biphasic patterns of change. Mean arterial blood pressure and cerebral perfusion as monitored by laser Doppler flowmetry always showed small, but coincident increases following median nerve stimulation regardless of the type of response detected by the NIRS in the contralateral cortex. Hypercapnia significantly increased the baseline total haemoglobin and deoxyhaemoglobin, and in 7 of 8 fetal sheep positively increased the changes in contralateral total haemoglobin and oxyhaemoglobin in response to the 7.8 s stimulus train, compared to the response recorded during normocapnia. These results show that activity‐driven changes in cerebral perfusion and oxygen delivery are present in the fetal brain, and persist even during periods of hypercapnia‐induced cerebral vasodilatation.

  December 11, 2016   doi: 10.1113/JP273163   open full text
• Effect of maternal position on fetal behavioural state and heart rate variability in healthy late gestation pregnancy.
  Peter R. Stone, Wendy Burgess, Jordan P. R. McIntyre, Alistair J. Gunn, Christopher A. Lear, Laura Bennet, Edwin A. Mitchell, John M. D. Thompson,.
  The Journal of Physiology. December 11, 2016
  Key points Fetal behavioural state in healthy late gestation pregnancy is affected by maternal position. Fetal state 1F is more likely to occur in maternal supine or right lateral positions. Fetal state 4F is less likely to occur when the woman lies supine or semi‐recumbent. Fetal state change is more likely when the woman is supine or semi‐recumbent. Fetal heart rate variability is affected by maternal position with variability reduced in supine and semi‐recumbent positions. Abstract Fetal behavioural states (FBS) are measures of fetal wellbeing. In acute hypoxaemia, the human fetus adapts to a lower oxygen consuming state with changes in the cardiotocograph and reduced fetal activity. Recent studies of late gestation stillbirth described the importance of sleep position in the risk of intrauterine death. We designed this study to assess the effects of different maternal positions on FBS in healthy late gestation pregnancies under controlled conditions. Twenty‐nine healthy women had continuous fetal ECG recordings under standardized conditions in four randomly allocated positions, left lateral, right lateral, supine and semi‐recumbent. Two blinded observers, assigned fetal states in 5 min blocks. Measures of fetal heart rate variability were calculated from ECG beat to beat data. Compared to state 2F, state 4F was less likely to occur when women were semi‐recumbent [odds ratio (OR) = 0.11, 95% confidence interval (95% CI) 0.02, 0.55], and supine (OR = 0.27, 95% CI 0.07, 1.10). State 1F was more likely on the right (OR = 2.36, 95% CI 1.11, 5.04) or supine (OR = 4.99, 95% CI 2.41, 10.43) compared to the left. State change was more likely when the mother was semi‐recumbent (OR = 2.17, 95% CI 1.19, 3.95) or supine (OR = 2.67, 95% CI 1.46, 4.85). There was a significant association of maternal position to mean fetal heart rate. The measures of heart rate variability (SDNN and RMSSD) were reduced in both semi‐recumbent and supine positions. In healthy late gestation pregnancy, maternal position affects FBS and heart rate variability. These effects are likely fetal adaptations to positions which may produce a mild hypoxic stress.

  December 11, 2016   doi: 10.1113/JP273201   open full text
• Acute and chronic effects of noradrenergic enhancement on transcranial direct current stimulation‐induced neuroplasticity in humans.
  Hsiao‐I. Kuo, Walter Paulus, Giorgi Batsikadze, Asif Jamil, Min‐Fang Kuo, Michael A. Nitsche.
  The Journal of Physiology. December 07, 2016
  Key points Chronic administration of the selective noradrenaline reuptake inhibitor (NRI) reboxetine (RBX) increased and prolonged the long‐term potentiation‐like plasticity induced by anodal transcranial direct current stimulation (tDCS) for over 24 h. Chronic administration of RBX converted cathodal tDCS‐induced long‐term depression‐like plasticity into facilitation for 120 min. Chronic noradrenergic activity enhancement on plasticity of the human brain might partially explain the delayed therapeutic impact of selective NRIs in depression and other neuropsychiatric diseases. Abstract Noradrenaline affects cognition and motor learning processes via its impact on long‐term potentiation (LTP) and depression (LTD). We aimed to explore the impact of single dose and chronic administration of the selective noradrenaline reuptake inhibitor (NRI) reboxetine (RBX) on plasticity induced by transcranial direct current stimulation (tDCS) in healthy humans via a double‐blinded, placebo‐controlled, randomized crossover study. Sixteen healthy volunteers received placebo or single dose RBX (8 mg) before anodal or cathodal tDCS of the primary motor cortex. Afterwards, the same subjects took RBX (8 mg day−1) consecutively for 21 days. During this period, two additional interventions were performed (RBX with anodal or cathodal tDCS), to explore the impact of chronic RBX treatment on plasticity. Plasticity was monitored by motor‐evoked potential amplitudes elicited by transcranial magnetic stimulation. Chronic administration of RBX increased and prolonged the LTP‐like plasticity induced by anodal tDCS for over 24 h. Chronic RBX significantly converted cathodal tDCS‐induced LTD‐like plasticity into facilitation, as compared to the single dose condition, for 120 min after stimulation. The results show a prominent impact of chronic noradrenergic enhancement on plasticity of the human brain that might partially explain the delayed therapeutic impact of selective NRIs in depression and other neuropsychiatric diseases.

  December 07, 2016   doi: 10.1113/JP273137   open full text
• Distinct subcellular mechanisms for the enhancement of the surface membrane expression of SK2 channel by its interacting proteins, α‐actinin2 and filamin A.
  Zheng Zhang, Hannah A. Ledford, Seojin Park, Wenying Wang, Sassan Rafizadeh, Hyo Jeong Kim, Wilson Xu, Ling Lu, Victor C. Lau, Anne A. Knowlton, Xiao‐Dong Zhang, Ebenezer N. Yamoah, Nipavan Chiamvimonvat.
  The Journal of Physiology. December 07, 2016
  Key points Ion channels are transmembrane proteins that are synthesized within the cells but need to be trafficked to the cell membrane for the channels to function. Small‐conductance, Ca2+‐activated K+ channels (SK, KCa2) are unique subclasses of K+ channels that are regulated by Ca2+ inside the cells; they are expressed in human atrial myocytes and responsible for shaping atrial action potentials. We have previously shown that interacting proteins of SK2 channels are important for channel trafficking to the membrane. Using total internal reflection fluorescence (TIRF) and confocal microscopy, we studied the mechanisms by which the surface membrane localization of SK2 (KCa2.2) channels is regulated by their interacting proteins. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. Abstract The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small‐conductance, Ca2+‐activated K+ channels (SK, KCa2) are expressed in human atrial myocytes and are responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C‐ and N‐termini of SK2 channels interact with the actin‐binding proteins α‐actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (KCa2.2) channels. When SK2 channels were co‐expressed with filamin A or α‐actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively active or dominant‐negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α‐Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes.

  December 07, 2016   doi: 10.1113/JP272942   open full text
• Effects of postural change from supine to head‐up tilt on the skin sympathetic nerve activity component synchronised with the cardiac cycle in warmed men.
  Yu Ogawa, Yoshi‐ichiro Kamijo, Shigeki Ikegawa, Shizue Masuki, Hiroshi Nose.
  The Journal of Physiology. December 07, 2016
  Key points Humans are unique in controlling body temperature in a hot environment by a large amount of skin blood flow; however, the decrease in total peripheral resistance due to systemic cutaneous vasodilatation and the reduction of venous return to the heart due to blood pooling in the cutaneous vein threatens blood pressure maintenance in the upright position, and occasionally causes heat syncope. Against this condition, cutaneous vasodilatation is reportedly suppressed to maintain arterial pressure; however, the nerve activity responsible for this phenomenon has not been identified. In the present study, we found that the skin sympathetic nerve activity component that was synchronised with the cardiac cycle increased in hyperthermia, but the increase was suppressed when the posture was changed from supine to head‐up tilt. The profile of the component agreed with that of cutaneous vasodilatation. Thus, the component might contribute to the prevention of heat syncope in humans. Abstract In humans, the cutaneous vasodilatation response to hyperthermia has been suggested to be suppressed by baroreflexes to maintain arterial pressure when the posture is changed from supine to upright, and if the reflexes do not function sufficiently, it can cause heat syncope. However, the efferent signals of the reflexes have not been identified. To identify the signals, we continuously measured skin sympathetic nerve activity (SSNA; microneurography), right atrial volume (RAV; echocardiography, the baroreceptors for the reflexes are reportedly located in the right atrium), cutaneous vascular conductance on the chest (CVCchest; laser Doppler flowmetry), and oesophageal temperature (Toes; thermocouple) in young men before and after passive warming with a perfusion suit, during which periods the posture was changed from supine to 30 deg head‐up tilt positions. During these periods, we also simultaneously measured muscle sympathetic nerve activity (MSNA) to distinguish the SSNA from MSNA. We found that an increase in Toes by ∼0.7°C (P < 0.0001) increased the total SSNA (P < 0.005); however, the head‐up tilt in hyperthermia did not change the total SSNA (P > 0.26) although an increase in CVCchest (P < 0.019) was suppressed and RAV was reduced (P < 0.008). In contrast, the SSNA component synchronised with the cardiac cycle increased in hyperthermia (P < 0.015), but decreased with the postural change (P < 0.017). The SSNA component during the postural change before and after warming was highly correlated with the CVCchest (r = 0.817, P < 0.0001), but the MSNA component was not (r = 0.359, P = 0.085). Thus, the SSNA component synchronised with the cardiac cycle appeared to be involved in suppressing cutaneous vasodilatation during postural changes.

  December 07, 2016   doi: 10.1113/JP273281   open full text
• Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.
  Jian Shi, Francesc Miralles, Lutz Birnbaumer, William A. Large, Anthony P. Albert.
  The Journal of Physiology. December 07, 2016
  Key points Depletion of Ca2+ stores activates store‐operated channels (SOCs), which mediate Ca2+ entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP‐PLCδ1‐PH imaging and co‐localization techniques. Store‐operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1−/− cells, and store‐operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1‐based SOCs in VSMCs, and a novel role for STIM1, where store‐operated STIM1‐TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating. Abstract In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein‐based store‐operated channels (SOCs) mediates Ca2+ entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1‐based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca2+ sensor. Store‐operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild‐type VSMCs, and was absent in TRPC1−/− VSMCs. Store‐operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store‐operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5‐bisphosphate/inositol 1,4,5‐trisphosphate biosensor GFP‐PLCδ1‐PH was reduced by STIM1 shRNA and absent in TRPC1−/− cells. Immunocytochemistry, co‐immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1‐TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N‐terminal and C‐terminal STIM1 antibodies on TRPC1‐based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1‐based SOCs in VSMCs, and a novel role for STIM1, in which store‐operated STIM1‐TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating.

  December 07, 2016   doi: 10.1113/JP273302   open full text
• N‐Acetylcysteine, a glutathione precursor, reverts vascular dysfunction and endothelial epigenetic programming in intrauterine growth restricted guinea pigs.
  Emilio A. Herrera, Francisca Cifuentes‐Zúñiga, Esteban Figueroa, Cristian Villanueva, Cherie Hernández, René Alegría, Viviana Arroyo‐Jousse, Estefania Peñaloza, Marcelo Farías, Ricardo Uauy, Paola Casanello, Bernardo J. Krause.
  The Journal of Physiology. December 04, 2016
  Key points Intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial epigenetic programming of the umbilical vessels. There is no evidence that this epigenetic programming is occurring on systemic fetal arteries. In IUGR guinea pigs we studied the functional and epigenetic programming of endothelial nitric oxide synthase (eNOS) (Nos3 gene) in umbilical and systemic fetal arteries, addressing the role of oxidative stress in this process by maternal treatment with N‐acetylcysteine (NAC) during the second half of gestation. The present study suggests that IUGR endothelial cells have common molecular markers of programming in umbilical and systemic arteries. Notably, maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium in IUGR guinea pigs. Abstract In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N‐acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire‐myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P < 0.05) and recovered fetal weight (P < 0.05), increasing fetal‐to‐placental ratio at term (∼40%) (P < 0.001). In IUGR, NAC treatment restored eNOS‐dependent relaxation in aorta and umbilical arteries (P < 0.05), normalizing eNOS mRNA levels in EC fetal and umbilical arteries (P < 0.05). IUGR‐derived ECs had a decreased DNA methylation (∼30%) at CpG −170 (from the transcription start site) and this epigenetic signature was absent in NAC‐treated fetuses (P < 0.001). These data show that IUGR‐ECs have common molecular markers of eNOS programming in umbilical and systemic arteries and this effect is prevented by maternal treatment with antioxidants.

  December 04, 2016   doi: 10.1113/JP273396   open full text
• Molecular mechanisms of Slo2 K+ channel closure.
  M. Hunter Giese, Alison Gardner, Angela Hansen, Michael C. Sanguinetti.
  The Journal of Physiology. December 02, 2016
  Key points Intracellular Na+‐activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood. A cryo‐electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss‐crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue. Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non‐conducting state. Mutation of the conserved Met residues in the S6 segments to the negatively‐charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing. Abstract Large conductance K+‐selective Slo2 channels are in a closed state unless activated by elevated [Na+]i. Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss‐crossing of the cytoplasmic ends of the S6 α‐helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo‐electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss‐crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively‐charged Glu did not induce constitutive channel opening.

  December 02, 2016   doi: 10.1113/JP273225   open full text
• Synaptic vesicle pool‐specific modification of neurotransmitter release by intravesicular free radical generation.
  Olusoji A. T. Afuwape, Catherine R. Wasser, Thomas Schikorski, Ege T. Kavalali.
  The Journal of Physiology. December 02, 2016
  Key points Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Abstract Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) – a haem‐containing plant enzyme – or antibodies against synaptotagmin‐1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2O2) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization‐induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP‐tagged syt1 construct and subsequent H2O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss‐of‐function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

  December 02, 2016   doi: 10.1113/JP273115   open full text
• Synaptic reliability and temporal precision are achieved via high quantal content and effective replenishment: auditory brainstem versus hippocampus.
  Elisa G Krächan, Alexander U Fischer, Jürgen Franke, Eckhard Friauf.
  The Journal of Physiology. December 02, 2016
  Key points Auditory brainstem neurons involved in sound source localization are equipped with several morphological and molecular features that enable them to compute interaural level and time differences. As sound source localization works continually, synaptic transmission between these neurons should be reliable and temporally precise, even during sustained periods of high‐frequency activity. Using patch‐clamp recordings in acute brain slices, we compared synaptic reliability and temporal precision in the seconds–minute range between auditory and two types of hippocampal synapses; the latter are less confronted with temporally precise high‐frequency transmission than the auditory ones. We found striking differences in synaptic properties (e.g. continually high quantal content) that allow auditory synapses to reliably release vesicles at much higher rate than their hippocampal counterparts. Thus, they are indefatigable and also in a position to transfer information with exquisite temporal precision and their performance appears to be supported by very efficient replenishment mechanisms. Abstract At early stations of the auditory pathway, information is encoded by precise signal timing and rate. Auditory synapses must maintain the relative timing of events with submillisecond precision even during sustained and high‐frequency stimulation. In non‐auditory brain regions, e.g. telencephalic ones, synapses are activated at considerably lower frequencies. Central to understanding the heterogeneity of synaptic systems is the elucidation of the physical, chemical and biological factors that determine synapse performance. In this study, we used slice recordings from three synapse types in the mouse auditory brainstem and hippocampus. Whereas the auditory brainstem nuclei experience high‐frequency activity in vivo, the hippocampal circuits are activated at much lower frequencies. We challenged the synapses with sustained high‐frequency stimulation (up to 200 Hz for 60 s) and found significant performance differences. Our results show that auditory brainstem synapses differ considerably from their hippocampal counterparts in several aspects, namely resistance to synaptic fatigue, low failure rate and exquisite temporal precision. Their high‐fidelity performance supports the functional demands and appears to be due to the large size of the readily releasable pool and a high release probability, which together result in a high quantal content. In conjunction with very efficient vesicle replenishment mechanisms, these properties provide extremely rapid and temporally precise signalling required for neuronal communication at early stations of the auditory system, even during sustained activation in the minute range.

  December 02, 2016   doi: 10.1113/JP272799   open full text
• TRPV4 participates in pressure‐induced inhibition of renin secretion by juxtaglomerular cells.
  François Seghers, Xavier Yerna, Nadège Zanou, Olivier Devuyst, Rudi Vennekens, Bernd Nilius, Philippe Gailly.
  The Journal of Physiology. December 02, 2016
  Key points Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+]i) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin. Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA‐mediated repression of TRPV4. Inhibition of TRPV4 impairs pressure‐induced decrease in renin secretion. Compared to wild‐type mice, Trpv4−/− mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure–renin relationship. We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus. Abstract The renin–angiotensin system is a crucial blood pressure regulation system. It consists of a hormonal cascade where the rate‐limiting enzyme is renin, which is secreted into the blood flow by renal juxtaglomerular (JG) cells in response to low pressure in the renal afferent arteriole. In contrast, an increase in blood pressure results in a decreased renin secretion. This is accompanied by a transitory increase in [Ca2+]i of JG cells. The inverse relationship between [Ca2+]i and renin secretion has been called the ‘calcium paradox’ of renin release. How increased pressure induces a [Ca2+]i transient in JG cells, is however, unknown. We observed that [Ca2+]i transients induced by mechanical stimuli in JG As4.1 cells were completely abolished by HC067047 and RN1734, two inhibitors of TRPV4. They were also reduced by half by siRNA‐mediated repression of TRPV4 but not after repression or inhibition of TRPV2 or Piezo1 ion channels. Interestingly, the stimulation of renin secretion by the adenylate cyclase activator forskolin was totally inhibited by cyclic stretching of the cells. This effect was mimicked by stimulation with GSK1016790A and 4αPDD, two activators of TRPV4 and inhibited in the presence of HC067047. Moreover, in isolated perfused kidneys from Trpv4−/− mice, the pressure–renin relationship was significantly altered. In vivo, Trpv4−/− mice presented increased plasma levels of renin and aldosterone compared to wild‐type mice. Altogether, our results suggest that TRPV4 is involved in the pressure‐induced entry of Ca2+ in JG cells, which inhibits renin release and allows the negative feedback regulation on blood pressure.

  December 02, 2016   doi: 10.1113/JP273595   open full text
• Effects of continuous positive airway pressure and isocapnic‐hypoxia on cerebral autoregulation in patients with obstructive sleep apnoea.
  Xavier Waltz, Andrew E. Beaudin, Patrick J. Hanly, Georgios D. Mitsis, Marc J. Poulin.
  The Journal of Physiology. December 01, 2016
  Key points Altered cerebral autoregulation (CA) in obstructive sleep apnoea (OSA) patients may contribute to increased stroke risk in this population; the gold standard treatment for OSA is continuous positive airway pressure, which improves cerebrovascular regulation and may decrease the risk of stroke. Isocapnic‐hypoxia impairs CA in healthy subjects, but it remains unknown in OSA whether impaired CA is further exacerbated by isocapnic‐hypoxia and whether it is improved by treatment with continuous positive airway pressure. During normoxia, CA was altered in the more severe but not in the less severe OSA patients, while, in contrast, during isocapnic‐hypoxia, CA was similar between groups and tended to improve in patients with more severe OSA compared to normoxia. From a clinical perspective, one month of continuous positive airway pressure treatment does not improve CA. From a physiological perspective, this study suggests that sympathetic overactivity may be responsible for altered CA in the more severe OSA patients. Abstract Cerebral autoregulation (CA) impairment may contribute to the increased risk of stroke associated with obstructive sleep apnoea (OSA). It is unknown if impaired CA is further exacerbated by isocapnic‐hypoxia and whether it is improved by treatment of OSA with continuous positive airway pressure (CPAP). CA was assessed during wakefulness in 53 OSA patients (50.3 ± 9.3 years) and 21 controls (49.8 ± 8.6 years) at baseline and following a minimum of 1 month of effective CPAP therapy (OSA patients, n = 40). Control participants (n = 21) performed a follow‐up visit to control for time effects within OSA patients between baseline and the post‐CPAP visit. Beat‐by‐beat middle cerebral artery blood flow velocity and mean arterial blood pressure (MBP), and breath‐by‐breath end‐tidal partial pressure of CO2 (P ET ,CO2) were monitored. CA was determined during normoxia and isocapnic‐hypoxia using transfer function (phase and gain) and coherence analysis (including multiple and partial coherence (using MBP and P ET ,CO2 as inputs)) in the very low frequency range (0.03–0.07 Hz). OSA patients were divided into two subgroups (less severe and more severe) based upon the median respiratory disturbance index (RDI). During normoxia, the more severe OSA patients (RDI 45.9 ± 10.3) exhibited altered CA compared to controls and the less severe OSA patients (RDI 24.5 ± 5.9). In contrast, during isocapnic‐hypoxia, CA was similar between groups. CPAP had no effect on CA. In conclusion, CA is altered in the more severe OSA patients during normoxia but not during isocapnic‐hypoxia and CPAP treatment does not impact CA.

  December 01, 2016   doi: 10.1113/JP272967   open full text
• Magnetic resonance imaging biomarkers of exercise‐induced improvement of oxidative stress and inflammation in the brain of old high‐fat‐fed ApoE−/− mice.
  Erica N. Chirico, Vanessa Di Cataldo, Fabien Chauveau, Alain Geloën, David Patsouris, Benoît Thézé, Cyril Martin, Hubert Vidal, Jennifer Rieusset, Vincent Pialoux, Emmanuelle Canet‐Soulas.
  The Journal of Physiology. December 01, 2016
  Key points Vascular brain lesions and atherosclerosis are two similar conditions that are characterized by increased inflammation and oxidative stress. Non‐invasive imaging in a murine model of atherosclerosis showed vascular brain damage and peripheral inflammation. In this study, exercise training reduced magnetic resonance imaging‐detected abnormalities, insulin resistance and markers of oxidative stress and inflammation in old ApoE−/− mice. Our results demonstrate the protective effect of exercise on neurovascular damage in the ageing brain of ApoE−/− mice. Abstract Vascular brain lesions, present in advanced atherosclerosis, share pathological hallmarks with peripheral vascular lesions, such as increased inflammation and oxidative stress. Physical activity reduces these peripheral risk factors, but its cerebrovascular effect is less documented, especially by non‐invasive imaging. Through a combination of in vivo and post‐mortem techniques, we aimed to characterize vascular brain damage in old ApoE−/− mice fed a high‐cholesterol (HC) diet with dietary controlled intake. We then sought to determine the beneficial effects of exercise training on oxidative stress and inflammation in the brain as a treatment option in an ageing atherosclerosis mouse model. Using in vivo magnetic resonance imaging (MRI) and biological markers of oxidative stress and inflammation, we evaluated the occurrence of vascular abnormalities in the brain of HC‐diet fed ApoE−/− mice >70 weeks old, its association with local and systemic oxidative stress and inflammation, and whether both can be modulated by exercise. Exercise training significantly reduced both MRI‐detected abnormalities (present in 71% of untrained vs. 14% of trained mice) and oxidative stress (lipid peroxidation, 9.1 ± 1.4 vs. 5.2 ± 0.9 μmol mg−1; P < 0.01) and inflammation (interleukin‐1β, 226.8 ± 27.1 vs. 182.5 ± 21.5 pg mg−1; P < 0.05) in the brain, and the mortality rate. Exercise also decreased peripheral insulin resistance, oxidative stress and inflammation, but significant associations were seen only within brain markers. Highly localized vascular brain damage is a frequent finding in this ageing atherosclerosis model, and exercise is able to reduce this outcome and improve lifespan. In vivo MRI evaluated both the neurovascular damage and the protective effect of exercise.

  December 01, 2016   doi: 10.1113/JP271903   open full text
• Peroxisome proliferator‐activated receptor‐γ coactivator 1 α1 induces a cardiac excitation–contraction coupling phenotype without metabolic remodelling.
  Maija Mutikainen, Tomi Tuomainen, Nikolay Naumenko, Jenni Huusko, Boris Smirin, Svetlana Laidinen, Krista Kokki, Heidi Hynynen, Seppo Ylä‐Herttuala, Merja Heinäniemi, Jorge L. Ruas, Pasi Tavi.
  The Journal of Physiology. December 01, 2016
  Key points Transcriptional co‐activator PGC‐1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC‐1α1 expression are not known. Transgenic PGC‐1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC‐1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise‐induced heart phenotype, but does not protect the heart against load‐induced pathological hypertrophy. Transcriptional effects of PGC‐1α1 show clear dose‐dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC‐1α1 expression levels. These results imply that the physiological role of PGC‐1α1 is to promote a beneficial excitation–contraction coupling phenotype in the heart. Abstract The transcriptional coactivator PGC‐1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC‐1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC‐1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC‐1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation–contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC‐1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus‐induced PGC‐1α1 expression, highlights PGC‐1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose‐dependent fashion. At low levels of overexpression PGC‐1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC‐1α1 are recruited at higher PGC‐1α1 expression levels. Together these findings demonstrate that PGC‐1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC‐1α1 is to promote a beneficial EC coupling phenotype in the heart.

  December 01, 2016   doi: 10.1113/JP272847   open full text
• Active integration of glutamatergic input to the inferior olive generates bidirectional postsynaptic potentials.
  Derek L. F. Garden, Arianna Rinaldi, Matthew F. Nolan.
  The Journal of Physiology. November 29, 2016
  Key points We establish experimental preparations for optogenetic investigation of glutamatergic input to the inferior olive. Neurones in the principal olivary nucleus receive monosynaptic extra‐somatic glutamatergic input from the neocortex. Glutamatergic inputs to neurones in the inferior olive generate bidirectional postsynaptic potentials (PSPs), with a fast excitatory component followed by a slower inhibitory component. Small conductance calcium‐activated potassium (SK) channels are required for the slow inhibitory component of glutamatergic PSPs and oppose temporal summation of inputs at intervals ≤ 20 ms. Active integration of synaptic input within the inferior olive may play a central role in control of olivo‐cerebellar climbing fibre signals. Abstract The inferior olive plays a critical role in motor coordination and learning by integrating diverse afferent signals to generate climbing fibre inputs to the cerebellar cortex. While it is well established that climbing fibre signals are important for motor coordination, the mechanisms by which neurones in the inferior olive integrate synaptic inputs and the roles of particular ion channels are unclear. Here, we test the hypothesis that neurones in the inferior olive actively integrate glutamatergic synaptic inputs. We demonstrate that optogenetically activated long‐range synaptic inputs to the inferior olive, including projections from the motor cortex, generate rapid excitatory potentials followed by slower inhibitory potentials. Synaptic projections from the motor cortex preferentially target the principal olivary nucleus. We show that inhibitory and excitatory components of the bidirectional synaptic potentials are dependent upon AMPA (GluA) receptors, are GABAA independent, and originate from the same presynaptic axons. Consistent with models that predict active integration of synaptic inputs by inferior olive neurones, we find that the inhibitory component is reduced by blocking large conductance calcium‐activated potassium channels with iberiotoxin, and is abolished by blocking small conductance calcium‐activated potassium channels with apamin. Summation of excitatory components of synaptic responses to inputs at intervals ≤ 20 ms is increased by apamin, suggesting a role for the inhibitory component of glutamatergic responses in temporal integration. Our results indicate that neurones in the inferior olive implement novel rules for synaptic integration and suggest new principles for the contribution of inferior olive neurones to coordinated motor behaviours.

  November 29, 2016   doi: 10.1113/JP273424   open full text
• Rate of rise in diastolic blood pressure influences vascular sympathetic response to mental stress.
  Khadigeh El Sayed, Vaughan G. Macefield, Sarah L. Hissen, Michael J. Joyner, Chloe E. Taylor.
  The Journal of Physiology. November 29, 2016
  Key points Research indicates that individuals may experience a rise (positive responders) or fall (negative responders) in muscle sympathetic nerve activity (MSNA) during mental stress. In this study, we examined the early blood pressure responses (including the peak, time of peak and rate of rise in blood pressure) to mental stress in positive and negative responders. Negative MSNA responders to mental stress exhibit a more rapid rise in diastolic pressure at the onset of the stressor, suggesting a baroreflex‐mediated suppression of MSNA. In positive responders there is a more sluggish rise in blood pressure during mental stress, which appears to be MSNA‐driven. This study suggests that whether MSNA has a role in the pressor response is dependent upon the reactivity of blood pressure early in the task. Abstract Research indicates that individuals may experience a rise (positive responders) or fall (negative responders) in muscle sympathetic nerve activity (MSNA) during mental stress. The aim was to examine the early blood pressure response to stress in positive and negative responders and thus its influence on the direction of change in MSNA. Blood pressure and MSNA were recorded continuously in 21 healthy young males during 2 min mental stressors (mental arithmetic, Stroop test) and physical stressors (cold pressor, handgrip exercise, post‐exercise ischaemia). Participants were classified as negative or positive responders according to the direction of the mean change in MSNA during the stressor tasks. The peak changes, time of peak and rate of changes in blood pressure were compared between groups. During mental arithmetic negative responders experienced a significantly greater rate of rise in diastolic blood pressure in the first minute of the task (1.3 ± 0.5 mmHg s−1) compared with positive responders (0.4 ± 0.1 mmHg s−1; P = 0.03). Similar results were found for the Stroop test. Physical tasks elicited robust parallel increases in blood pressure and MSNA across participants. It is concluded that negative MSNA responders to mental stress exhibit a more rapid rise in diastolic pressure at the onset of the stressor, suggesting a baroreflex‐mediated suppression of MSNA. In positive responders there is a more sluggish rise in blood pressure during mental stress, which appears to be MSNA‐driven. This study suggests that whether MSNA has a role in the pressor response is dependent upon the reactivity of blood pressure early in the task.

  November 29, 2016   doi: 10.1113/JP272963   open full text
• Role of dynamin‐related protein 1‐mediated mitochondrial fission in resistance of mouse C2C12 myoblasts to heat injury.
  Tianzheng Yu, Patricia Deuster, Yifan Chen.
  The Journal of Physiology. November 29, 2016
  Key points Understanding how skeletal muscles respond to high temperatures may help develop strategies for improving exercise tolerance and preventing heat injury. Mitochondria regulate cell survival by constantly changing their morphology through fusion and fission in response to environmental stimuli. Little is known about the involvement of mitochondrial dynamics in tolerance of skeletal muscle against heat stress. Mild heat acclimation and moderate heat shock appear to have different effects on the mitochondrial morphology and fission protein Drp1 in skeletal muscle cells. Mitochondrial integrity plays a key role in cell survival under heat stress. Abstract The regulation of mitochondrial morphology is closely coupled to cell survival during stress. We examined changes in the mitochondrial morphology of mouse C2C12 skeletal muscle cells in response to heat acclimation and heat shock exposure. Acclimated cells showed a greater survival rate during heat shock exposure than non‐acclimated cells, and were characterized by long interconnected mitochondria and reduced expression of dynamin‐related protein 1 (Drp1) for their mitochondrial fractions. Exposure of C2C12 muscle cells to heat shock led to apoptotic death featuring activation of caspase 3/7, release of cytochrome c and loss of cell membrane integrity. Heat shock also caused excessive mitochondrial fragmentation, loss of mitochondrial membrane potential and production of reactive oxygen species in C2C12 cells. Western blot and immunofluorescence image analysis revealed translocation of Drp1 to mitochondria from the cytosol in C2C12 cells exposed to heat shock. Mitochondrial division inhibitor 1 or Drp1 gene silencer reduced mitochondrial fragmentation and increased cell viability during exposure to heat shock. These results suggest that Drp1‐dependent mitochondrial fission may regulate susceptibility to heat‐induced apoptosis in muscle cells and that Drp1 may serve as a target for the prevention of heat‐related injury.

  November 29, 2016   doi: 10.1113/JP272885   open full text
• Impact of vesicular glutamate leakage on synaptic transmission at the calyx of Held.
  Chihiro Takami, Kohgaku Eguchi, Tetsuya Hori, Tomoyuki Takahashi.
  The Journal of Physiology. November 29, 2016
  Key points It is controversial whether glutamate can leak out of vesicles in the nerve terminal. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate or by blocking vacuolar ATPase activity using bafilomycin A1. In the absence of vesicular glutamate uptake, both spontaneous and nerve‐evoked EPSCs underwent a rundown, suggesting that vesicular glutamate can leak out of vesicles. However, the rundown of evoked EPSCs was caused mainly by accumulation of unfilled vesicles after exocytic release of glutamate, suggesting a minor influence of glutamate leakage on synaptic transmission. Abstract Glutamate leaks out of synaptic vesicles when the transvesicular proton gradient is dissipated in isolated vesicle preparations. In the nerve terminal, however, it is controversial whether glutamate can leak out of vesicles. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate in whole‐cell dialysis or by blocking vacuolar ATPase using bafilomycin A1 (Baf) at the calyx of Held in mouse brainstem slices. Presynaptic glutamate washout or Baf application reduced the mean amplitude and frequency of spontaneous miniature (m)EPSCs and the mean amplitude of EPSCs evoked every 10 min. The percentage reduction of mEPSC amplitude was much less than that of EPSC amplitude or mEPSC frequency, and tended to reach a plateau. The mean amplitude of mEPSCs after glutamate washout or Baf application remained high above the detection limit, deduced from the reduction of mEPSC amplitude by the AMPA receptor blocker 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione. Membrane capacitance measurements from presynaptic terminals indicated no effect of glutamate washout on exocytosis or endocytosis of synaptic vesicles. We conclude that glutamate can leak out of vesicles unless it is continuously taken up from presynaptic cytosol. However, the magnitude of glutamate leakage was small and had only a minor effect on synaptic responses. In contrast, prominent rundowns of EPSC amplitude and mEPSC frequency observed after glutamate washout or Baf application are likely to be caused by accumulation of unfilled vesicles in presynaptic terminals retrieved after spontaneous and evoked glutamate release.

  November 29, 2016   doi: 10.1113/JP273467   open full text
• Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin‐1E.
  Christoph Hönigsperger, Maximiliano J. Nigro, Johan F. Storm.
  The Journal of Physiology. November 13, 2016
  Key points Kv2 channels underlie delayed‐rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia. Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space‐representing grid cells. We used the new Kv2 blocker Guangxitoxin‐1E (GTx) to study Kv2 functions in these neurons. Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed‐rectifier K+ current but not transient A‐type current. In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after‐depolarizations; (iii) reducing the fast and medium after‐hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering. GTx is a useful tool for studying Kv2 channels and their functions in neurons. Abstract The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin‐1E (GTx; 10–100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond –30 mV but not transient A‐type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after‐depolarization (ADP); (iii) reduced fast and medium after‐hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after‐potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells.

  November 13, 2016   doi: 10.1113/JP273024   open full text
• The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.
  Christopher L. Riley, Christine Dao, M. Alexander Kenaston, Luigina Muto, Shohei Kohno, Sara M. Nowinski, Ashley D. Solmonson, Matthew Pfeiffer, Michael N. Sack, Zhongping Lu, Giuseppe Fiermonte, Jon E. Sprague, Edward M. Mills.
  The Journal of Physiology. November 13, 2016
  Key points Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline‐induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. Abstract Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue‐dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine‐induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle‐specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

  November 13, 2016   doi: 10.1113/JP272971   open full text
• Potassium channels in the heart: structure, function and regulation.
  Eleonora Grandi, Michael C. Sanguinetti, Daniel C. Bartos, Donald M. Bers, Ye Chen‐Izu, Nipavan Chiamvimonvat, Henry M. Colecraft, Brian P. Delisle, Jordi Heijman, Manuel F. Navedo, Sergei Noskov, Catherine Proenza, Jamie I. Vandenberg, Vladimir Yarov‐Yarovoy.
  The Journal of Physiology. November 13, 2016
  Abstract This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation–Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was ‘K+ Channels and Regulation’. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K+ channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage‐gated K+ channel function, as well as the mechanisms involved in regulation of K+ channel gating, expression and membrane localization. Given the critical role for K+ channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K+ channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K+ channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K+ channels in cardiac electrophysiology, i.e. how K+ currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K+ channel‐based therapeutics. A fundamental understanding of K+ channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

  November 13, 2016   doi: 10.1113/JP272864   open full text
• Na+/H+ exchange via the Drosophila vesicular glutamate transporter mediates activity‐induced acid efflux from presynaptic terminals.
  Adam J. Rossano, Akira Kato, Karyl I. Minard, Michael F. Romero, Gregory T. Macleod.
  The Journal of Physiology. November 13, 2016
  Key points Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid‐extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals. Abstract Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named ‘pHerry’, in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity‐induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+/Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid‐extruding protein when deposited on the plasmamembrane.

  November 13, 2016   doi: 10.1113/JP273105   open full text
• Vestibular feedback maintains reaching accuracy during body movement.
  Craig P. Smith, Raymond F. Reynolds.
  The Journal of Physiology. November 13, 2016
  Key points Reaching movements can be perturbed by vestibular input, but the function of this response is unclear. Here, we applied galvanic vestibular stimulation concurrently with real body movement while subjects maintained arm position either fixed in space or fixed with respect to their body. During the fixed‐in‐space conditions, galvanic vestibular stimulation caused large changes in arm trajectory consistent with a compensatory response to maintain upper‐limb accuracy in the face of body movement. Galvanic vestibular stimulation responses were absent during the body‐fixed task, demonstrating task dependency in vestibular control of the upper limb. The results suggest that the function of vestibular‐evoked arm movements is to maintain the accuracy of the upper limb during unpredictable body movement, but only when reaching in an earth‐fixed reference frame. Abstract When using our arms to interact with the world, unintended body motion can introduce movement error. A mechanism that could detect and compensate for such motion would be beneficial. Observations of arm movements evoked by vestibular stimulation provide some support for this mechanism. However, the physiological function underlying these artificially evoked movements is unclear from previous research. For such a mechanism to be functional, it should operate only when the arm is being controlled in an earth‐fixed rather than a body‐fixed reference frame. In the latter case, compensation would be unnecessary and even deleterious. To test this hypothesis, subjects were gently rotated in a chair while being asked to maintain their outstretched arm pointing towards either earth‐fixed or body‐fixed memorized targets. Galvanic vestibular stimulation was applied concurrently during rotation to isolate the influence of vestibular input, uncontaminated by inertial factors. During the earth‐fixed task, galvanic vestibular stimulation produced large polarity‐dependent corrections in arm position. These corrections mimicked those evoked when chair velocity was altered without any galvanic vestibular stimulation, indicating a compensatory arm response to a sensation of altered body motion. In stark contrast, corrections were completely absent during the body‐fixed task, despite the same chair movement profile and arm posture. These effects persisted when we controlled for differences in limb kinematics between the two tasks. Our results demonstrate that vestibular control of the upper limb maintains reaching accuracy during unpredictable body motion. The observation that such responses occurred only when reaching within an earth‐fixed reference frame confirms the functional nature of vestibular‐evoked arm movement.

  November 13, 2016   doi: 10.1113/JP273125   open full text
• Plasticity in mitochondrial cristae density allows metabolic capacity modulation in human skeletal muscle.
  Joachim Nielsen, Kasper D. Gejl, Martin Hey‐Mogensen, Hans‐Christer Holmberg, Charlotte Suetta, Peter Krustrup, Coen P. H. Elemans, Niels Ørtenblad.
  The Journal of Physiology. November 13, 2016
  Key points In human skeletal muscles, the current view is that the capacity for mitochondrial energy production, and thus endurance capacity, is set by the mitochondria volume. However, increasing the mitochondrial inner membrane surface comprises an alternative mechanism for increasing the energy production capacity. In the present study, we show that mitochondrial inner membranes in leg muscles of endurance‐trained athletes have an increased ratio of surface per mitochondrial volume. We show a positive correlation between this ratio and whole body oxygen uptake and muscle fibre mitochondrial content. The results obtained in the present study help us to understand modulation of mitochondrial function, as well as how mitochondria can increase their oxidative capacity with increased demand. Abstract Mitochondrial energy production involves the movement of protons down a large electrochemical gradient via ATP synthase located on the folded inner membrane, known as cristae. In mammalian skeletal muscle, the density of cristae in mitochondria is assumed to be constant. However, recent experimental studies have shown that respiration per mitochondria varies. Modelling studies have hypothesized that this variation in respiration per mitochondria depends on plasticity in cristae density, although current evidence for such a mechanism is lacking. In the present study, we confirm this hypothesis by showing that, in human skeletal muscle, and in contrast to the current view, the mitochondrial cristae density is not constant but, instead, exhibits plasticity with long‐term endurance training. Furthermore, we show that frequently recruited mitochondria‐enriched fibres have significantly increased cristae density and that, at the whole‐body level, muscle mitochondrial cristae density is a better predictor of maximal oxygen uptake rate than muscle mitochondrial volume. Our findings establish an elevating mitochondrial cristae density as a regulatory mechanism for increasing metabolic power in human skeletal muscle. We propose that this mechanism allows evasion of the trade‐off between cell occupancy by mitochondria and other cellular constituents, as well as improved metabolic capacity and fuel catabolism during prolonged elevated energy requirements.

  November 13, 2016   doi: 10.1113/JP273040   open full text
• The effects of cold water immersion and active recovery on inflammation and cell stress responses in human skeletal muscle after resistance exercise.
  Jonathan M. Peake, Llion A. Roberts, Vandre C. Figueiredo, Ingrid Egner, Simone Krog, Sigve N. Aas, Katsuhiko Suzuki, James F. Markworth, Jeff S. Coombes, David Cameron‐Smith, Truls Raastad.
  The Journal of Physiology. November 13, 2016
  Key points Cold water immersion and active recovery are common post‐exercise recovery treatments. A key assumption about the benefits of cold water immersion is that it reduces inflammation in skeletal muscle. However, no data are available from humans to support this notion. We compared the effects of cold water immersion and active recovery on inflammatory and cellular stress responses in skeletal muscle from exercise‐trained men 2, 24 and 48 h during recovery after acute resistance exercise. Exercise led to the infiltration of inflammatory cells, with increased mRNA expression of pro‐inflammatory cytokines and neurotrophins, and the subcellular translocation of heat shock proteins in muscle. These responses did not differ significantly between cold water immersion and active recovery. Our results suggest that cold water immersion is no more effective than active recovery for minimizing the inflammatory and stress responses in muscle after resistance exercise. Abstract Cold water immersion and active recovery are common post‐exercise recovery treatments. However, little is known about whether these treatments influence inflammation and cellular stress in human skeletal muscle after exercise. We compared the effects of cold water immersion versus active recovery on inflammatory cells, pro‐inflammatory cytokines, neurotrophins and heat shock proteins (HSPs) in skeletal muscle after intense resistance exercise. Nine active men performed unilateral lower‐body resistance exercise on separate days, at least 1 week apart. On one day, they immersed their lower body in cold water (10°C) for 10 min after exercise. On the other day, they cycled at a low intensity for 10 min after exercise. Muscle biopsies were collected from the exercised leg before, 2, 24 and 48 h after exercise in both trials. Exercise increased intramuscular neutrophil and macrophage counts, MAC1 and CD163 mRNA expression (P < 0.05). Exercise also increased IL1β, TNF, IL6, CCL2, CCL4, CXCL2, IL8 and LIF mRNA expression (P < 0.05). As evidence of hyperalgesia, the expression of NGF and GDNF mRNA increased after exercise (P < 0.05). The cytosolic protein content of αB‐crystallin and HSP70 decreased after exercise (P < 0.05). This response was accompanied by increases in the cytoskeletal protein content of αB‐crystallin and the percentage of type II fibres stained for αB‐crystallin. Changes in inflammatory cells, cytokines, neurotrophins and HSPs did not differ significantly between the recovery treatments. These findings indicate that cold water immersion is no more effective than active recovery for reducing inflammation or cellular stress in muscle after a bout of resistance exercise.

  November 13, 2016   doi: 10.1113/JP272881   open full text
• Excitation of lateral habenula neurons as a neural mechanism underlying ethanol‐induced conditioned taste aversion.
  Shashank Tandon, Kristen A. Keefe, Sharif A. Taha.
  The Journal of Physiology. November 08, 2016
  Key points The lateral habenula (LHb) has been implicated in regulation of drug‐seeking behaviours through aversion‐mediated learning. In this study, we recorded neuronal activity in the LHb of rats during an operant task before and after ethanol‐induced conditioned taste aversion (CTA) to saccharin. Ethanol‐induced CTA caused significantly higher baseline firing rates in LHb neurons, as well as elevated firing rates in response to cue presentation, lever press and saccharin taste. In a separate cohort of rats, we found that bilateral LHb lesions blocked ethanol‐induced CTA. Our results strongly suggest that excitation of LHb neurons is required for ethanol‐induced CTA, and point towards a mechanism through which LHb firing may regulate voluntary ethanol consumption. Abstract Ethanol, like other drugs of abuse, has both rewarding and aversive properties. Previous work suggests that sensitivity to ethanol's aversive effects negatively modulates voluntary alcohol intake and thus may be important in vulnerability to developing alcohol use disorders. We previously found that rats with lesions of the lateral habenula (LHb), which is implicated in aversion‐mediated learning, show accelerated escalation of voluntary ethanol consumption. To understand neural encoding in the LHb contributing to ethanol‐induced aversion, we recorded neural firing in the LHb of freely behaving, water‐deprived rats before and after an ethanol‐induced (1.5 g kg−1 20% ethanol, i.p.) conditioned taste aversion (CTA) to saccharin taste. Ethanol‐induced CTA strongly decreased motivation for saccharin in an operant task to obtain the tastant. Comparison of LHb neural firing before and after CTA induction revealed four main differences in firing properties. First, baseline firing after CTA induction was significantly higher. Second, firing evoked by cues signalling saccharin availability shifted from a pattern of primarily inhibition before CTA to primarily excitation after CTA induction. Third, CTA induction reduced the magnitude of lever press‐evoked inhibition. Finally, firing rates were significantly higher during consumption of the devalued saccharin solution after CTA induction. Next, we studied sham‐ and LHb‐lesioned rats in our operant CTA paradigm and found that LHb lesion significantly attenuated CTA effects in the operant task. Our data demonstrate the importance of LHb excitation in regulating expression of ethanol‐induced aversion and suggest a mechanism for its role in modulating escalation of voluntary ethanol intake.

  November 08, 2016   doi: 10.1113/JP272994   open full text
• Systematic evaluation of the impact of stimulation intensity on neuroplastic after‐effects induced by transcranial direct current stimulation.
  Asif Jamil, Giorgi Batsikadze, Hsiao‐I. Kuo, Ludovica Labruna, Alkomiet Hasan, Walter Paulus, Michael A. Nitsche.
  The Journal of Physiology. November 08, 2016
  Key points Applications of transcranial direct current stimulation to modulate human neuroplasticity have increased in research and clinical settings. However, the need for longer‐lasting effects, combined with marked inter‐individual variability, necessitates a deeper understanding of the relationship between stimulation parameters and physiological effects. We systematically investigated the full DC intensity range (0.5–2.0 mA) for both anodal and cathodal tDCS in a sham‐controlled repeated measures design, monitoring changes in motor‐cortical excitability via transcranial magnetic stimulation up to 2 h after stimulation. For both tDCS polarities, the excitability after‐effects did not linearly correlate with increasing DC intensity; effects of lower intensities (0.5, 1.0 mA) showed equal, if not greater effects in motor‐cortical excitability. Further, while intra‐individual responses showed good reliability, inter‐individual sensitivity to TMS accounted for a modest percentage of the variance in the early after‐effects of 1.0 mA anodal tDCS, which may be of practical relevance for future optimizations. Abstract Contemporary non‐invasive neuromodulatory techniques, such as transcranial direct current stimulation (tDCS), have shown promising potential in both restituting impairments in cortical physiology in clinical settings, as well as modulating cognitive abilities in the healthy population. However, neuroplastic after‐effects of tDCS are highly dependent on stimulation parameters, relatively short lasting, and not expectedly uniform between individuals. The present study systematically investigates the full range of current intensity between 0.5 and 2.0 mA on left primary motor cortex (M1) plasticity, as well as the impact of individual‐level covariates on explaining inter‐individual variability. Thirty‐eight healthy subjects were divided into groups of anodal and cathodal tDCS. Five DC intensities (sham, 0.5, 1.0, 1.5 and 2.0 mA) were investigated in separate sessions. Using transcranial magnetic stimulation (TMS), 25 motor‐evoked potentials (MEPs) were recorded before, and 10 time points up to 2 h following 15 min of tDCS. Repeated‐measures ANOVAs indicated a main effect of intensity for both anodal and cathodal tDCS. With anodal tDCS, all active intensities resulted in equivalent facilitatory effects relative to sham while for cathodal tDCS, only 1.0 mA resulted in sustained excitability diminution. An additional experiment conducted to assess intra‐individual variability revealed generally good reliability of 1.0 mA anodal tDCS (ICC(2,1) = 0.74 over the first 30 min). A post hoc analysis to discern sources of inter‐individual variability confirmed a previous finding in which individual TMS SI1mV (stimulus intensity for 1 mV MEP amplitude) sensitivity correlated negatively with 1.0 mA anodal tDCS effects on excitability. Our study thus provides further insights on the extent of non‐linear intensity‐dependent neuroplastic after‐effects of anodal and cathodal tDCS.

  November 08, 2016   doi: 10.1113/JP272738   open full text
• Local depletion of glycogen with supramaximal exercise in human skeletal muscle fibres.
  Kasper D. Gejl, Niels Ørtenblad, Erik Andersson, Peter Plomgaard, Hans‐Christer Holmberg, Joachim Nielsen.
  The Journal of Physiology. November 08, 2016
  Key points Glycogen is stored in local spatially distinct compartments within skeletal muscle fibres and is the main energy source during supramaximal exercise. Using quantitative electron microscopy, we show that supramaximal exercise induces a differential depletion of glycogen from these compartments and also demonstrate how this varies with fibre types. Repeated exercise alters this compartmentalized glycogen depletion. The results obtained in the present study help us understand the muscle metabolic dynamics of whole body repeated supramaximal exercise, and suggest that the muscle has a compartmentalized local adaptation to repeated exercise, which affects glycogen depletion. Abstract Skeletal muscle glycogen is heterogeneously distributed in three separated compartments (intramyofibrillar, intermyofibrillar and subsarcolemmal). Although only constituting 3–13% of the total glycogen volume, the availability of intramyofibrillar glycogen is of particular importance to muscle function. The present study aimed to investigate the depletion of these three subcellular glycogen compartments during repeated supramaximal exercise in elite athletes. Ten elite cross‐country skiers (aged 25 ± 4 years, V̇O2 max : 65 ± 4 ml kg−1 min−1; mean ± SD) performed four ∼4 min supramaximal sprint time trials (STT 1–4) with 45 min of recovery. The subcellular glycogen volumes in musculus triceps brachii were quantified from electron microscopy images before and after both STT 1 and 4. During STT 1, the depletion of intramyofibrillar glycogen was higher in type 1 fibres [−52%; (−89:−15%)] than type 2 fibres [−15% (−52:22%)] (P = 0.02), whereas the depletion of intermyofibrillar glycogen [main effect: −19% (−33:0%), P = 0.006] and subsarcolemmal glycogen [main effect: −35% (−66:0%), P = 0.03] was similar between fibre types. By contrast, only intermyofibrillar glycogen volume was significantly reduced during STT 4, in both fibre types [main effect: −31% (−50:−11%), P = 0.002]. Furthermore, for each of the subcellular compartments, the depletion of glycogen during STT 1 was associated with the volumes of glycogen before STT 1. In conclusion, the depletion of spatially distinct glycogen compartments differs during supramaximal exercise. Furthermore, the depletion changes with repeated exercise and is fibre type‐dependent.

  November 08, 2016   doi: 10.1113/JP273109   open full text
• Central control of interlimb coordination and speed‐dependent gait expression in quadrupeds.
  Simon M. Danner, Simon D. Wilshin, Natalia A. Shevtsova, Ilya A. Rybak.
  The Journal of Physiology. November 08, 2016
  Key points Quadrupeds express different gaits depending on speed of locomotion. Central pattern generators (one per limb) within the spinal cord generate locomotor oscillations and control limb movements. Neural interactions between these generators define interlimb coordination and gait. We present a computational model of spinal circuits representing four rhythm generators with left–right excitatory and inhibitory commissural and fore–hind inhibitory interactions within the cord. Increasing brainstem drive to all rhythm generators and excitatory commissural interneurons induces an increasing frequency of locomotor oscillations accompanied by speed‐dependent gait changes from walk to trot and to gallop and bound. The model closely reproduces and suggests explanations for multiple experimental data, including speed‐dependent gait transitions in intact mice and changes in gait expression in mutants lacking certain types of commissural interneurons. The model suggests the possible circuit organization in the spinal cord and proposes predictions that can be tested experimentally. Abstract As speed of locomotion is increasing, most quadrupeds, including mice, demonstrate sequential gait transitions from walk to trot and to gallop and bound. The neural mechanisms underlying these transitions are poorly understood. We propose that the speed‐dependent expression of different gaits results from speed‐dependent changes in the interactions between spinal circuits controlling different limbs and interlimb coordination. As a result, the expression of each gait depends on (1) left–right interactions within the spinal cord mediated by different commissural interneurons (CINs), (2) fore–hind interactions on each side of the spinal cord and (3) brainstem drives to rhythm‐generating circuits and CIN pathways. We developed a computational model of spinal circuits consisting of four rhythm generators (RGs) with bilateral left–right interactions mediated by V0 CINs (V0D and V0V sub‐types) providing left–right alternation, and conditional V3 CINs promoting left–right synchronization. Fore and hind RGs mutually inhibited each other. We demonstrate that linearly increasing excitatory drives to the RGs and V3 CINs can produce a progressive increase in the locomotor speed accompanied by sequential changes of gaits from walk to trot and to gallop and bound. The model closely reproduces and suggests explanations for the speed‐dependent gait expression observed in vivo in intact mice and in mutants lacking V0V or all V0 CINs. Specifically, trot is not expressed after removal of V0V CINs, and only bound is expressed after removal of all V0 CINs. The model provides important insights into the organization of spinal circuits and neural control of locomotion.

  November 08, 2016   doi: 10.1113/JP272787   open full text
• Long‐chain acyl‐CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.
  Bruno G. Teodoro, Igor H. Sampaio, Lucas H. M. Bomfim, André L. Queiroz, Leonardo R. Silveira, Anderson O. Souza, Anna M. A. P. Fernandes, Marcos N. Eberlin, Tai‐Yu Huang, Donghai Zheng, P. Darrell Neufer, Ronald N. Cortright, Luciane C. Alberici.
  The Journal of Physiology. November 08, 2016
  Key points Long‐chain acyl‐CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1‐α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. Abstract Long‐chain acyl‐CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl‐CoA species toward different metabolic fates such as lipid synthesis or β‐oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up‐regulated by acute high‐fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p‐AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC‐1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC‐1α mRNA. In conclusion, ACSL6 drives acyl‐CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1‐α pathway.

  November 08, 2016   doi: 10.1113/JP272962   open full text
• Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate.
  Isabella Salzer, Fatma Asli Erdem, Wei‐Qiang Chen, Seok Heo, Xaver Koenig, Klaus W. Schicker, Helmut Kubista, Gert Lubec, Stefan Boehm, Jae‐Won Yang.
  The Journal of Physiology. November 07, 2016
  Key points Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2. Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors. Abstract The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.

  November 07, 2016   doi: 10.1113/JP273274   open full text
• Large‐scale analysis reveals populational contributions of cortical spike rate and synchrony to behavioural functions.
  Rie Kimura, Akiko Saiki, Yoko Fujiwara‐Tsukamoto, Yutaka Sakai, Yoshikazu Isomura.
  The Journal of Physiology. November 07, 2016
  Key points There have been few systematic population‐wide analyses of relationships between spike synchrony within a period of several milliseconds and behavioural functions. In this study, we obtained a large amount of spike data from > 23,000 neuron pairs by multiple single‐unit recording from deep layer neurons in motor cortical areas in rats performing a forelimb movement task. The temporal changes of spike synchrony in the whole neuron pairs were statistically independent of behavioural changes during the task performance, although some neuron pairs exhibited correlated changes in spike synchrony. Mutual information analyses revealed that spike synchrony made a smaller contribution than spike rate to behavioural functions. The strength of spike synchrony between two neurons was statistically independent of the spike rate‐based preferences of the pair for behavioural functions. Abstract Spike synchrony within a period of several milliseconds in presynaptic neurons enables effective integration of functional information in the postsynaptic neuron. However, few studies have systematically analysed the population‐wide relationships between spike synchrony and behavioural functions. Here we obtained a sufficiently large amount of spike data among regular‐spiking (putatively excitatory) and fast‐spiking (putatively inhibitory) neuron subtypes (> 23,000 pairs) by multiple single‐unit recording from deep layers in motor cortical areas (caudal forelimb area, rostral forelimb area) in rats performing a forelimb movement task. After holding a lever, rats pulled the lever either in response to a cue tone (external‐trigger trials) or spontaneously without any cue (internal‐trigger trials). Many neurons exhibited functional spike activity in association with forelimb movements, and the preference of regular‐spiking neurons in the rostral forelimb area was more biased toward externally triggered movement than that in the caudal forelimb area. We found that a population of neuron pairs with spike synchrony does exist, and that some neuron pairs exhibit a dependence on movement phase during task performance. However, the population‐wide analysis revealed that spike synchrony was statistically independent of the movement phase and the spike rate‐based preferences of the pair for behavioural functions, whereas spike rates were clearly dependent on the movement phase. In fact, mutual information analyses revealed that the contribution of spike synchrony to the behavioural functions was small relative to the contribution of spike rate. Our large‐scale analysis revealed that cortical spike rate, rather than spike synchrony, contributes to population coding for movement.

  November 07, 2016   doi: 10.1113/JP272794   open full text
• Synchronous deficits in cumulative muscle protein synthesis and ribosomal biogenesis underlie age‐related anabolic resistance to exercise in humans.
  Matthew S. Brook, Daniel J. Wilkinson, William K. Mitchell, Jonathan N. Lund, Bethan E. Phillips, Nathaniel J. Szewczyk, Paul L. Greenhaff, Kenneth Smith, Philip J. Atherton.
  The Journal of Physiology. November 07, 2016
  Key points Resistance exercise training (RET) is one of the most effective strategies for preventing declines in skeletal muscle mass and strength with age. Hypertrophic responses to RET with age are diminished compared to younger individuals. In response to 6 weeks RET, we found blunted hypertrophic responses with age are underpinned by chronic deficits in long‐term muscle protein synthesis. We show this is likely to be the result of multifactorial deficits in anabolic hormones and blunted translational efficiency and capacity. These results provide great insight into age‐related exercise adaptations and provide a platform on which to devise appropriate nutritional and exercise interventions on a longer term basis. Abstract Ageing is associated with impaired hypertrophic responses to resistance exercise training (RET). Here we investigated the aetiology of ‘anabolic resistance’ in older humans. Twenty healthy male individuals, 10 younger (Y; 23 ± 1 years) and 10 older (O; 69 ± 3 years), performed 6 weeks unilateral RET (6 × 8 repetitions, 75% of one repetition maximum (1‐RM), 3 times per week). After baseline bilateral vastus lateralis (VL) muscle biopsies, subjects consumed 150 ml D2O (70 atom%; thereafter 50 ml week−1), further bilateral VL muscle biopsies were taken at 3 and 6 weeks to quantify muscle protein synthesis (MPS) via gas chromatography–pyrolysis–isotope ratio mass spectrometry. After RET, 1‐RM increased in Y (+35 ± 4%) and O (+25 ± 3%; P < 0.01), while MVC increased in Y (+21 ± 5%; P < 0.01) but not O (+6 ± 3%; not significant (NS)). In comparison to Y, O displayed blunted RET‐induced increases in muscle thickness (at 3 and 6 weeks, respectively, Y: +8 ± 1% and +11 ± 2%, P < 0.01; O: +2.6 ± 1% and +3.5 ± 2%, NS). While ‘basal’ longer term MPS was identical between Y and O (∼1.35 ± 0.1% day−1), MPS increased in response to RET only in Y (3 weeks, Y: 1.61 ± 0.1% day−1; O: 1.49 ± 0.1% day−1). Consistent with this, O exhibited inferior ribosomal biogenesis (RNA:DNA ratio and c‐MYC induction: Y: +4 ± 2 fold change; O: +1.9 ± 1 fold change), translational efficiency (S6K1 phosphorylation, Y: +10 ± 4 fold change; O: +4 ± 2 fold change) and anabolic hormone milieu (testosterone, Y: 367 ± 19; O: 274 ± 19 ng dl−1 (all P < 0.05). Anabolic resistance is thus multifactorial.

  November 07, 2016   doi: 10.1113/JP272857   open full text
• Role of Cl−–HCO3− exchanger AE3 in intracellular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent astrocytes.
  Ahlam I. Salameh, Christian A. Hübner, Walter F. Boron.
  The Journal of Physiology. November 06, 2016
  Key points A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive. Comparisons of cells from wild‐type vs. AE3–/– mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3– efflux) enhances intracellular pH (pHi) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes. During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes. AE3 speeds re‐alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse‐induced acid load. We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl– loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization‐induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. Abstract The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi) regulation by facilitating the exchange of extracellular Cl– for intracellular HCO3–. The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3–/–) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3–/– and wild‐type neurons is indistinguishable. The purpose of the present study was to use AE3–/– mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co‐cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2]. The presence of AE3 also speeds intracellular acidification during the early phase of metabolic acidosis (MAc), not just in neurons but, surprisingly, in adjacent astrocytes. Additionally, AE3 contributes to braking the decrease in pHi later during MAc in both neurons and astrocytes. Paradoxically, AE3 enhances intracellular re‐alkalization after MAc removal in neurons and astrocytes, and pHi recovery from an ammonium prepulse‐induced acid load in neurons. The effects of AE3 knockout on astrocytic pHi homeostasis in MAc‐related assays require the presence of neurons, and are consistent with the hypothesis that the AE3 knockout reduces functional expression of astrocytic NBCe1. These findings suggest a new type of neuron–astrocyte communication, based on the expression of AE3 in neurons, which could explain how AE3 reduces seizure susceptibility.

  November 06, 2016   doi: 10.1113/JP272470   open full text
• Accumulation of K+ in the synaptic cleft modulates activity by influencing both vestibular hair cell and calyx afferent in the turtle.
  Donatella Contini, Steven D. Price, Jonathan J. Art.
  The Journal of Physiology. November 04, 2016
  Key points In the synaptic cleft between type I hair cells and calyceal afferents, K+ ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft. High‐fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance. Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion. Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential. With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K+]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs. Abstract Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch‐clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High‐fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA‐dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high‐speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells.

  November 04, 2016   doi: 10.1113/JP273060   open full text
• Cannabinoid signalling inhibits sarcoplasmic Ca2+ release and regulates excitation–contraction coupling in mammalian skeletal muscle.
  Tamás Oláh, Dóra Bodnár, Adrienn Tóth, János Vincze, János Fodor, Barbara Reischl, Adrienn Kovács, Olga Ruzsnavszky, Beatrix Dienes, Péter Szentesi, Oliver Friedrich, László Csernoch.
  The Journal of Physiology. October 27, 2016
  Key points Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation–contraction coupling (ECC) of mammalian skeletal muscle remains unknown. We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+‐sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue. We demonstrate that CB1Rs are not connected to the inositol 1,4,5‐trisphosphate pathway either in myotubes or in adult muscle fibres. By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein‐mediated way in adult skeletal muscle fibres but not in myotubes. These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs. Abstract Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R‐mediated signalling affects the contraction and Ca²⁺ homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+‐sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ transients, nor did they alter excitation–contraction coupling. By contrast, in isolated muscle fibres of wild‐type mice, although CB1R agonists did not evoke IP3‐mediated Ca2+ transients too, they significantly reduced the amplitude of the depolarization‐evoked transients in a pertussis‐toxin sensitive manner, indicating a Gi/o protein‐dependent mechanism. Concurrently, on skeletal muscle fibres isolated from CB1R‐knockout animals, depolarization‐evoked Ca2+ transients, as well qas Ca2+ release flux via ryanodine receptors (RyRs), and the total amount of released Ca2+ was significantly greater than that from wild‐type mice. Our results show that CB1R‐mediated signalling exerts both a constitutive and an agonist‐mediated inhibition on the Ca2+ transients via RyR, regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase and enhances muscle fatigability, which might decrease exercise performance, thus playing a role in myopathies, and therefore should be considered during the development of new cannabinoid drugs.

  October 27, 2016   doi: 10.1113/JP272449   open full text
• Impaired central respiratory chemoreflex in an experimental genetic model of epilepsy.
  Leonardo T. Totola, Ana C. Takakura, José Antonio C. Oliveira, Norberto Garcia‐Cairasco, Thiago S. Moreira.
  The Journal of Physiology. October 27, 2016
  Key points It is recognized that seizures commonly cause apnoea and oxygen desaturation, but there is still a lack in the literature about the respiratory impairments observed ictally and in the post‐ictal period. Respiratory disorders may involve changes in serotonergic transmission at the level of the retrotrapezoid nucleus (RTN). In this study, we evaluated breathing activity and the role of serotonergic transmission in the RTN with a rat model of tonic–clonic seizures, the Wistar audiogenic rat (WAR). We conclude that the respiratory impairment in the WAR could be correlated to an overall decrease in the number of neurons located in the respiratory column. Abstract Respiratory disorders may involve changes in serotonergic neurotransmission at the level of the chemosensitive neurons located in the retrotrapezoid nucleus (RTN). Here, we investigated the central respiratory chemoreflex and the role of serotonergic neurotransmission in the RTN with a rat model of tonic–clonic seizures, the Wistar audiogenic rat (WAR). We found that naive or kindled WARs have reduced resting ventilation and ventilatory response to hypercapnia (7% CO2). The number of chemically coded (Phox2b+/TH−) RTN neurons, as well as the serotonergic innervation to the RTN, was reduced in WARs. We detected that the ventilatory response to serotonin (1 mm, 50 nl) within the RTN region was significantly reduced in WARs. Our results uniquely demonstrated a respiratory impairment in a genetic model of tonic–clonic seizures, the WAR strain. More importantly, we demonstrated an overall decrease in the number of neurons located in the ventral respiratory column (VRC), as well as a reduction in serotonergic neurons in the midline medulla. This is an important step forward to demonstrate marked changes in neuronal activity and breathing impairment in the WAR strain, a genetic model of epilepsy.

  October 27, 2016   doi: 10.1113/JP272822   open full text
• Dedicated C‐fibre viscerosensory pathways to central nucleus of the amygdala.
  Stuart J. McDougall, Haoyao Guo, Michael C. Andresen.
  The Journal of Physiology. October 25, 2016
  Key points Emotions are accompanied by concordant changes in visceral function, including cardiac output, respiration and digestion. One major forebrain integrator of emotional responses, the amygdala, is considered to rely on embedded visceral afferent information, although few details are known. In the present study, we retrogradely transported dye from the central nucleus of the amygdala (CeA) to identify CeA‐projecting nucleus of the solitary tract (NTS) neurons for synaptic characterization and compared them with unlabelled, near‐neighboor NTS neurons. Solitary tract (ST) afferents converged onto NTS‐CeA second‐order sensory neurons in greater numbers, as well as indirectly via polysynaptic pathways. Unexpectedly, all mono‐ and polysynaptic ST afferent pathways to NTS‐CeA neurons were organized exclusively as either transient receptor potential cation channel subfamily V member 1 (TRPV1)‐sensitive or TRPV1‐resistant, regardless of whether intervening neurons were excitatory or inhibitory. This strict sorting provides viscerosensory signals to CeA about visceral conditions with respect to being either ‘normal’ via A‐fibres or ‘alarm’ via TRPV1 expressing C‐fibres and, accordingly, this pathway organization probably encodes interoceptive status. Abstract Emotional state is impacted by changes in visceral function, including blood pressure, breathing and digestion. A main line of viscerosensory information processing occurs first in the nucleus of the solitary tract (NTS). In the present study conducted in rats, we examined the synaptic characteristics of visceral afferent pathways to the central nucleus of the amygdala (CeA) in brainstem slices by recording from retrogradely labelled NTS projection neurons. We simultaneously recorded neuron pairs: one dye positive (i.e. NTS‐CeA) and a second unlabelled neighbour. Graded shocks to the solitary tract (ST) always (93%) triggered EPSCs at CeA projecting NTS neurons. Half of the NTS‐CeA neurons received at least one primary afferent input (classed ‘second order’) indicating that viscerosensory information arrives at the CeA conveyed via a pathway involving as few as two synapses. The remaining NTS‐CeA neurons received viscerosensory input only via polysynaptic pathways. By contrast, ∼3/4 of unlabelled neighbouring neurons were directly connected to ST. NTS‐CeA neurons received greater numbers of ST‐related inputs compared to unlabelled NTS neurons, indicating that highly convergent viscerosensory signals reach the CeA. Remarkably, despite multifibre convergence, all single NTS‐CeA neurons received inputs derived from only unmyelinated afferents [transient receptor potential cation channel subfamily V member 1 (TRPV1) expressing C‐fibres] or only non‐TRPV1 ST afferent inputs, and never a combination of both. Such segregation means that visceral afferent information followed separate lines to reach the CeA. Their very different physiological activation profiles mean that these parallel visceral afferent pathways encode viscerosensory signals to the amygdala that may provide interoceptive assessments to impact on behaviours.

  October 25, 2016   doi: 10.1113/JP272898   open full text
• Increased sympathetic nerve activity and reduced cardiac baroreflex sensitivity in rheumatoid arthritis.
  Ahmed M. Adlan, Julian F. R. Paton, Gregory Y. H. Lip, George D. Kitas, James P. Fisher.
  The Journal of Physiology. October 24, 2016
  Key points Rheumatoid arthritis (RA) is a chronic inflammatory condition associated with an increased risk of cardiovascular mortality. Increased sympathetic nerve activity and reduced cardiac baroreflex sensitivity heighten cardiovascular risk, althogh whether such autonomic dysfunction is present in RA is not known. In the present study, we observed an increased sympathetic nerve activity and reduced cardiac baroreflex sensitivity in patients with RA compared to matched controls. Pain was positively correlated with sympathetic nerve activity and negatively correlated with cardiac baroreflex sensitivity. The pattern of autonomic dysfunction that we describe may help to explain the increased cardiovascular risk in RA, and raises the possibility that optimizing pain management may resolve autonomic dysfunction in RA. Abstract Rheumatoid arthritis (RA) is a chronic inflammatory condition associated with increased cardiovascular morbidity/mortality and an incompletely understood pathophysiology. In animal studies, central and blood borne inflammatory cytokines that can be elevated in RA evoke pathogenic increases in sympathetic activity and reductions in baroreflex sensitivity (BRS). We hypothesized that muscle sympathetic nerve activity (MSNA) was increased and BRS decreased in RA. MSNA, blood pressure and heart rate (HR) were recorded in age‐ and sex‐matched RA‐normotensive (n = 13), RA‐hypertensive patients (RA‐HTN; n = 17), normotensive (NC; n = 17) and hypertensive controls (HTN; n = 16). BRS was determined using the modified Oxford technique. Inflammation and pain were determined using serum high sensitivity C‐reactive protein (hs‐CRP) and a visual analogue scale (VAS), respectively. MSNA was elevated similarly in RA, RA‐HTN and HTN patients (32 ± 9, 35 ± 14, 37 ± 8 bursts min–1) compared to NC (22 ± 9 bursts min–1; P = 0.004). Sympathetic BRS was similar between groups (P = 0.927), whereas cardiac BRS (cBRS) was reduced in RA, RA‐HTN and HTN patients [5(3–8), 4 (2–7), 6 (4–9) ms mmHg–1] compared to NC [11 (8–15) ms mmHg–1; P = 0.002]. HR was independently associated with hs‐CRP. Increased MSNA and reduced cBRS were associated with hs‐CRP although confounded in multivariable analysis. VAS was independently associated with MSNA burst frequency, cBRS and HR. We provide the first evidence for heightened sympathetic outflow and reduced cBRS in RA that can be independent of hypertension. In RA patients, reported pain was positively correlated with MSNA and negatively correlated with cBRS. Future studies should assess whether therapies to ameliorate pain and inflammation in RA restores autonomic balance and reduces cardiovascular events.

  October 24, 2016   doi: 10.1113/JP272944   open full text
• Hyperammonaemia‐induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress.
  Gangarao Davuluri, Allawy Allawy, Samjhana Thapaliya, Julie H. Rennison, Dharmvir Singh, Avinash Kumar, Yana Sandlers, David R. Wagoner, Chris A. Flask, Charles Hoppel, Takhar Kasumov, Srinivasan Dasarathy.
  The Journal of Physiology. October 23, 2016
  Key points Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia. We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content. During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids. Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content. Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. Abstract Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non‐hepatic ammonia disposal. Non‐hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α‐ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+/NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell‐permeable ester of α‐ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia‐induced mitochondrial dysfunction is a novel therapeutic approach.

  October 23, 2016   doi: 10.1113/JP272796   open full text
• Denervation drives mitochondrial dysfunction in skeletal muscle of octogenarians.
  Sally Spendiff, Madhusudanarao Vuda, Gilles Gouspillou, Sudhakar Aare, Anna Perez, José A. Morais, Robert T. Jagoe, Marie‐Eve Filion, Robin Glicksman, Sophia Kapchinsky, Norah J. MacMillan, Charlotte H. Pion, Mylène Aubertin‐Leheudre, Stefan Hettwer, José A. Correa, Tanja Taivassalo, Russell T. Hepple.
  The Journal of Physiology. October 23, 2016
  Key points Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown. We show that increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting. However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target. Abstract Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. Because this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean ± SD age: 23.7 ± 2.7 years and 71.2 ± 4.9 years) and two inactive groups (64.8 ± 3.1 years and 82.5 ± 4.8 years), and compared this with a murine model of sporadic denervation. We tested the hypothesis that, although some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, mitochondrial dysfunction would be impacted by persistent denervation in advanced age. Both ageing in humans and sporadic denervation in mice increased mitochondrial sensitivity to permeability transition (humans, P = 0.004; mice, P = 0.01). To determine the contribution of sporadic denervation to mitochondrial function, we pharmacologically inhibited the denervation‐induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, which is similar to that seen in humans older than 75 years (–66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age, when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target.

  October 23, 2016   doi: 10.1113/JP272487   open full text
• The impacts of age and frailty on heart rate and sinoatrial node function.
  Motahareh Moghtadaei, Hailey J. Jansen, Martin Mackasey, Sara A. Rafferty, Oleg Bogachev, John L. Sapp, Susan E. Howlett, Robert A. Rose.
  The Journal of Physiology. October 17, 2016
  Key points Sinoatrial node (SAN) function declines with age; however, not all individuals age at the same rate and health status can vary from fit to frail. Frailty was quantified in young and aged mice using a non‐invasive frailty index so that the impacts of age and frailty on heart rate and SAN function could be assessed. SAN function was impaired in aged mice due to alterations in electrical conduction, changes in SAN action potential morphology and fibrosis in the SAN. Changes in SAN function, electrical conduction, action potential morphology and fibrosis were correlated with, and graded by, frailty. This study shows that mice of the same chronological age have quantifiable differences in health status that impact heart rate and SAN function and that these differences in health status can be identified using our frailty index. Abstract Sinoatrial node (SAN) dysfunction increases with age, although not all older adults are affected in the same way. This is because people age at different rates and individuals of the same chronological age vary in health status from very fit to very frail. Our objective was to determine the impacts of age and frailty on heart rate (HR) and SAN function using a new model of frailty in ageing mice. Frailty, which was quantified in young and aged mice using a frailty index (FI), was greater in aged vs. young mice. Intracardiac electrophysiology demonstrated that HR was reduced whereas SAN recovery time (SNRT) was prolonged in aged mice; however, both parameters showed heteroscedasticity suggesting differences in health status among mice of similar chronological age. Consistent with this, HR and corrected SNRT were correlated with, and graded by, FI score. Optical mapping of the SAN demonstrated that conduction velocity (CV) was reduced in aged hearts in association with reductions in diastolic depolarization (DD) slope and action potential (AP) duration. In agreement with in vivo results, SAN CV, DD slope and AP durations all correlated with FI score. Finally, SAN dysfunction in aged mice was associated with increased interstitial fibrosis and alterations in expression of matrix metalloproteinases, which also correlated with frailty. These findings demonstrate that age‐related SAN dysfunction occurs in association with electrical and structural remodelling and that frailty is a critical determinant of health status of similarly aged animals that correlates with changes in HR and SAN function.

  October 17, 2016   doi: 10.1113/JP272979   open full text
• Eliminating Nox2 reactive oxygen species production protects dystrophic skeletal muscle from pathological calcium influx assessed in vivo by manganese‐enhanced magnetic resonance imaging.
  James A. Loehr, Gary R. Stinnett, Mayra Hernández‐Rivera, Wesley T. Roten, Lon J. Wilson, Robia G. Pautler, George G. Rodney.
  The Journal of Physiology. October 17, 2016
  Key points Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese‐enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non‐invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy. Abstract Duchenne muscular dystrophy (DMD) is an X‐linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese‐enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long‐term effects on animal health or muscle function. We conclude that MEMRI is a viable, non‐invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.

  October 17, 2016   doi: 10.1113/JP272907   open full text
• Endothelium‐dependent vasodilatory signalling modulates α1‐adrenergic vasoconstriction in contracting skeletal muscle of humans.
  Christopher M. Hearon, Brett S. Kirby, Gary J. Luckasen, Dennis G. Larson, Frank A. Dinenno.
  The Journal of Physiology. October 13, 2016
  Key points ‘Functional sympatholysis’ describes the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and is critical to ensure proper blood flow and oxygen delivery to metabolically active skeletal muscle. The signalling mechanism responsible for sympatholysis in healthy humans is unknown. Evidence from animal models has identified endothelium‐derived hyperpolarization (EDH) as a potential mechanism capable of attenuating sympathetic vasoconstriction. In this study, increasing endothelium‐dependent signalling during exercise significantly enhanced the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction in humans. This is the first study in humans to identify endothelium‐dependent regulation of sympathetic vasoconstriction in contracting skeletal muscle, and specifically supports a role for EDH‐like vasodilatory signalling. Impaired functional sympatholysis is a common feature of cardiovascular ageing, hypertension and heart failure, and thus identifying fundamental mechanisms responsible for sympatholysis is clinically relevant. Abstract Stimulation of α‐adrenoceptors elicits vasoconstriction in resting skeletal muscle that is blunted during exercise in an intensity‐dependent manner. In humans, the underlying mechanisms remain unclear. We tested the hypothesis that stimulating endothelium‐dependent vasodilatory signalling will enhance the ability of contracting skeletal muscle to blunt α1‐adrenergic vasoconstriction. Changes in forearm vascular conductance (FVC; Doppler ultrasound, brachial intra‐arterial pressure via catheter) to local intra‐arterial infusion of phenylephrine (PE; α1‐adrenoceptor agonist) were calculated during (1) infusion of the endothelium‐dependent vasodilators acetylcholine (ACh) and adenosine triphosphate (ATP), the endothelium‐independent vasodilator (sodium nitroprusside, SNP), or potassium chloride (KCl) at rest; (2) mild or moderate intensity handgrip exercise; and (3) combined mild exercise + ACh, ATP, SNP, or KCl infusions in healthy adults. Robust vasoconstriction to PE was observed during vasodilator infusion alone and mild exercise, and this was blunted during moderate intensity exercise (ΔFVC: −34 ± 4 and −34 ± 3 vs. −13 ± 2%, respectively, P < 0.05). Infusion of ACh or ATP during mild exercise significantly attenuated PE vasoconstriction similar to levels observed during moderate exercise (ACh: −3 ± 4; ATP: −18 ± 4%). In contrast, infusion of SNP or KCl during mild exercise did not attenuate PE‐mediated vasoconstriction (−32 ± 5 and −46 ± 3%). To further study the role of endothelium‐dependent hyperpolarization (EDH), ACh trials were repeated with combined nitric oxide synthase and cyclooxygenase inhibition. Here, PE‐mediated vasoconstriction was blunted at rest (blockade: −20 ± 5 vs. control: −31 ± 3% vs.; P < 0.05) and remained blunted during exercise (blockade: −15 ± 5 vs. control: −14 ± 5%). We conclude that stimulation of EDH‐like vasodilatation can blunt α1‐adrenergic vasoconstriction in contracting skeletal muscle of humans.

  October 13, 2016   doi: 10.1113/JP272829   open full text
• Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.
  Kwangseok Hong, Guiling Zhao, Zhongkui Hong, Zhe Sun, Yan Yang, Philip S. Clifford, Michael J. Davis, Gerald A. Meininger, Michael A. Hill.
  The Journal of Physiology. October 13, 2016
  Key points Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1R), causes a concentration‐dependent inhibition of pressure‐dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein‐coupled receptor. Mechanoactivation of the AT1R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1R stimulates actin polymerization by a protein kinase C‐dependent mechanism, but independently of a change in intracellular Ca2+. Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1R. Abstract The Gq/11 protein‐coupled angiotensin II type 1 receptor (AT1R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first‐order cremaster muscle arterioles the AT1R inhibitor candesartan (10−7–10−5 m) showed partial but concentration‐dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure–diameter relationship over the intraluminal pressure range, 30–110 mmHg. Pressure‐induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura‐2) were similar in the absence or presence of candesartan, indicating that AT1R‐mediated myogenic constriction relies on Ca2+‐independent downstream signalling. The diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G‐actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1R in arterioles and suggest that mechanically activated AT1R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure‐induced vasoconstriction.

  October 13, 2016   doi: 10.1113/JP272834   open full text
• Supraspinal control of spinal reflex responses to body bending during different behaviours in lampreys.
  Li‐Ju Hsu, Pavel V. Zelenin, Grigori N. Orlovsky, Tatiana G. Deliagina.
  The Journal of Physiology. October 13, 2016
  Key points Spinal reflexes are substantial components of the motor control system in all vertebrates and centrally driven reflex modifications are essential to many behaviours, but little is known about the neuronal mechanisms underlying these modifications. To study this issue, we took advantage of an in vitro brainstem–spinal cord preparation of the lamprey (a lower vertebrate), in which spinal reflex responses to spinal cord bending (caused by signals from spinal stretch receptor neurons) can be evoked during different types of fictive behaviour. Our results demonstrate that reflexes observed during fast forward swimming are reversed during escape behaviours, with the reflex reversal presumably caused by supraspinal commands transmitted by a population of reticulospinal neurons. NMDA receptors are involved in the formation of these commands, which are addressed primarily to the ipsilateral spinal networks. In the present study the neuronal mechanisms underlying reflex reversal have been characterized for the first time. Abstract Spinal reflexes can be modified during different motor behaviours. However, our knowledge about the neuronal mechanisms underlying these modifications in vertebrates is scarce. In the lamprey, a lower vertebrate, body bending causes activation of intraspinal stretch receptor neurons (SRNs) resulting in spinal reflexes: activation of motoneurons (MNs) with bending towards either the contralateral or ipsilateral side (a convex or concave response, respectively). The present study had two main aims: (i) to investigate how these spinal reflexes are modified during different motor behaviours, and (ii) to reveal reticulospinal neurons (RSNs) transmitting commands for the reflex modification. For this purpose in in vitro brainstem–spinal cord preparation, RSNs and reflex responses to bending were recorded during different fictive behaviours evoked by supraspinal commands. We found that during fast forward swimming MNs exhibited convex responses. By contrast, during escape behaviours, MNs exhibited concave responses. We found RSNs that were activated during both stimulation causing reflex reversal without initiation of any specific behaviour, and stimulation causing reflex reversal during escape behaviour. We suggest that these RSNs transmit commands for the reflex modification. Application of the NMDA antagonist (AP‐5) to the brainstem significantly decreased the reversed reflex, suggesting involvement of NMDA receptors in the formation of these commands. Longitudinal split of the spinal cord did not abolish the reflex reversal caused by supraspinal commands, suggesting an important role for ipsilateral networks in determining this type of motor response. This is the first study to reveal the neuronal mechanisms underlying supraspinal control of reflex reversal.

  October 13, 2016   doi: 10.1113/JP272714   open full text
• Synaptic transmission at the endbulb of Held deteriorates during age‐related hearing loss.
  Ruili Xie, Paul B. Manis.
  The Journal of Physiology. October 10, 2016
  Key points Synaptic transmission at the endbulb of Held was assessed by whole‐cell patch clamp recordings from auditory neurons in mature (2–4 months) and aged (20–26 months) mice. Synaptic transmission is degraded in aged mice, which may contribute to the decline in neural processing of the central auditory system during age‐related hearing loss. The changes in synaptic transmission in aged mice can be partially rescued by improving calcium buffering, or decreasing action potential‐evoked calcium influx. These experiments suggest potential mechanisms, such as regulating intraterminal calcium, that could be manipulated to improve the fidelity of transmission at the aged endbulb of Held. Abstract Age‐related hearing loss (ARHL) is associated with changes to the auditory periphery that raise sensory thresholds and alter coding, and is accompanied by alterations in excitatory and inhibitory synaptic transmission, and intrinsic excitability in the circuits of the central auditory system. However, it remains unclear how synaptic transmission changes at the first central auditory synapses during ARHL. Using mature (2–4 months) and old (20–26 months) CBA/CaJ mice, we studied synaptic transmission at the endbulb of Held. Mature and old mice showed no difference in either spontaneous quantal synaptic transmission or low frequency evoked synaptic transmission at the endbulb of Held. However, when challenged with sustained high frequency stimulation, synapses in old mice exhibited increased asynchronous transmitter release and reduced synchronous release. This suggests that the transmission of temporally precise information is degraded at the endbulb during ARHL. Increasing intraterminal calcium buffering with EGTA‐AM or decreasing calcium influx with ω‐agatoxin IVA decreased the amount of asynchronous release and restored synchronous release in old mice. In addition, recovery from depression following high frequency trains was faster in old mice, but was restored to a normal time course by EGTA‐AM treatment. These results suggest that intraterminal calcium in old endbulbs may rise to abnormally high levels during high rates of auditory nerve firing, or that calcium‐dependent processes involved in release are altered with age. These observations suggest that ARHL is associated with a decrease in temporal precision of synaptic release at the first central auditory synapse, which may contribute to perceptual deficits in hearing.

  October 10, 2016   doi: 10.1113/JP272683   open full text
• Obesity‐induced lymphatic dysfunction is reversible with weight loss.
  Matthew D. Nitti, Geoffrey E. Hespe, Raghu P. Kataru, Gabriela D. García Nores, Ira L. Savetsky, Jeremy S. Torrisi, Jason C. Gardenier, Andrew J. Dannenberg, Babak J. Mehrara.
  The Journal of Physiology. October 09, 2016
  Key points Obesity induces lymphatic leakiness, decreases initial lymphatic vessel density, impairs collecting vessel pumping and decreases transport of macromolecules. Obesity results in perilymphatic inducible nitric oxide synthase (iNOS) expression and accumulation of T cells and macrophages. Deleterious effects of obesity on the lymphatic system correlate with weight gain. Weight loss restores lymphatic function in obese animals and decreases perilymphatic iNOS and inflammatory cell accumulation. Abstract Although clinical and experimental studies have shown that obesity results in lymphatic dysfunction, it remains unknown whether these changes are permanent or reversible with weight loss. In the current study, we used a mouse model of diet‐induced obesity to identify putative cellular mechanisms of obesity‐induced lymphatic dysfunction, determine whether there is a correlation between these deleterious effects and increasing weight gain, and finally examine whether lymphatic dysfunction is reversible with diet‐induced weight loss. We report that obesity is negatively correlated with cutaneous lymphatic collecting vessel pumping rate (r = –0.9812, P < 0.0005) and initial lymphatic vessel density (r = –0.9449, P < 0.005). In addition, we show a significant positive correlation between weight gain and accumulation of perilymphatic inflammatory cells (r = 0.9872, P < 0.0005) and expression of inducible nitric oxide synthase (iNOS; r = 0.9986, P < 0.0001). Weight loss resulting from conversion to a normal chow diet for 8 weeks resulted in more than a 25% decrease in body weight and normalized cutaneous lymphatic collecting vessel pumping rate, lymphatic vessel density, lymphatic leakiness, and lymphatic macromolecule clearance (all P < 0.05). In addition, weight loss markedly decreased perilymphatic inflammation and iNOS expression. Taken together, our findings show that obesity is linearly correlated with lymphatic dysfunction, perilymphatic inflammation and iNOS expression, and that weight loss via dietary modification effectively reverses these deleterious effects.

  October 09, 2016   doi: 10.1113/JP273061   open full text
• Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.
  Patricia Ortega‐Sáenz, David Macías, Konstantin L. Levitsky, José A. Rodríguez‐Gómez, Patricia González‐Rodríguez, Victoria Bonilla‐Henao, Ignacio Arias‐Mayenco, José López‐Barneo.
  The Journal of Physiology. October 09, 2016
  Key points Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin‐deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin‐deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Abstract Biotin is a water‐soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin‐deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin‐deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin‐deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin‐deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

  October 09, 2016   doi: 10.1113/JP272961   open full text
• Altered corticospinal function during movement preparation in humans with spinal cord injury.
  Paolo Federico, Monica A. Perez.
  The Journal of Physiology. October 07, 2016
  Key points In uninjured humans, transmission in the corticospinal pathway changes in a task‐dependent manner during movement preparation. We investigated whether this ability is preserved in humans with incomplete chronic cervical spinal cord injury (SCI). Our results show that corticospinal excitability is altered in the preparatory phase of an upcoming movement when there is a need to suppress but not to execute rapid index finger voluntary contractions in individuals with SCI compared with controls. This is probably related to impaired transmission at a cortical and spinal level after SCI. Overall our findings indicate that deficits in corticospinal transmission in humans with chronic incomplete SCI are also present in the preparatory phase of upcoming movements. Abstract Corticospinal output is modulated in a task‐dependent manner during the preparatory phase of upcoming movements in humans. Whether this ability is preserved after spinal cord injury (SCI) is unknown. In this study, we examined motor evoked potentials elicited by cortical (MEPs) and subcortical (CMEPs) stimulation of corticospinal axons and short‐interval intracortical inhibition in the first dorsal interosseous muscle in the preparatory phase of a reaction time task where individuals with chronic incomplete cervical SCI and age‐matched controls needed to suppress (NOGO) or initiate (GO) ballistic index finger isometric voluntary contractions. Reaction times were prolonged in SCI participants compared with control subjects and stimulation was provided ∼90 ms prior to movement onset in each group. During NOGO trials, both MEPs and CMEPs remained unchanged compared to baseline in SCI participants but were suppressed in control subjects. Notably, during GO trials, MEPs increased to a similar extent in both groups but CMEPs increased only in controls. The magnitude of short‐interval intracortical inhibition increased in controls but not in SCI subjects during NOGO trials and decreased in both groups in GO trials. These novel observations reveal that humans with incomplete cervical SCI have an altered ability to modulate corticospinal excitability during movement preparation when there is a need to suppress but not to execute upcoming rapid finger movements, which is probably related to impaired transmission at a cortical and spinal level. Thus, deficits in corticospinal transmission after human SCI extend to the preparatory phase of upcoming movements.

  October 07, 2016   doi: 10.1113/JP272266   open full text
• Hyperphagia in male melanocortin 4 receptor deficient mice promotes growth independently of growth hormone.
  H. Y. Tan, F. J. Steyn, L. Huang, M. Cowley, J. D. Veldhuis, C. Chen.
  The Journal of Physiology. October 02, 2016
  Key points Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth. Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth. We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone–insulin‐like growth factor‐1 (GH–IGF‐1) axis. We propose that hyperinsulinaemia promotes growth while suppressing the GH–IGF‐1 axis. It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Abstract Defects in melanocortin‐4‐receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)‐mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin‐like growth factor‐1 (IGF‐1) production and/or release relative to pubertal growth. We demonstrate early‐onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH–IGF‐1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia‐associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild‐type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair‐fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia‐associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia‐associated childhood‐onset obesity.

  October 02, 2016   doi: 10.1113/JP272770   open full text
• A simulation study on the constancy of cardiac energy metabolites during workload transition.
  Ryuta Saito, Ayako Takeuchi, Yukiko Himeno, Nobuya Inagaki, Satoshi Matsuoka.
  The Journal of Physiology. October 02, 2016
  Key points The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition. How this is accomplished is not yet clarified, though Ca2+ has been suggested to be one of the possible mechanisms. We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca2+‐dependent regulation mechanisms play roles in the metabolite constancy. Model simulations revealed that the Ca2+‐dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist. These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition. Abstract The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca2+, because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca2+‐dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca2+ and inorganic phosphate (Pi) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca2+‐dependent activations of the aspartate/glutamate carrier and the F1Fo‐ATPase, and the Pi‐dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca2+ concentration ([Ca2+]cyt) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2‐oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca2+]cyt. It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca2+ has only a minor role under physiological conditions.

  October 02, 2016   doi: 10.1113/JP272598   open full text
• Transient cerebellar alterations during development prior to obvious motor phenotype in a mouse model of spinocerebellar ataxia type 6.
  Sriram Jayabal, Lovisa Ljungberg, Alanna J. Watt.
  The Journal of Physiology. October 02, 2016
  Key points Spinocerebellar ataxia type 6 (SCA6) is a midlife‐onset neurodegenerative disease caused by a CACNA1A mutation; CACNA1A is also implicated in cerebellar development. We have previously shown that when disease symptoms are present in midlife in SCA684Q/84Q mice, cerebellar Purkinje cells spike with reduced rate and precision. In contrast, we find that during postnatal development (P10–13), SCA684Q/84Q Purkinje cells spike with elevated rate and precision. Although surplus climbing fibres are linked to ataxia in other mouse models, we found surplus climbing fibre inputs on developing (P10–13) SCA684Q/84Q Purkinje cells when motor deficits were not detected. Developmental alterations were transient and were no longer observed in weanling (P21–24) SCA684Q/84Q Purkinje cells. Our results suggest that changes in the developing cerebellar circuit can occur without detectable motor abnormalities, and that changes in cerebellar development may not necessarily persist into adulthood. Abstract Although some neurodegenerative diseases are caused by mutations in genes that are known to regulate neuronal development, surprisingly, patients may not present disease symptoms until adulthood. Spinocerebellar ataxia type 6 (SCA6) is one such midlife‐onset disorder in which the mutated gene, CACNA1A, is implicated in cerebellar development. We wondered whether changes were observed in the developing cerebellum in SCA6 prior to the detection of motor deficits. To address this question, we used a transgenic mouse with a hyper‐expanded triplet repeat (SCA684Q/84Q) that displays late‐onset motor deficits at 7 months, and measured cerebellar Purkinje cell synaptic and intrinsic properties during postnatal development. We found that firing rate and precision were enhanced during postnatal development in P10–13 SCA684Q/84Q Purkinje cells, and observed surplus multiple climbing fibre innervation without changes in inhibitory input or dendritic structure during development. Although excess multiple climbing fibre innervation has been associated with ataxic symptoms in several adult transgenic mice, we observed no detectable changes in cerebellar‐related motor behaviour in developing SCA684Q/84Q mice. Interestingly, we found that developmental alterations were transient, as both Purkinje cell firing properties and climbing fibre innervation from weanling‐aged (P21–24) SCA684Q/84Q mice were indistinguishable from litter‐matched control mice. Our results demonstrate that significant alterations in neuronal circuit development may be observed without any detectable behavioural read‐out, and that early changes in brain development may not necessarily persist into adulthood in midlife‐onset diseases.

  October 02, 2016   doi: 10.1113/JP273184   open full text
• Caloric restriction selectively reduces the GABAergic phenotype of mouse hypothalamic proopiomelanocortin neurons.
  Brooke C. Jarvie, Connie M. King, Alexander R. Hughes, Matthew S. Dicken, Christina S. Dennison, Shane T. Hentges.
  The Journal of Physiology. October 02, 2016
  Key points Hypothalamic proopiomelanocortin (POMC) neurons release peptide products that potently inhibit food intake and reduce body weight. These neurons also release the amino acid transmitter GABA, which can inhibit downstream neurons. Although the release of peptide transmitters from POMC neurons is regulated by energy state, whether similar regulation of GABA release might occur had not been examined. The present results show that the GABAergic phenotype of POMC neurons is decreased selectively by caloric deficit and not altered by high‐fat diet or stress. The fact the GABAergic phenotype of POMC neurons is sensitive to energy state suggests a dynamic physiological role for this transmitter and highlights the importance of determining the functional consequence of GABA released from POMC neurons in terms of the regulation of normal energy balance. Abstract In addition to peptide transmitters, hypothalamic neurons, including proopiomelanocortin (POMC) and agouti‐related peptide (AgRP) neurons, also release amino acid transmitters that can alter energy balance regulation. While recent studies show that the GABAergic nature of AgRP neurons is increased by caloric restriction, whether the GABAergic phenotype of POMC neurons is also regulated in an energy‐state‐dependent manner has not been previously examined. The present studies used fluorescence in situ hybridization to detect Gad1 and Gad2 mRNA in POMC neurons, as these encode the glutamate decarboxylase enzymes GAD67 and GAD65, respectively. The results show that both short‐term fasting and chronic caloric restriction significantly reduce the percentage of POMC neurons expressing Gad1 mRNA in both male and female mice, with less of an effect on Gad2 expression. Neither acute nor chronic intermittent restraint stress altered Gad1 expression in POMC neurons. Maintenance on a high‐fat diet also did not affect the portion POMC neurons expressing Gad1, suggesting that the GABAergic phenotype of POMC neurons is particularly sensitive to energy deficit. Because changes in Gad1 expression have been previously shown to correlate with altered terminal GABA release, fasting is likely to cause a decrease in GABA release from POMC neurons. Altogether, the present results show that the GABAergic nature of POMC neurons can be dynamically regulated by energy state in a manner opposite to that in AgRP neurons and suggest the importance of considering the functional role of GABA release in addition to the peptide transmitters from POMC neurons.

  October 02, 2016   doi: 10.1113/JP273020   open full text
• Development of an excitatory kisspeptin projection to the oxytocin system in late pregnancy.
  Alexander J. Seymour, Victoria Scott, Rachael A. Augustine, Gregory T. Bouwer, Rebecca E. Campbell, Colin H. Brown.
  The Journal of Physiology. October 02, 2016
  Key points Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized. We found that that kisspeptin fibre density around oxytocin neurones increases in late‐pregnant rats. These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy. Oxytocin neurones were excited by central kisspeptin administration in late‐pregnant rats but not in non‐pregnant rats or early‐ to mid‐pregnant rats. Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth. Abstract The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day 21 of gestation) had a higher density of kisspeptin‐positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non‐pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT‐PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone–supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late‐pregnant rats (days 18–21 of gestation) but not in non‐pregnant rats, or in early‐ or mid‐pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition.

  October 02, 2016   doi: 10.1113/JP273051   open full text
• Epigenetic regulation of redox state mediates persistent cardiorespiratory abnormalities after long‐term intermittent hypoxia.
  Jayasri Nanduri, Ying‐Jie Peng, Ning Wang, Shakil A. Khan, Gregg L. Semenza, Ganesh K. Kumar, Nanduri R. Prabhakar.
  The Journal of Physiology. October 02, 2016
  Key points The effects of short‐term (ST; 10 days) and long‐term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats. ST‐ and LT‐IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST‐ but not from LT‐IH. The persistent cardiorespiratory responses to LT‐IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation‐dependent suppression of genes encoding anti‐oxidant enzymes (AOEs). Treating rats with decitabine either during LT‐IH or during recovery from LT‐IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing. Abstract Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O2 saturation profiles during obstructive sleep apnoea (OSA), have been shown to exhibit a heightened carotid body (CB) chemosensory reflex and hypertension. CB chemosensory reflex activation also results in unstable breathing with apnoeas. However, the effect of chronic IH on breathing is not known. In the present study, we examined the effects of chronic IH on breathing along with blood pressure (BP) and assessed whether the autonomic responses are normalized after recovery from chronic IH. Studies were performed on adult, male, Sprague–Dawley rats exposed to either short‐term (ST; 10 days) or long‐term (LT, 30 days) IH. Rats exposed to either ST‐ or LT‐IH exhibited hypertension, irregular breathing with apnoeas, an augmented CB chemosensory reflex as indicated by elevated CB neural activity and plasma catecholamine levels, and elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla (AM). All these effects were normalized after recovery from ST‐IH but not from LT‐IH. Analysis of the molecular mechanisms underlying the persistent effects of LT‐IH revealed increased DNA methylation of genes encoding anti‐oxidant enzymes (AOEs). Treatment with decitabine, a DNA methylation inhibitor, either during LT‐IH or during recovery from LT‐IH, prevented DNA methylation, normalized the expression of AOE genes, ROS levels, CB chemosensory reflex and BP, and also stabilized breathing. These results suggest that persistent cardiorespiratory abnormalities caused by LT‐IH are mediated by epigenetic re‐programming of the redox state in the CB chemosensory reflex pathway.

  October 02, 2016   doi: 10.1113/JP272346   open full text
• KV7/M channels as targets for lipopolysaccharide‐induced inflammatory neuronal hyperexcitability.
  Arik Tzour, Hodaya Leibovich, Omer Barkai, Yoav Biala, Shaya Lev, Yoel Yaari, Alexander M. Binshtok.
  The Journal of Physiology. October 02, 2016
  Key points Neuroinflammation associated with CNS insults leads to neuronal hyperexcitability, which may culminate in epileptiform discharges. Application of the endotoxin lipopolysaccharide (LPS) to brain tissue initiates a neuroinflammatory cascade, providing an experimental model to study the mechanisms of neuroinflammatory neuronal hyperexcitability. Here we show that LPS application to hippocampal slices markedly enhances the excitability of CA1 pyramidal cells by inhibiting a specific potassium current, the M‐current, generated by KV7/M channels, which controls the excitability of almost every neuron in the CNS. The LPS‐induced M‐current inhibition is triggered by sequential activation of microglia, astrocytes and pyramidal cells, mediated by metabotropic purinergic and glutamatergic transmission, leading to blockade of KV7/M channels by calcium released from intracellular stores. The identification of the downstream molecular target of neuroinflammation, namely the KV7/M channel, potentially has far reaching implications for the understanding and treatment of many acute and chronic brain disorders. Abstract Acute brain insults and many chronic brain diseases manifest an innate inflammatory response. The hallmark of this response is glia activation, which promotes repair of damaged tissue, but also induces structural and functional changes that may lead to an increase in neuronal excitability. We have investigated the mechanisms involved in the modulation of neuronal activity by acute inflammation. Initiating inflammatory responses in hippocampal tissue rapidly led to neuronal depolarization and repetitive firing even in the absence of active synaptic transmission. This action was mediated by a complex metabotropic purinergic and glutamatergic glia‐to‐neuron signalling cascade, leading to the blockade of neuronal KV7/M channels by Ca2+ released from internal stores. These channels generate the low voltage‐activating, non‐inactivating M‐type K+ current (M‐current) that controls intrinsic neuronal excitability, and its inhibition was the predominant cause of the inflammation‐induced hyperexcitability. Our discovery that the ubiquitous KV7/M channels are the downstream target of the inflammation‐induced cascade, has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.

  October 02, 2016   doi: 10.1113/JP272547   open full text
• Roadmap for cardiovascular circulation model.
  Soroush Safaei, Christopher P. Bradley, Vinod Suresh, Kumar Mithraratne, Alexandre Muller, Harvey Ho, David Ladd, Leif R. Hellevik, Stig W. Omholt, J. Geoffrey Chase, Lucas O. Müller, Sansuke M. Watanabe, Pablo J. Blanco, Bernard Bono, Peter J. Hunter.
  The Journal of Physiology. September 29, 2016
  Abstract Computational models of many aspects of the mammalian cardiovascular circulation have been developed. Indeed, along with orthopaedics, this area of physiology is one that has attracted much interest from engineers, presumably because the equations governing blood flow in the vascular system are well understood and can be solved with well‐established numerical techniques. Unfortunately, there have been only a few attempts to create a comprehensive public domain resource for cardiovascular researchers. In this paper we propose a roadmap for developing an open source cardiovascular circulation model. The model should be registered to the musculo‐skeletal system. The computational infrastructure for the cardiovascular model should provide for near real‐time computation of blood flow and pressure in all parts of the body. The model should deal with vascular beds in all tissues, and the computational infrastructure for the model should provide links into CellML models of cell function and tissue function. In this work we review the literature associated with 1D blood flow modelling in the cardiovascular system, discuss model encoding standards, software and a model repository. We then describe the coordinate systems used to define the vascular geometry, derive the equations and discuss the implementation of these coupled equations in the open source computational software OpenCMISS. Finally, some preliminary results are presented and plans outlined for the next steps in the development of the model, the computational software and the graphical user interface for accessing the model.

  September 29, 2016   doi: 10.1113/JP272660   open full text
• HCN channels segregate stimulation‐evoked movement responses in neocortex and allow for coordinated forelimb movements in rodents.
  Jeffery A. Boychuk, Jordan S. Farrell, Laura A. Palmer, Anna C. Singleton, Quentin J. Pittman, G. Campbell Teskey.
  The Journal of Physiology. September 27, 2016
  Key points The present study tested whether HCN channels contribute to the organization of motor cortex and to skilled motor behaviour during a forelimb reaching task. Experimental reductions in HCN channel signalling increase the representation of complex multiple forelimb movements in motor cortex as assessed by intracortical microstimulation. Global HCN1KO mice exhibit reduced reaching accuracy and atypical movements during a single‐pellet reaching task relative to wild‐type controls. Acute pharmacological inhibition of HCN channels in forelimb motor cortex decreases reaching accuracy and increases atypical movements during forelimb reaching. Abstract The mechanisms by which distinct movements of a forelimb are generated from the same area of motor cortex have remained elusive. Here we examined a role for HCN channels, given their ability to alter synaptic integration, in the expression of forelimb movement responses during intracortical microstimulation (ICMS) and movements of the forelimb on a skilled reaching task. We used short‐duration high‐resolution ICMS to evoke forelimb movements following pharmacological (ZD7288), experimental (electrically induced cortical seizures) or genetic approaches that we confirmed with whole‐cell patch clamp to substantially reduce Ih current. We observed significant increases in the number of multiple movement responses evoked at single sites in motor maps to all three experimental manipulations in rats or mice. Global HCN1 knockout mice were less successful and exhibited atypical movements on a skilled‐motor learning task relative to wild‐type controls. Furthermore, in reaching‐proficient rats, reaching accuracy was reduced and forelimb movements were altered during infusion of ZD7288 within motor cortex. Thus, HCN channels play a critical role in the separation of overlapping movement responses and allow for successful reaching behaviours. These data provide a novel mechanism for the encoding of multiple movement responses within shared networks of motor cortex. This mechanism supports a viewpoint of primary motor cortex as a site of dynamic integration for behavioural output.

  September 27, 2016   doi: 10.1113/JP273068   open full text
• Left ventricular vascular and metabolic adaptations to high‐intensity interval and moderate intensity continuous training: a randomized trial in healthy middle‐aged men.
  Jari‐Joonas Eskelinen, Ilkka Heinonen, Eliisa Löyttyniemi, Juuso Hakala, Marja A. Heiskanen, Kumail K. Motiani, Kirsi Virtanen, Jussi P. Pärkkä, Juhani Knuuti, Jarna C. Hannukainen, Kari K. Kalliokoski.
  The Journal of Physiology. September 27, 2016
  Key points High‐intensity interval training (HIIT) has become popular, time‐sparing alternative to moderate intensity continuous training (MICT), although the cardiac vascular and metabolic effects of HIIT are incompletely known. We compared the effects of 2‐week interventions with HIIT and MICT on myocardial perfusion and free fatty acid and glucose uptake. Insulin‐stimulated myocardial glucose uptake was decreased by training without any significantly different response between the groups, whereas free fatty acid uptake remained unchanged. Adenosine‐stimulated myocardial perfusion responded differently to the training modes (change in mean HIIT: –19%; MICT: +9%; P = 0.03 for interaction) and was correlated with myocardial glucose uptake for the entire dataset and especially after HIIT training. HIIT and MICT induce similar metabolic and functional changes in the heart, although myocardial vascular hyperaemic reactivity is impaired after HIIT, and this should be considered when prescribing very intense HIIT for previously untrained subjects. Abstract High‐intensity interval training (HIIT) is a time‐efficient way of obtaining the health benefits of exercise, although the cardiac effects of this training mode are incompletely known. We compared the effects of short‐term HIIT and moderate intensity continuous training (MICT) interventions on myocardial perfusion and metabolism and cardiac function in healthy, sedentary, middle‐aged men. Twenty‐eight healthy, middle‐aged men were randomized to either HIIT or MICT groups (n = 14 in both) and underwent six cycle ergometer training sessions within 2 weeks (HIIT session: 4–6 × 30 s all‐out cycling/4 min recovery, MICT session 40–60 min at 60% V̇O2 peak ). Cardiac magnetic resonance imaging (CMRI) was performed to measure cardiac structure and function and positron emission tomography was used to measure myocardial perfusion at baseline and during adenosine stimulation, insulin‐stimulated glucose uptake (MGU) and fasting free fatty acid uptake (MFFAU). End‐diastolic and end‐systolic volumes increased and ejection fraction slightly decreased with both training modes, although no other changes in CMRI were observed. MFFAU and basal myocardial perfusion remained unchanged. MGU was decreased by training (HIIT from 46.5 to 35.9; MICT from 47.4 to 44.4 mmol 100 g–1 min–1, P = 0.007 for time, P = 0.11 for group × time). Adenosine‐stimulated myocardial perfusion responded differently to the training modes (change in mean HIIT: –19%; MICT: +9%; P = 0.03 for group × time interaction). HIIT and MICT induce similar metabolic and functional changes in the heart, although myocardial vascular hyperaemic reactivity is impaired after HIIT. This should be taken into account when prescribing very intense HIIT for previously untrained subjects.

  September 27, 2016   doi: 10.1113/JP273089   open full text
• Unexpected reductions in regional cerebral perfusion during prolonged hypoxia.
  Justin S. Lawley, Jamie H. Macdonald, Samuel J. Oliver, Paul G. Mullins.
  The Journal of Physiology. September 24, 2016
  Key points Cognitive performance is impaired by hypoxia despite global cerebral oxygen delivery and metabolism being maintained. Using arterial spin labelled (ASL) magnetic resonance imaging, this is the first study to show regional reductions in cerebral blood flow (CBF) in response to decreased oxygen supply (hypoxia) at 2 h that increased in area and became more pronounced at 10 h. Reductions in CBF were seen in brain regions typically associated with the ‘default mode’ or ‘task negative’ network. Regional reductions in CBF, and associated vasoconstriction, within the default mode network in hypoxia is supported by increased vasodilatation in these regions to a subsequent hypercapnic (5% CO2) challenge. These results suggest an anatomical mechanism through which hypoxia may cause previously reported deficits in cognitive performance. Abstract Hypoxia causes an increase in global cerebral blood flow, which maintains global cerebral oxygen delivery and metabolism. However, neurological deficits are abundant under hypoxic conditions. We investigated regional cerebral microvascular responses to acute (2 h) and prolonged (10 h) poikilocapnic normobaric hypoxia. We found that 2 h of hypoxia caused an expected increase in frontal cortical grey matter perfusion but unexpected perfusion decreases in regions of the brain normally associated with the ‘default mode’ or ‘task negative’ network. After 10 h in hypoxia, decreased blood flow to the major nodes of the default mode network became more pronounced and widespread. The use of a hypercapnic challenge (5% CO2) confirmed that these reductions in cerebral blood flow from hypoxia were related to vasoconstriction. Our findings demonstrate steady‐state deactivation of the default network under acute hypoxia, which become more pronounced over time. Moreover, these data provide a unique insight into the nuanced localized cerebrovascular response to hypoxia that is not attainable through traditional methods. The observation of reduced perfusion in the posterior cingulate and cuneal cortex, which are regions assumed to play a role in declarative and procedural memory, provides an anatomical mechanism through which hypoxia may cause deficits in working memory.

  September 24, 2016   doi: 10.1113/JP272557   open full text
• Fetal programming of blood pressure in a transgenic mouse model of altered intrauterine environment.
  Giuseppe Chiossi, Maged M. Costantine, Esther Tamayo, Gary D. V. Hankins, George R. Saade, Monica Longo.
  The Journal of Physiology. September 24, 2016
  Key points Nitric oxide is essential in the vascular adaptation to pregnancy, as knockout mice lacking nitric oxide synthase (NOS3) have abnormal utero‐placental perfusion, hypertension and growth restriction. We previously showed with ex vivo studies on transgenic animals lacking NOS3 that adverse intrauterine environment alters fetal programming of vascular reactivity in adult offspring. The current research shows that altered vascular reactivity correlates with higher blood pressure in vivo. Our data suggest that higher blood pressure depends on both genetic background (NOS3 deficiency) and uterine environment, becomes more evident with age (> 7 postnatal weeks), activity and stress, is gender specific (preponderant among males), and can be affected by the sleep–awake cycle. In utero or early postnatal life (< 7 weeks), before onset of hypertension, may represent a potential window for intervention to prevent future cardiovascular disorders. Abstract Nitric oxide is involved in the vascular adaptation to pregnancy. Using transgenic animals, we previously showed that adverse intrauterine environment alters vascular reactivity in adult offspring. The aim of our study was to determine if altered vascular programming is associated with abnormal blood pressure (BP) profiles in vivo. Mice lacking a functional endothelial nitric oxide synthase (KO, NOS3−/−) and wild‐type mice (WT, NOS3+/+) were crossbred to generate homozygous NOS3−/− (KO), maternally derived heterozygous NOS3+/− (KOM: mother with adverse intrauterine environment from NOS3 deficiency), paternally derived heterozygous NOS3+/− (KOP: mother with normal in utero milieu) and NOS3+/+ (WT) litters. BP was measured in vivo at 7, 14 and 21 weeks of age. After univariate analysis, multivariate population‐averaged linear regression models were used to identify factors affecting BP. When compared to WT offspring, systolic (SBP), diastolic (DBP) and mean (MAP) BP progressively increased from KOP, to KOM, and peaked among KO (P < 0.001), although significance was not reached for KOP. Higher BP was also associated with male gender, older age (> 7 postnatal weeks), higher locomotor activity, daytime recordings, and recent blood pressure transducer insertion (P < 0.001). Post hoc analysis showed that KOM had higher SBP than KOP (P < 0.05). Our study indicates that adverse intrauterine environment contributes, along with multiple other factors, to account for hypertension; moreover, in utero or early postnatal life may represent a possible therapeutic window for prevention of cardiovascular disease later in life.

  September 24, 2016   doi: 10.1113/JP272602   open full text
• Differential α‐adrenergic modulation of rapid onset vasodilatation along resistance networks of skeletal muscle in old versus young mice.
  Shenghua Y. Sinkler, Charmain A. Fernando, Steven S. Segal.
  The Journal of Physiology. September 23, 2016
  Key points Rapid onset vasodilatation (ROV) initiates functional hyperaemia upon skeletal muscle contraction and is attenuated during ageing via α‐adrenoreceptor (αAR) stimulation, but it is unknown where this effect predominates in resistance networks. In gluteus maximus muscles of young (4 months) and old (24 months) male C57BL/6 mice, tetanic contraction while observing feed arteries and arterioles initiated ROV, which increased with contraction duration, peaked later in upstream versus downstream vessel branches and was attenuated throughout networks with advanced age. With no effect on muscle force production, inhibiting αARs improved ROV in old mice while activating αARs attenuated ROV in young mice. Modulating ROV through αARs was greater in upstream feed arteries and arterioles compared to downstream arterioles, with α2ARs more effective than α1ARs. ROV is coordinated along resistance networks and modulated differentially between young and old mice via αARs; with advanced age, attenuated dilatation of upstream branches will restrict muscle blood flow. Abstract Rapid onset vasodilatation (ROV) in skeletal muscle is attenuated during advanced age via α‐adrenoreceptor (αAR) activation, but it is unknown where such effects predominate in the resistance vasculature. Studying the gluteus maximus muscle (GM) of anaesthetized young (4 months) and old (24 months) male C57BL/6 mice, we tested the hypothesis that attenuation of ROV during advanced age is most effective in proximal branches of microvascular resistance networks. Diameters of a feed artery (FA) and first‐ (1A), second‐ (2A) and third‐ (3A) order arterioles were studied in response to single tetanic contractions (100 Hz, 100–1000 ms). ROV began within 1 s and peaked sooner in 2A and 3A (∼3 s) than in 1A or FA (∼4 s). Relative amplitudes of dilatation increased with contraction duration and with vessel branch order (FA<1A<2A<3A). In old mice, attenuation of ROV was greater in FA and 1A compared to 2A and 3A. With no effect on muscle force production, inhibiting αARs (phentolamine; 10−6 m) improved ROV in FA and 1A of old mice while subthreshold stimulation of αARs in young mice (noradrenaline; 10−9 m) depressed ROV most effectively in FA and 1A. In young mice, stimulating α1ARs (phenylephrine; 10−7 m) and α2ARs (UK 14304; 10−7 m) attenuated ROV primarily in FA. In old mice, inhibiting α2ARs (rauwolscine; 10−7 m) restored ROV more effectively in FA and 1A than did inhibiting α1ARs (prazosin; 10−8 m). We conclude that, with temporal and spatial coordination along resistance networks, attenuation of ROV with advanced age is most effective in proximal branches via constitutive activation of α2ARs.

  September 23, 2016   doi: 10.1113/JP272409   open full text
• Carotid sinus denervation ameliorates renovascular hypertension in adult Wistar rats.
  Wioletta Pijacka, Fiona D. McBryde, Paul J. Marvar, Gisele S. Lincevicius, Ana P. L. Abdala, Lavinia Woodward, Dan Li, David J. Paterson, Julian F. R. Paton.
  The Journal of Physiology. September 23, 2016
  Key points Peripheral chemoreflex sensitization is a feature of renovascular hypertension. Carotid sinus nerve denervation (CSD) has recently been shown to relieve hypertension and reduce sympathetic activity in other rat models of hypertension. We show that CSD in renovascular hypertension halts further increases in blood pressure. Possible mechanisms include improvements in baroreceptor reflex sensitivity and renal function, restoration of cardiac calcium signalling towards control levels, and reduced neural inflammation. Our data suggest that the peripheral chemoreflex may be a viable therapeutic target for renovascular hypertension. Abstract The peripheral chemoreflex is known to be hyper‐responsive in both spontaneously hypertensive (SHR) and Goldblatt hypertensive (two kidney one clip; 2K1C) rats. We have previously shown that carotid sinus nerve denervation (CSD) reduces arterial blood pressure (ABP) in SHR. In the present study, we show that CSD ameliorates 2K1C hypertension and reveal the potential underlying mechanisms. Adult Wistar rats were instrumented to record ABP via telemetry, and then underwent CSD (n = 9) or sham CSD (n = 9) 5 weeks after renal artery clipping, in comparison with normal Wistar rats (n = 5). After 21 days, renal function was assessed, and tissue was collected to assess sympathetic postganglionic intracellular calcium transients ([Ca2+]i) and immune cell infiltrates. Hypertensive 2K1C rats showed a profound elevation in ABP (Wistar: 98 ± 4 mmHg vs. 2K1C: 147 ± 8 mmHg; P < 0.001), coupled with impairments in renal function and baroreflex sensitivity, increased neuroinflammatory markers and enhanced [Ca2+]I in stellate neurons (P < 0.05). CSD reduced ABP in 2K1C+CSD rats and prevented the further progressive increase in ABP seen in 2K1C+sham CSD rats, with a between‐group difference of 14 ± 2 mmHg by week 3 (P < 0.01), which was accompanied by improvements in both baroreflex control and spectral indicators of cardiac sympatho‐vagal balance. Furthermore, CSD improved protein and albuminuria, decreased [Ca2+]i evoked responses from stellate neurons, and also reduced indicators of brainstem inflammation. In summary, CSD in 2K1C rats reduces the hypertensive burden and improves renal function. This may be mediated by improvements in autonomic balance, functional remodelling of post‐ganglionic neurons and reduced inflammation. Our results suggest that the peripheral chemoreflex may be considered as a potential therapeutic target for controlling renovascular hypertension.

  September 23, 2016   doi: 10.1113/JP272708   open full text
• Administration of prostacyclin modulates cutaneous blood flow but not sweating in young and older males: roles for nitric oxide and calcium‐activated potassium channels.
  Naoto Fujii, Sean R. Notley, Christopher T. Minson, Glen P. Kenny.
  The Journal of Physiology. September 23, 2016
  Key points In young adults, cyclooxygenase (COX) contributes to the heat loss responses of cutaneous vasodilatation and sweating, and this may be mediated by prostacyclin‐induced activation of nitric oxide synthase (NOS) and calcium‐activated potassium (KCa) channels. This prostacyclin‐induced response may be diminished in older relative to young adults because ageing is known to attenuate COX‐dependent heat loss responses. We observed that, although prostacyclin does not mediate sweating in young and older males, it does modulate cutaneous vasodilatation, although the magnitude of increase is similar between groups. We also found that, although NOS and KCa channels contribute to prostacyclin‐induced cutaneous vasodilatation in young males, these contributions are diminished in older males. Our findings provide new insight into the mechanisms governing heat loss responses and suggest that the age‐related diminished COX‐dependent heat loss responses reported in previous studies may be a result of the reduced COX‐derived production of prostanoids (e.g., prostacyclin) rather than the decreased sensitivity of prostanoid receptors. Abstract Cyclooxygenase (COX) contributes to the regulation of cutaneous vasodilatation and sweating; however, the mechanism(s) underpinning this response remain unresolved. We hypothesized that prostacyclin (a COX‐derived product) may directly mediate cutaneous vasodilatation and sweating through nitric oxide synthase (NOS) and calcium‐activated potassium (KCa) channels in young adults. However, these responses would be diminished in older adults because ageing attenuates COX‐dependent cutaneous vasodilatation and sweating. In young (25 ± 4 years) and older (60 ± 6 years) males (nine per group), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites: (i) control; (ii) 10 mm NG‐nitro‐l‐arginine (l‐NNA), a non‐specific NOS inhibitor; (iii) 50 mm tetraethylammonium (TEA), a non‐specific KCa channel blocker; and (iv) 10 mm l‐NNA + 50 mm TEA. All four sites were coadministered with prostacyclin in an incremental manner (0.04, 0.4, 4, 40 and 400 μm each for 25 min). Prostacyclin‐induced increases in CVC were similar between groups (all concentrations, P > 0.05). l‐NNA and TEA, as well as their combination, lowered CVC in young males at all prostacyclin concentrations (P ≤ 0.05), with the exception of l‐NNA at 0.04 μm (P > 0.05). In older males, CVC during prostacyclin administration was not influenced by l‐NNA (all concentrations), TEA (4‐400 μm) or their combination (400 μm) (P > 0.05). No effect on sweat rate was observed in either group (all concentrations, P > 0.05). We conclude that, although prostacyclin does not mediate sweating, it modulates cutaneous vasodilatation to a similar extent in young and older males. Furthermore, although NOS and KCa channels contribute to the prostacyclin‐induced cutaneous vasodilatation in young males, these contributions are diminished in older males.

  September 23, 2016   doi: 10.1113/JP273174   open full text
• Sequence determinants of subtype‐specific actions of KCNQ channel openers.
  Alice W. Wang, Runying Yang, Harley T. Kurata.
  The Journal of Physiology. September 23, 2016
  Key points Retigabine is a KCNQ voltage‐gated potassium channel opener that was recently approved as an add‐on therapeutic for patients with drug‐resistant epilepsy. Retigabine exhibits very little specificity between most KCNQ channel subtypes, and there is interest in generating more potent and specific KCNQ channel openers. The present study describes the marked specificity of ICA069673 for KCNQ2 vs. KCNQ3, and exploits this property to investigate determinants of KCNQ subtype specificity. ICA069673 acts on a binding site in the voltage‐sensing domain that is distinct from the putative retigabine site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity. We identify two channel residues required for subtype specificity of KCNQ channel openers and show that these are sufficient to generate ICA069673 sensitivity in KCNQ3. Abstract Retigabine (RTG) is the first approved anti‐epileptic drug that acts via activation of voltage‐gated potassium channels, targeting KCNQ channels that underlie the neuronal M‐current. RTG exhibits little specificity between KCNQ2–5 as a result of conservation of a Trp residue in the pore domain that binds to the drug. The RTG analogue ICA‐069673 (‘ICA73’) exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage‐dependence of activation, an ∼2‐fold enhancement of peak current and pronounced subtype specificity for KCNQ2 over KCNQ3. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1–S4) rather than the pore region targeted by RTG. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73‐mediated gating shift, but preserve RTG sensitivity. Interestingly, A181P mutant channels show little ICA73‐mediated gating shift but retain current potentiation by the drug. Mutations (L198F and P211A), which introduce these critical KCNQ2 residues at corresponding positions in KCNQ3, transplant partial ICA73 sensitivity. These findings demonstrate that RTG and ICA73 act via distinct mechanisms, and also reveal specific residues that underlie subtype specificity of KCNQ channel openers.

  September 23, 2016   doi: 10.1113/JP272762   open full text
• Metaboreflex activation delays heart rate recovery after aerobic exercise in never‐treated hypertensive men.
  Tiago Peçanha, Leandro Campos Brito, Rafael Yokoyama Fecchio, Patricia Nascimento Sousa, Natan Daniel da Silva Junior, Andrea Pio Abreu, Giovanio Vieira Silva, Décio Mion‐Junior, Cláudia Lúcia de Moraes Forjaz.
  The Journal of Physiology. September 21, 2016
  Key points Recent evidence indicates that metaboreflex regulates heart rate recovery after exercise (HRR). An increased metaboreflex activity during the post‐exercise period might help to explain the reduced HRR observed in hypertensive subjects. Using lower limb circulatory occlusion, the present study showed that metaboreflex activation during the post‐exercise period delayed HRR in never‐treated hypertensive men compared to normotensives. These findings may be relevant for understanding the physiological mechanisms associated with autonomic dysfunction in hypertensive men. Abstract Muscle metaboreflex influences heart rate (HR) regulation after aerobic exercise. Therefore, increased metaboreflex sensitivity may help to explain the delayed HR recovery (HRR) reported in hypertension. The present study assessed and compared the effect of metaboreflex activation after exercise on HRR, cardiac baroreflex sensitivity (cBRS) and heart rate variability (HRV) in normotensive (NT) and hypertensive (HT) men. Twenty‐three never‐treated HT and 25 NT men randomly underwent two‐cycle ergometer exercise sessions (30 min, 70% V̇O2 peak ) followed by 5 min of inactive recovery performed with (occlusion) or without (control) leg circulatory occlusion (bilateral thigh cuffs inflated to a suprasystolic pressure). HRR was assessed via HR reduction after 30, 60 and 300 s of recovery (HRR30s, HRR60s and HRR300s), as well as by the analysis of short‐ and long‐term time constants of HRR. cBRS was assessed by sequence technique and HRV by the root mean square residual and the root mean square of successive differences between adjacent RR intervals on subsequent 30 s segments. Data were analysed using two‐ and three‐way ANOVA. HRR60s and cBRS were significant and similarly reduced in both groups in the occlusion compared to the control session (combined values: 20 ± 10 vs. 26 ± 9 beats min–1 and 2.1 ± 1.2 vs. 3.2 ± 2.4 ms mmHg−1, respectively, P < 0.05). HRR300s and HRV were also reduced in the occlusion session, although these reductions were significantly greater in HT compared to NT (−16 ± 11 vs. −8 ± 15 beats min–1 for HRR300s, P < 0.05). The results support the role of metaboreflex in HRR and suggest that increased metaboreflex sensitivity may partially explain the delayed HRR observed in HT men.

  September 21, 2016   doi: 10.1113/JP272851   open full text
• The relative contributions of store‐operated and voltage‐gated Ca2+ channels to the control of Ca2+ oscillations in airway smooth muscle.
  Sebastian Boie, Jun Chen, Michael J. Sanderson, James Sneyd.
  The Journal of Physiology. September 21, 2016
  Key points Agonist‐dependent oscillations in the concentration of free cytosolic calcium are a vital mechanism for the control of airway smooth muscle contraction and thus are a critical factor in airway hyper‐responsiveness. Using a mathematical model, closely tied to experimental work, we show that the oscillations in membrane potential accompanying the calcium oscillations have no significant effect on the properties of the calcium oscillations. In addition, the model shows that calcium entry through store‐operated calcium channels is critical for calcium oscillations, but calcium entry through voltage‐gated channels has much less effect. The model predicts that voltage‐gated channels are less important than store‐operated channels in the control of airway smooth muscle tone. Abstract Airway smooth muscle contraction is typically the key mechanism underlying airway hyper‐responsiveness, and the strength of muscle contraction is determined by the frequency of oscillations of intracellular calcium (Ca2+) concentration. In airway smooth muscle cells, these Ca2+ oscillations are caused by cyclic Ca2+ release from the sarcoplasmic reticulum, although Ca2+ influx via plasma membrane channels is also necessary to sustain the oscillations over longer times. To assess the relative contributions of store‐operated and voltage‐gated Ca2+ channels to this Ca2+ influx, we generated a comprehensive mathematical model, based on experimental Ca2+ measurements in mouse precision‐cut lung slices, to simulate Ca2+ oscillations and changes in membrane potential. Agonist‐induced Ca2+ oscillations are accompanied by oscillations in membrane potential, although the membrane potential oscillations are too small to generate large Ca2+ currents through voltage‐gated Ca2+ channels, and thus have little effect on the Ca2+ oscillations. Ca2+ entry through voltage‐gated channels only becomes important when the cell is depolarized (e.g. by a high external K+ concentration). As a result, agonist‐induced Ca2+ oscillations are critically dependent on Ca2+ entry through store‐operated channels but do not depend strongly on Ca2+ entry though voltage‐gated channels.

  September 21, 2016   doi: 10.1113/JP272996   open full text
• Inhibition linearizes firing rate responses in human motor units: implications for the role of persistent inward currents.
  Ann L. Revill, Andrew J. Fuglevand.
  The Journal of Physiology. September 20, 2016
  Key points Motor neurons are the output neurons of the central nervous system and are responsible for controlling muscle contraction. When initially activated during voluntary contraction, firing rates of motor neurons increase steeply but then level out at modest rates. Activation of an intrinsic source of excitatory current at recruitment onset may underlie the initial steep increase in firing rate in motor neurons. We attempted to disable this intrinsic excitatory current by artificially activating an inhibitory reflex. When motor neuron activity was recorded while the inhibitory reflex was engaged, firing rates no longer increased steeply, suggesting that the intrinsic excitatory current was probably responsible for the initial sharp rise in motor neuron firing rate. Abstract During graded isometric contractions, motor unit (MU) firing rates increase steeply upon recruitment but then level off at modest rates even though muscle force continues to increase. The mechanisms underlying such firing behaviour are not known although activation of persistent inward currents (PICs) might be involved. PICs are intrinsic, voltage‐dependent currents that activate strongly when motor neurons (MNs) are first recruited. Such activation might cause a sharp escalation in depolarizing current and underlie the steep initial rise in MU firing rate. Because PICs can be disabled with synaptic inhibition, we hypothesized that artificial activation of an inhibitory pathway might curb this initial steep rise in firing rate. To test this, human subjects performed slow triangular ramp contractions of the ankle dorsiflexors in the absence and presence of tonic synaptic inhibition delivered to tibialis anterior (TA) MNs by sural nerve stimulation. Firing rate profiles (expressed as a function of contraction force) of TA MUs recorded during these tasks were compared for control and stimulation conditions. Under control conditions, during the ascending phase of the triangular contractions, 93% of the firing rate profiles were best fitted by rising exponential functions. With stimulation, however, firing rate profiles were best fitted with linear functions or with less steeply rising exponentials. Firing rate profiles for the descending phases of the contractions were best fitted with linear functions for both control and stimulation conditions. These results seem consistent with the idea that PICs contribute to non‐linear firing rate profiles during ascending but not descending phases of contractions.

  September 20, 2016   doi: 10.1113/JP272823   open full text
• Mitochondria‐specific antioxidant supplementation does not influence endurance exercise training‐induced adaptations in circulating angiogenic cells, skeletal muscle oxidative capacity or maximal oxygen uptake.
  Daniel D. Shill, W. Michael Southern, T. Bradley Willingham, Kasey A. Lansford, Kevin K. McCully, Nathan T. Jenkins.
  The Journal of Physiology. September 18, 2016
  Key points Reducing excessive oxidative stress, through chronic exercise or antioxidants, can decrease the negative effects induced by excessive amounts of oxidative stress. Transient increases in oxidative stress produced during acute exercise facilitate beneficial vascular training adaptations, but the effects of non‐specific antioxidants on exercise training‐induced vascular adaptations remain elusive. Circulating angiogenic cells (CACs) are an exercise‐inducible subset of white blood cells that maintain vascular integrity. We investigated whether mitochondria‐specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training in CACs, muscle mitochondrial capacity and maximal oxygen uptake in young healthy men. We show that endurance exercise training increases multiple CAC types, an adaptation that is not altered by MitoQ supplementation. Additionally, MitoQ does not affect skeletal muscle or whole‐body aerobic adaptations to exercise training. These results indicate that MitoQ supplementation neither enhances nor attenuates endurance training adaptations in young healthy men. Abstract Antioxidants have been shown to improve endothelial function and cardiovascular outcomes. However, the effects of antioxidants on exercise training‐induced vascular adaptations remain elusive. General acting antioxidants combined with exercise have not impacted circulating angiogenic cells (CACs). We investigated whether mitochondria‐specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training on CD3+, CD3+/CD31+, CD14+/CD31+, CD31+, CD34+/VEGFR2+ and CD62E+ peripheral blood mononuclear cells (PBMCs), muscle mitochondrial capacity, and maximal oxygen uptake (VO2 max ) in healthy men aged 22.1 ± 0.7 years, with a body mass index of 26.9 ± 0.9 kg m–2, and 24.8 ± 1.3% body fat. Analysis of main effects revealed that training induced 33, 105 and 285% increases in CD14+/CD31+, CD62E+ and CD34+/VEGFR2+ CACs, respectively, and reduced CD3+/CD31− PBMCs by 14%. There was no effect of MitoQ on CAC levels. Also independent of MitoQ supplementation, exercise training significantly increased quadriceps muscle mitochondrial capacity by 24% and VO2 max by roughly 7%. In conclusion, endurance exercise training induced increases in multiple CAC types, and this adaptation is not modified by MitoQ supplementation. Furthermore, we demonstrate that a mitochondrial‐targeted antioxidant does not influence skeletal muscle or whole‐body aerobic adaptations to exercise training.

  September 18, 2016   doi: 10.1113/JP272491   open full text
• Systemic availability and metabolism of colonic‐derived short‐chain fatty acids in healthy subjects: a stable isotope study.
  Eef Boets, Sara V. Gomand, Lise Deroover, Tom Preston, Karen Vermeulen, Vicky Preter, Henrike M. Hamer, Guy den Mooter, Luc Vuyst, Christophe M. Courtin, Pieter Annaert, Jan A. Delcour, Kristin A. Verbeke.
  The Journal of Physiology. September 18, 2016
  Key points The short‐chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. Abstract The short‐chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13C‐labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13C‐SCFAs 13C‐glucose, 13C‐cholesterol and 13C‐fatty acids were measured. The butyrate‐producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic‐administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate‐producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13CO2, whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13C‐labelled fibres in the human colon by measurement of 13C‐labelled SCFA concentrations in blood.

  September 18, 2016   doi: 10.1113/JP272613   open full text
• Environmentally induced return to juvenile‐like chemosensitivity in the respiratory control system of adult bullfrog, Lithobates catesbeianus.
  Joseph M. Santin, Lynn K. Hartzler.
  The Journal of Physiology. September 15, 2016
  Key points The degree to which developmental programmes or environmental signals determine physiological phenotypes remains a major question in physiology. Vertebrates change environments during development, confounding interpretation of the degree to which development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) produces phenotypes. Tadpoles mainly breathe water for gas exchange and frogs may breathe water or air depending on their environment and are, therefore, exemplary models to differentiate the degree to which life‐stage vs. environmental context drives developmental phenotypes associated with neural control of lung breathing. Using isolated brainstem preparations and patch clamp electrophysiology, we demonstrate that adult bullfrogs acclimatized to water‐breathing conditions do not exhibit CO2 and O2 chemosensitivity of lung breathing, similar to water‐breathing tadpoles. Our results establish that phenotypes associated with developmental stage may arise from plasticity per se and suggest that a developmental trajectory coinciding with environmental change obscures origins of stage‐dependent physiological phenotypes by masking plasticity. Abstract An unanswered question in developmental physiology is to what extent does the environment vs. a genetic programme produce phenotypes? Developing animals inhabit different environments and switch from one to another. Thus a developmental time course overlapping with environmental change confounds interpretations as to whether development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) generates phenotypes. Tadpoles of the American bullfrog, Lithobates catesbeianus, breathe water at early life‐stages and minimally use lungs for gas exchange. As adults, bullfrogs rely on lungs for gas exchange, but spend months per year in ice‐covered ponds without lung breathing. Aquatic submergence, therefore, removes environmental pressures requiring lung breathing and enables separation of adulthood from environmental factors associated with adulthood that necessitate control of lung ventilation. To test the hypothesis that postmetamorphic respiratory control phenotypes arise through permanent developmental changes vs. reversible environmental signals, we measured respiratory‐related nerve discharge in isolated brainstem preparations and action potential firing from CO2‐sensitive neurons in bullfrogs acclimatized to semi‐terrestrial (air‐breathing) and aquatic‐overwintering (no air‐breathing) habitats. We found that aquatic overwintering significantly reduced neuroventilatory responses to CO2 and O2 involved in lung breathing. Strikingly, this gas sensitivity profile reflects that of water‐breathing tadpoles. We further demonstrated that aquatic overwintering reduced CO2‐induced firing responses of chemosensitive neurons. In contrast, respiratory rhythm generating processes remained adult‐like after submergence. Our results establish that phenotypes associated with life‐stage can arise from phenotypic plasticity per se. This provides evidence that developmental time courses coinciding with environmental changes obscure interpretations regarding origins of stage‐dependent physiological phenotypes by masking plasticity.

  September 15, 2016   doi: 10.1113/JP272777   open full text
• Convergence of visual and whisker responses in the primary somatosensory thalamus (ventral posterior medial region) of the mouse.
  Annette E Allen, Christopher A. Procyk, Timothy M. Brown, Robert J. Lucas.
  The Journal of Physiology. September 15, 2016
  Key points Using in vivo electrophysiology, we find that a subset of whisker‐responsive neurons in the ventral posterior medial region (VPM) respond to visual stimuli. These light‐responsive neurons in the VPM are particularly sensitive to optic flow. Presentation of optic flow stimuli modulates the amplitude of concurrent whisker responses. Visual information reaches the VPM via a circuit encompassing the visual cortex. These data represent a new example of cross‐modal integration in the primary sensory thalamus. Abstract Sensory signals reach the cortex via sense‐specific thalamic nuclei. Here we report that neurons in the primary sensory thalamus of the mouse vibrissal system (the ventral posterior medial region; VPM) can be excited by visual as well as whisker stimuli. Using extracellular electrophysiological recordings from anaesthetized mice we first show that simple light steps can excite a subset of VPM neurons. We then test the ability of the VPM to respond to spatial patterns and show that many units are excited by visual motion in a direction‐selective manner. Coherent movement of multiple objects (an artificial recreation of ‘optic flow’ that would usually occur during head rotations or body movements) best engages this visual motion response. We next show that, when co‐applied with visual stimuli, the magnitude of responses to whisker deflections is highest in the presence of optic flow going in the opposite direction. Importantly, whisker response amplitude is also modulated by presentation of a movie recreating the mouse's visual experience during natural exploratory behaviour. We finally present functional and anatomical data indicating a functional connection (probably multisynaptic) from the primary visual cortex to VPM. These data provide a rare example of multisensory integration occurring at the level of the sensory thalamus, and provide evidence for dynamic regulation of whisker responses according to visual experience.

  September 15, 2016   doi: 10.1113/JP272791   open full text
• Endocannabinoids control vesicle release mode at midbrain periaqueductal grey inhibitory synapses.
  Karin R. Aubrey, Geoffrey M. Drew, Hyo‐Jin Jeong, Benjamin K. Lau, Christopher W. Vaughan.
  The Journal of Physiology. September 15, 2016
  Key points The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. Abstract The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress‐induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)‐3,5‐dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+, and when release probability was reduced by lowering Ca2+. However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+. Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+. The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+. Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.

  September 15, 2016   doi: 10.1113/JP272292   open full text
• Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age‐related declines in voluntary physical activity.
  Gregory N. Ruegsegger, Ryan G. Toedebusch, Thomas E. Childs, Kolter B. Grigsby, Frank W. Booth.
  The Journal of Physiology. September 15, 2016
  Key points Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases. The nucleus accumbens (the pleasure and reward ‘hub’ in the brain) influences wheel running behaviour in rodents. RNA‐sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age‐related reductions in wheel running. Upon completion of follow‐up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age‐related changes in voluntary running. Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running. The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age‐dependent reductions in the motivation to be physically active. Abstract Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA‐sequencing was used to interrogate transcriptomic changes between 8‐ and 14‐week‐old wheel running rats, and select transcripts were later analysed by quantitative RT‐PCR in age‐matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP‐mediated signalling, dopamine‐ and cAMP‐regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8‐ vs. 14‐week‐old rats. In depth analysis of these networks showed significant (∼20–30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin‐dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14‐ vs. 8‐week‐old sedentary rats (P = 0.03). Intriguingly, intra‐NAc injection of the Cdk5 inhibitor roscovitine, dose‐dependently decreased wheel running. Collectively, these experiments suggest that an age‐dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age‐related declines in voluntary running behaviour.

  September 15, 2016   doi: 10.1113/JP272489   open full text
• In vivo matching of postsynaptic excitability with spontaneous synaptic inputs during formation of the rat calyx of Held synapse.
  Martijn C. Sierksma, Milly S. Tedja, J. Gerard G. Borst.
  The Journal of Physiology. September 15, 2016
  Key points Neurons in the medial nucleus of the trapezoid body of anaesthetized rats of postnatal day (P)2–6 showed burst firing with a preferred interval of about 100 ms, which was stable, and a second preferred interval of 5–30 ms, which shortened during development. In 3 out of 132 cases, evidence for the presence of two large inputs was found. In vivo whole‐cell recordings revealed that the excitability of the principal neuron and the size of its largest synaptic inputs were developmentally matched. At P2–4, action potentials were triggered by barrages of small synaptic events that summated to plateau potentials, while at later stages firing depended on a single, large and often prespike‐associated input, which is probably the nascent calyx of Held. Simulations with a Hodgkin–Huxley‐like model, which was based on fits of the intrinsic postsynaptic properties, suggested an essential role for the low‐threshold potassium conductance in this transition. Abstract In the adult, principal neurons of the medial nucleus of the trapezoid body (MNTB) are typically contacted by a single, giant terminal called the calyx of Held, whereas during early development a principal neuron receives inputs from many axons. How these changes in innervation impact the postsynaptic activity has not yet been studied in vivo. We therefore recorded spontaneous inputs and intrinsic properties of principal neurons in anaesthetized rat pups during the developmental period in which the calyx forms. A characteristic bursting pattern could already be observed at postnatal day (P)2, before formation of the calyx. At this age, action potentials (APs) were triggered by barrages of summating EPSPs causing plateau depolarizations. In contrast, at P5, a single EPSP reliably triggered APs, resulting in a close match between pre‐ and postsynaptic firing. Postsynaptic excitability and the size of the largest synaptic events were developmentally matched. The developmental changes in intrinsic properties were estimated by fitting in vivo current injections to a Hodgkin–Huxley‐type model of the principal neuron. Our simulations indicated that the developmental increases in Ih, low‐threshold K+ channels and leak currents contributed to the reduction in postsynaptic excitability, but that low‐threshold K+ channels specifically functioned as a dampening influence in the near‐threshold range, thus precluding small inputs from triggering APs. Together, these coincident changes help to propagate bursting activity along the auditory brainstem, and are essential steps towards establishing the relay function of the calyx of Held synapse.

  September 15, 2016   doi: 10.1113/JP272780   open full text
• Progressive impairment of cerebellar mGluR signalling and its therapeutic potential for cerebellar ataxia in spinocerebellar ataxia type 1 model mice.
  Anton N. Shuvaev, Nobutake Hosoi, Yamato Sato, Dai Yanagihara, Hirokazu Hirai.
  The Journal of Physiology. September 15, 2016
  Key points Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by a gene defect, leading to movement disorder such as cerebellar ataxia. It remains largely unknown which functional defect contributes to the cerebellar ataxic phenotype in SCA1. In this study, we report progressive dysfunction of metabotropic glutamate receptor (mGluR) signalling, which leads to smaller slow synaptic responses, reduced dendritic Ca2+ signals and impaired synaptic plasticity at cerebellar synapses, in the early disease stage of SCA1 model mice. We also show that enhancement of mGluR signalling by a clinically available drug, baclofen, leads to improvement of motor performance in SCA1 mice. SCA1 is an incurable disease with no effective treatment, and our results may provide mechanistic grounds for targeting mGluRs and a novel drug therapy with baclofen to treat SCA1 patients in the future. Abstract Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease that presents with cerebellar ataxia and motor learning defects. Previous studies have indicated that the pathology of SCA1, as well as other ataxic diseases, is related to signalling pathways mediated by the metabotropic glutamate receptor type 1 (mGluR1), which is indispensable for proper motor coordination and learning. However, the functional contribution of mGluR signalling to SCA1 pathology is unclear. In the present study, we show that SCA1 model mice develop a functional impairment of mGluR signalling which mediates slow synaptic responses, dendritic Ca2+ signals, and short‐ and long‐term synaptic plasticity at parallel fibre (PF)–Purkinje cell (PC) synapses in a progressive manner from the early disease stage (5 postnatal weeks) prior to PC death. Notably, impairment of mGluR‐mediated dendritic Ca2+ signals linearly correlated with a reduction of PC capacitance (cell surface area) in disease progression. Enhancement of mGluR signalling by baclofen, a clinically available GABAB receptor agonist, led to an improvement of motor performance in SCA1 mice and the improvement lasted ∼1 week after a single application of baclofen. Moreover, the restoration of motor performance in baclofen‐treated SCA1 mice matched the functional recovery of mGluR‐mediated slow synaptic currents and mGluR‐dependent short‐ and long‐term synaptic plasticity. These results suggest that impairment of synaptic mGluR cascades is one of the important contributing factors to cerebellar ataxia in early and middle stages of SCA1 pathology, and that modulation of mGluR signalling by baclofen or other clinical interventions may be therapeutic targets to treat SCA1.

  September 15, 2016   doi: 10.1113/JP272950   open full text
• Extracellular K+ rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl−‐dependent and independent mechanisms.
  David Penton, Jan Czogalla, Agnieszka Wengi, Nina Himmerkus, Dominique Loffing‐Cueni, Monique Carrel, Renuga Devi Rajaram, Olivier Staub, Markus Bleich, Frank Schweda, Johannes Loffing.
  The Journal of Physiology. September 11, 2016
  Key points High dietary potassium (K+) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K+, similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl− fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl−‐dependent. The Cl−‐dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl− independent pathway may include additional signalling cascades. Abstract A high dietary potassium (K+) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K+]ex) modulate NCC phosphorylation via a Cl−‐dependent modulation of the with no lysine (K) kinases (WNK)‐STE20/SPS‐1‐44 related proline‐alanine‐rich protein kinase (SPAK)/oxidative stress‐related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K+]ex, with the most prominent effects occurring around physiological plasma [K+]. Cellular Cl− conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K+]ex. However, NCC dephosphorylation triggered by high [K+]ex is neither blocked by removing extracellular Cl−, nor by the Cl− channel blocker 4,4′‐diisothiocyano‐2,2′‐stilbenedisulphonic acid. The response to [K+]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K+]ex directly and rapidly controls NCC phosphorylation by Cl−‐dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.

  September 11, 2016   doi: 10.1113/JP272504   open full text
• Effects of selective carotid body stimulation with adenosine in conscious humans.
  Stanislaw Tubek, Piotr Niewinski, Krzysztof Reczuch, Dariusz Janczak, Artur Rucinski, Bartlomiej Paleczny, Zoar J. Engelman, Waldemar Banasiak, Julian F. R. Paton, Piotr Ponikowski.
  The Journal of Physiology. September 11, 2016
  Key points In humans, excitation of peripheral chemoreceptors with systemic hypoxia causes hyperventilation, hypertension and tachycardia. However, the contribution of particular chemosensory areas (carotid vs. aortic bodies) to this response is unclear. We showed that selective stimulation of the carotid body by the injection of adenosine into the carotid artery causes a dose‐dependent increase in minute ventilation and blood pressure with a concomitant decrease in heart rate in conscious humans. The ventilatory response was abolished and the haemodynamic response was diminished following carotid body ablation. We found that the magnitude of adenosine evoked responses in minute ventilation and blood pressure was analogous to the responses evoked by hypoxia. By contrast, opposing heart rate responses were evoked by adenosine (bradycardia) vs. hypoxia (tachycardia). Intra‐carotid adenosine administration may provide a novel method for perioperative assessment of the effectiveness of carotid body ablation, which has been recently proposed as a treatment strategy for sympathetically‐mediated diseases. Abstract Stimulation of peripheral chemoreceptors by acute hypoxia causes an increase in minute ventilation (VI), heart rate (HR) and arterial blood pressure (BP). However, the contribution of particular chemosensory areas, such as carotid (CB) vs. aortic bodies, to this response in humans remains unknown. We performed a blinded, randomized and placebo‐controlled study in 11 conscious patients (nine men, two women) undergoing common carotid artery angiography. Doses of adenosine ranging from 4 to 512 μg or placebo solution of a matching volume were administered in randomized order via a diagnostic catheter located in a common carotid artery. Separately, ventilatory and haemodynamic responses to systemic hypoxia were also assessed. Direct excitation of a CB with intra‐arterial adenosine increased VI, systolic BP, mean BP and decreased HR. No responses in these variables were seen after injections of placebo. The magnitude of the ventilatory and haemodynamic responses depended on both the dose of adenosine used and on the level of chemosensitivity as determined by the ventilatory response to hypoxia. Percutaneous radiofrequency ablation of the CB abolished the adenosine evoked respiratory response and partially depressed the cardiovascular response in one participant. The results of the present study confirm the excitatory role of purines in CB physiology in humans and suggest that adenosine may be used for selective stimulation and assessment of CB activity. The trial is registered at ClinicalTrials.gov NCT01939912.

  September 11, 2016   doi: 10.1113/JP272109   open full text
• Modular modelling with Physiome standards.
  Michael T. Cooling, David P. Nickerson, Poul M. F. Nielsen, Peter J. Hunter.
  The Journal of Physiology. August 29, 2016
  Key points The complexity of computational models is increasing, supported by research in modelling tools and frameworks. But relatively little thought has gone into design principles for complex models. We propose a set of design principles for complex model construction with the Physiome standard modelling protocol CellML. By following the principles, models are generated that are extensible and are themselves suitable for reuse in larger models of increasing complexity. We illustrate these principles with examples including an architectural prototype linking, for the first time, electrophysiology, thermodynamically compliant metabolism, signal transduction, gene regulation and synthetic biology. The design principles complement other Physiome research projects, facilitating the application of virtual experiment protocols and model analysis techniques to assist the modelling community in creating libraries of composable, characterised and simulatable quantitative descriptions of physiology. Abstract The ability to produce and customise complex computational models has great potential to have a positive impact on human health. As the field develops towards whole‐cell models and linking such models in multi‐scale frameworks to encompass tissue, organ, or organism levels, reuse of previous modelling efforts will become increasingly necessary. Any modelling group wishing to reuse existing computational models as modules for their own work faces many challenges in the context of construction, storage, retrieval, documentation and analysis of such modules. Physiome standards, frameworks and tools seek to address several of these challenges, especially for models expressed in the modular protocol CellML. Aside from providing a general ability to produce modules, there has been relatively little research work on architectural principles of CellML models that will enable reuse at larger scales. To complement and support the existing tools and frameworks, we develop a set of principles to address this consideration. The principles are illustrated with examples that couple electrophysiology, signalling, metabolism, gene regulation and synthetic biology, together forming an architectural prototype for whole‐cell modelling (including human intervention) in CellML. Such models illustrate how testable units of quantitative biophysical simulation can be constructed. Finally, future relationships between modular models so constructed and Physiome frameworks and tools are discussed, with particular reference to how such frameworks and tools can in turn be extended to complement and gain more benefit from the results of applying the principles.

  August 29, 2016   doi: 10.1113/JP272633   open full text
• Exercise training preserves vagal preganglionic neurones and restores parasympathetic tonus in heart failure.
  Marcelo H. A. Ichige, Carla R. Santos, Camila P. Jordão, Alexandre Ceroni, Carlos E. Negrão, Lisete C. Michelini.
  The Journal of Physiology. August 29, 2016
  Key points Heart Failure (HF) is accompanied by reduced ventricular function, activation of compensatory neurohormonal mechanisms and marked autonomic dysfunction characterized by exaggerated sympathoexcitation and reduced parasympathetic activity. With 6 weeks of exercise training, HF‐related loss of choline acetyltransferase (ChAT)‐positive vagal preganglionic neurones is avoided, restoring the parasympathetic tonus to the heart, and the immunoreactivity of dopamine β‐hydroxylase‐positive premotor neurones that drive sympathetic outflow to the heart is reduced. Training‐induced correction of autonomic dysfunction occurs even with the persistence of abnormal ventricular function. Strong positive correlation between improved parasympathetic tonus to the heart and increased ChAT immunoreactivity in vagal preganglionic neurones after training indicates this is a crucial mechanism to restore autonomic function in heart failure. Abstract Exercise training is an efficient tool to attenuate sympathoexcitation, a hallmark of heart failure (HF). Although sympathetic modulation in HF is widely studied, information regarding parasympathetic control is lacking. We examined the combined effects of sympathetic and vagal tonus to the heart in sedentary (Sed) and exercise trained (ET) HF rats and the contribution of respective premotor and preganglionic neurones. Wistar rats submitted to coronary artery ligation or sham surgery were assigned to training or sedentary protocols for 6 weeks. After haemodynamic, autonomic tonus (atropine and atenolol i.v.) and ventricular function determinations, brains were collected for immunoreactivity assays (choline acetyltransferase, ChATir; dopamine β‐hydroxylase, DBHir) and neuronal counting in the dorsal motor nucleus of vagus (DMV), nucleus ambiguus (NA) and rostroventrolateral medulla (RVLM). HF‐Sed vs. SHAM‐Sed exhibited decreased exercise capacity, reduced ejection fraction, increased left ventricle end diastolic pressure, smaller positive and negative dP/dt, decreased intrinsic heart rate (IHR), lower parasympathetic and higher sympathetic tonus, reduced preganglionic vagal neurones and ChATir in the DMV/NA, and increased RVLM DBHir. Training increased treadmill performance, normalized autonomic tonus and IHR, restored the number of DMV and NA neurones and corrected ChATir without affecting ventricular function. There were strong positive correlations between parasympathetic tonus and ChATir in NA and DMV. RVLM DBHir was also normalized by training, but there was no change in neurone number and no correlation with sympathetic tonus. Training‐induced preservation of preganglionic vagal neurones is crucial to normalize parasympathetic activity and restore autonomic balance to the heart even in the persistence of cardiac dysfunction.

  August 29, 2016   doi: 10.1113/JP272730   open full text
• Fish oil prevents changes induced by a high‐fat diet on metabolism and adipokine secretion in mice subcutaneous and visceral adipocytes.
  Roberta D. C. da Cunha Sá, Amanda R. Crisma, Maysa M. Cruz, Amanda R. Martins, Laureane N. Masi, Catia L. do Amaral, R. Curi, Maria I. C. Alonso‐Vale.
  The Journal of Physiology. August 25, 2016
  Key points Fish oil (FO), rich in omega‐3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities. The effects of FO on adipocytes from different adipose tissue depots in high‐fat (HF) diet induced obese mice have not been uninvestigated. This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet‐induced obese mice. Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity. HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor‐α and interleukin‐6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro‐inflammatory cytokines) adipose depots. Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet. Abstract In the present study, we investigated the effect of fish oil (FO) on metabolism and adipokine production by adipocytes from s.c. (inguinal; ING) and visceral (retroperitoneal; RP) white adipose depots in high‐fat (HF) diet‐induced obese mice. Mice were divided into CO (control diet), CO+FO, HF and HF+FO groups. The HF group presented higher body weight, glucose intolerance, insulin resistance, higher plasma total and low‐density lipoprotein cholesterol levels, and greater weights of ING and RP adipose depots accompanied by hypertrophy of the adipocytes. FO exerted anti‐obesogenic effects associated with beneficial effects on dyslipidaemia and insulin resistance in mice fed a HF diet (HF+FO group). HF raised RP adipocyte lipolysis and the production of pro‐inflammatory cytokines and reduced de novo synthesis of fatty acids, whereas, in ING adipocytes, it decreased glucose uptake and adiponectin secretion but did not change lipolysis. Therefore, the adipose depots play different roles in HF diet‐induced insulin resistance according to their location in the body. Concerning cytokine secretion, adipocytes per se in addition to white adopise tissue infiltrated leukocytes have to be considered in the aetiology of the comorbidities associated with obesity. Evidence is presented showing that previous and concomitant administration of FO can prevent changes in metabolism and the secretion of hormones and cytokines in ING and RP adipocytes induced by HF.

  August 25, 2016   doi: 10.1113/JP272541   open full text
• A novel enteric neuron–glia coculture system reveals the role of glia in neuronal development.
  Catherine Berre‐Scoul, Julien Chevalier, Elena Oleynikova, François Cossais, Sophie Talon, Michel Neunlist, Hélène Boudin.
  The Journal of Physiology. August 18, 2016
  Key points Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron‐enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic‐ and glial cell line‐derived neurotrophic factor‐dependent pathways. Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation. Abstract In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron‐enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial‐derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y1 receptor‐ and glial cell line‐derived neurotrophic factor‐dependent pathways. Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation.

  August 18, 2016   doi: 10.1113/JP271989   open full text
• Thyroid hormone activation by type 2 deiodinase mediates exercise‐induced peroxisome proliferator‐activated receptor‐γ coactivator‐1α expression in skeletal muscle.
  Barbara M. L. C. Bocco, Ruy A. N. Louzada, Diego H. S. Silvestre, Maria C. S. Santos, Elena Anne‐Palmer, Igor F. Rangel, Sherine Abdalla, Andrea C. Ferreira, Miriam O. Ribeiro, Balázs Gereben, Denise P. Carvalho, Antonio C. Bianco, João P. Werneck‐de‐Castro.
  The Journal of Physiology. August 18, 2016
  Key points In skeletal muscle, physical exercise and thyroid hormone mediate the peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1a) expression that is crucial to skeletal muscle mitochondrial function. The expression of type 2 deiodinase (D2), which activates thyroid hormone in skeletal muscle is upregulated by acute treadmill exercise through a β‐adrenergic receptor‐dependent mechanism. Pharmacological block of D2 or disruption of the Dio2 gene in skeletal muscle fibres impaired acute exercise‐induced PGC‐1a expression. Dio2 disruption also impaired muscle PGC‐1a expression and mitochondrial citrate synthase activity in chronically exercised mice. Abstract Thyroid hormone promotes expression of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1a), which mediates mitochondrial biogenesis and oxidative capacity in skeletal muscle (SKM). Skeletal myocytes express the type 2 deiodinase (D2), which generates 3,5,3′‐triiodothyronine (T3), the active thyroid hormone. To test whether D2‐generated T3 plays a role in exercise‐induced PGC‐1a expression, male rats and mice with SKM‐specific Dio2 inactivation (SKM‐D2KO or MYF5‐D2KO) were studied. An acute treadmill exercise session (20 min at 70–75% of maximal aerobic capacity) increased D2 expression/activity (1.5‐ to 2.7‐fold) as well as PGC‐1a mRNA levels (1.5‐ to 5‐fold) in rat soleus muscle and white gastrocnemius muscle and in mouse soleus muscle, which was prevented by pretreatment with 1 mg (100 g body weight)−1 propranolol or 6 mg (100 g body weight)−1 iopanoic acid (5.9‐ vs. 2.8‐fold; P < 0.05), which blocks D2 activity . In the SKM‐D2KO mice, acute treadmill exercise failed to induce PGC‐1a fully in soleus muscle (1.9‐ vs. 2.8‐fold; P < 0.05), and in primary SKM‐D2KO myocytes there was only a limited PGC‐1a response to 1 μm forskolin (2.2‐ vs. 1.3‐fold; P < 0.05). Chronic exercise training (6 weeks) increased soleus muscle PGC‐1a mRNA levels (∼25%) and the mitochondrial enzyme citrate synthase (∼20%). In contrast, PGC‐1a expression did not change and citrate synthase decreased by ∼30% in SKM‐D2KO mice. The soleus muscle PGC‐1a response to chronic exercise was also blunted in MYF5‐D2KO mice. In conclusion, acute treadmill exercise increases SKM D2 expression through a β‐adrenergic receptor‐dependent mechanism. The accelerated conversion of T4 to T3 within myocytes mediates part of the PGC‐1a induction by treadmill exercise and its downstream effects on mitochondrial function.

  August 18, 2016   doi: 10.1113/JP272440   open full text
• Exogenous and endogenous angiotensin‐II decrease renal cortical oxygen tension in conscious rats by limiting renal blood flow.
  Tonja W. Emans, Ben J. Janssen, Maximilian I. Pinkham, Connie P. C. Ow, Roger G. Evans, Jaap A. Joles, Simon C. Malpas, C. T. Paul Krediet, Maarten P. Koeners.
  The Journal of Physiology. August 18, 2016
  Key points Our understanding of the mechanisms underlying the role of hypoxia in the initiation and progression of renal disease remains rudimentary. We have developed a method that allows wireless measurement of renal tissue oxygen tension in unrestrained rats. This method provides stable and continuous measurements of cortical tissue oxygen tension (PO2) for more than 2 weeks and can reproducibly detect acute changes in cortical oxygenation. Exogenous angiotensin‐II reduced renal cortical tissue PO2 more than equi‐pressor doses of phenylephrine, probably because it reduced renal oxygen delivery more than did phenylephrine. Activation of the endogenous renin–angiotensin system in transgenic Cyp1a1Ren2 rats reduced cortical tissue PO2; in this model renal hypoxia precedes the development of structural pathology and can be reversed acutely by an angiotensin‐II receptor type 1 antagonist. Angiotensin‐II promotes renal hypoxia, which may in turn contribute to its pathological effects during development of chronic kidney disease. Abstract We hypothesised that both exogenous and endogenous angiotensin‐II (AngII) can decrease the partial pressure of oxygen (PO2) in the renal cortex of unrestrained rats, which might in turn contribute to the progression of chronic kidney disease. Rats were instrumented with telemeters equipped with a carbon paste electrode for continuous measurement of renal cortical tissue PO2. The method reproducibly detected acute changes in cortical oxygenation induced by systemic hyperoxia and hypoxia. In conscious rats, renal cortical PO2 was dose‐dependently reduced by intravenous AngII. Reductions in PO2 were significantly greater than those induced by equi‐pressor doses of phenylephrine. In anaesthetised rats, renal oxygen consumption was not affected, and filtration fraction was increased only in the AngII infused animals. Oxygen delivery decreased by 50% after infusion of AngII and renal blood flow (RBF) fell by 3.3 ml min−1. Equi‐pressor infusion of phenylephrine did not significantly reduce RBF or renal oxygen delivery. Activation of the endogenous renin–angiotensin system in Cyp1a1Ren2 transgenic rats reduced cortical tissue PO2. This could be reversed within minutes by pharmacological angiotensin‐II receptor type 1 (AT1R) blockade. Thus AngII is an important modulator of renal cortical oxygenation via AT1 receptors. AngII had a greater influence on cortical oxygenation than did phenylephrine. This phenomenon appears to be attributable to the profound impact of AngII on renal oxygen delivery. We conclude that the ability of AngII to promote renal cortical hypoxia may contribute to its influence on initiation and progression of chronic kidney disease.

  August 18, 2016   doi: 10.1113/JP270731   open full text
• The renal cortical collecting duct: a secreting epithelium?
  Luciana Morla, Alain Doucet, Christine Lamouroux, Gilles Crambert, Aurélie Edwards.
  The Journal of Physiology. August 13, 2016
  Key points The cortical collecting duct (CCD) plays an essential role in sodium homeostasis by fine‐tuning the amount of sodium that is excreted in the urine. Ex vivo, the microperfused CCD reabsorbs sodium in the absence of lumen‐to‐bath concentration gradients. In the present study, we show that, in the presence of physiological lumen‐to‐bath concentration gradients, and in the absence of endocrine, paracrine and neural regulation, the mouse CCD secretes sodium, which represents a paradigm shift. This secretion occurs via the paracellular route, as well as a transcellular pathway that is energized by apical H+/K+‐ATPase type 2 pumps operating as Na+/K+ exchangers. The newly identified transcellular secretory pathway represents a physiological target for the regulation of sodium handling and for anti‐hypertensive therapeutic agents. Abstract In vitro microperfusion experiments have demonstrated that cortical collecting ducts (CCDs) reabsorb sodium via principal and type B intercalated cells under sodium‐depleted conditions and thereby contribute to sodium and blood pressure homeostasis. However, these experiments were performed in the absence of the transepithelial ion concentration gradients that prevail in vivo and determine paracellular transport. The present study aimed to characterize Na+, K+ and Cl− fluxes in the mouse CCD in the presence of physiological transepithelial concentration gradients. For this purpose, we combined in vitro measurements of ion fluxes across microperfused CCDs of sodium‐depleted mice with the predictions of a mathematical model. When NaCl transport was inhibited in all cells, CCDs secreted Na+ and reabsorbed K+; Cl− transport was negligible. Removing inhibitors of type A and B intercalated cells increased Na+ secretion in wild‐type (WT) mice but not in H+/K+‐ATPase type 2 (HKA2) knockout mice. Further inhibition of basolateral NaCl entry via the Na+‐K+‐2Cl− cotransporter in type A intercalated cells reduced Na+ secretion in WT mice to the levels observed in HKA2−/− mice. With no inhibitors, WT mouse CCDs still secreted Na+ and reabsorbed K+. In vivo, HKA2−/− mice excreted less Na+ than WT mice after switching to a high‐salt diet. Taken together, our results indicate that type A intercalated cells secrete Na+ via basolateral Na+‐K+‐2Cl− cotransporters in tandem with apical HKA2 pumps. They also suggest that the CCD can mediate overall Na+ secretion, and that its ability to reabsorb NaCl in vivo depends on the presence of acute regulatory factors.

  August 13, 2016   doi: 10.1113/JP272877   open full text
• Left–right coordination from simple to extreme conditions during split‐belt locomotion in the chronic spinal adult cat.
  Alain Frigon, Étienne Desrochers, Yann Thibaudier, Marie‐France Hurteau, Charline Dambreville.
  The Journal of Physiology. August 13, 2016
  Key points Coordination between the left and right sides is essential for dynamic stability during locomotion. The immature or neonatal mammalian spinal cord can adjust to differences in speed between the left and right sides during split‐belt locomotion by taking more steps on the fast side. We show that the adult mammalian spinal cord can also adjust its output so that the fast side can take more steps. During split‐belt locomotion, only certain parts of the cycle are modified to adjust left–right coordination, primarily those associated with swing onset. When the fast limb takes more steps than the slow limb, strong left–right interactions persist. Therefore, the adult mammalian spinal cord has a remarkable adaptive capacity for left–right coordination, from simple to extreme conditions. Abstract Although left–right coordination is essential for locomotion, its control is poorly understood, particularly in adult mammals. To investigate the spinal control of left–right coordination, a spinal transection was performed in six adult cats that were then trained to recover hindlimb locomotion. Spinal cats performed tied‐belt locomotion from 0.1 to 1.0 m s−1 and split‐belt locomotion with low to high (1:1.25–10) slow/fast speed ratios. With the left hindlimb stepping at 0.1 m s−1 and the right hindlimb stepping from 0.2 to 1.0 m s−1, 1:1, 1:2, 1:3, 1:4 and 1:5 left–right step relationships could appear. The appearance of 1:2+ relationships was not linearly dependent on the difference in speed between the slow and fast belts. The last step taken by the fast hindlimb displayed longer cycle, stance and swing durations and increased extensor activity, as the slow limb transitioned to swing. During split‐belt locomotion with 1:1, 1:2 and 1:3 relationships, the timing of stance onset of the fast limb relative to the slow limb and placement of both limbs at contact were invariant with increasing slow/fast speed ratios. In contrast, the timing of stance onset of the slow limb relative to the fast limb and the placement of both limbs at swing onset were modulated with slow/fast speed ratios. Thus, left–right coordination is adjusted by modifying specific parts of the cycle. Results highlight the remarkable adaptive capacity of the adult mammalian spinal cord, providing insight into spinal mechanisms and sensory signals regulating left–right coordination.

  August 13, 2016   doi: 10.1113/JP272740   open full text
• Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces.
  Fan Wang, Kaitlyn Knutson, Constanza Alcaino, David R. Linden, Simon J. Gibbons, Purna Kashyap, Madhusudan Grover, Richard Oeckler, Philip A. Gottlieb, Hui Joyce Li, Andrew B. Leiter, Gianrico Farrugia, Arthur Beyder.
  The Journal of Physiology. August 13, 2016
  Key points The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown. In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells. Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion. Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction. Abstract The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5‐hydroxytryptamine; 5‐HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5‐HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5‐HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically‐sensitive inward non‐selective cation current characteristic of Piezo2. Both inward currents and 5‐HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D‐GsMTx4). Jejunum mucosal pressure increased 5‐HT release and short‐circuit current via submucosal 5‐HT3 and 5‐HT4 receptors. Pressure‐induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D‐GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5‐HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.

  August 13, 2016   doi: 10.1113/JP272718   open full text
• The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype: direct demonstration of phenotypic modulation.
  Mairi E. Sandison, John Dempster, John G. McCarron.
  The Journal of Physiology. August 13, 2016
  Key points Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time‐lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly‐elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non‐contractile, migratory phenotype with phagocytic capacity that may act as a macrophage‐like cell. Abstract Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a ‘synthetic’ migratory phenotype, so‐called phenotypic modulation, whilst plaque macrophages are thought to derive from blood‐borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long‐term, high‐resolution, time‐lapse microscopy to track the fate of unambiguously identified, fully‐differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1–3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell‐cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage‐like behaviour was confirmed by their internalisation of 1 μm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low‐density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling.

  August 13, 2016   doi: 10.1113/JP272729   open full text
• Linear transformation of the encoding mechanism for light intensity underlies the paradoxical enhancement of cortical visual responses by sevoflurane.
  Alessandro Arena, Jacopo Lamanna, Marco Gemma, Maddalena Ripamonti, Giuliano Ravasio, Vincenzo Zimarino, Assunta Vitis, Luigi Beretta, Antonio Malgaroli.
  The Journal of Physiology. August 13, 2016
  Key points The mechanisms of action of anaesthetics on the living brain are still poorly understood. In this respect, the analysis of the differential effects of anaesthetics on spontaneous and sensory‐evoked cortical activity might provide important and novel cues. Here we show that the anaesthetic sevoflurane strongly silences the brain but potentiates in a dose‐ and frequency‐dependent manner the cortical visual response. Such enhancement arises from a linear scaling by sevoflurane of the power‐law relation between light intensity and the cortical response. The fingerprint of sevoflurane action suggests that circuit silencing can boost linearly synaptic responsiveness presumably by scaling the number of responding units and/or their correlation following a sensory stimulation. Abstract General anaesthetics, which are expected to silence brain activity, often spare sensory responses. To evaluate differential effects of anaesthetics on spontaneous and sensory‐evoked cortical activity, we characterized their modulation by sevoflurane and propofol. Power spectra and the bust‐suppression ratio from EEG data were used to evaluate anaesthesia depth. ON and OFF cortical responses were elicited by light pulses of variable intensity, duration and frequency, during light and deep states of anaesthesia. Both anaesthetics reduced spontaneous cortical activity but sevoflurane greatly enhanced while propofol diminished the ON visual response. Interestingly, the large potentiation of the ON visual response by sevoflurane was found to represent a linear scaling of the encoding mechanism for light intensity. To the contrary, the OFF cortical visual response was depressed by both anaesthetics. The selective depression of the OFF component by sevoflurane could be converted into a robust potentiation by the pharmacological blockade of the ON pathway, suggesting that the temporal order of ON and OFF responses leads to a depression of the latter. This hypothesis agrees with the finding that the enhancement of the ON response was converted into a depression by increasing the frequency of light‐pulse stimulation from 0.1 to 1 Hz. Overall, our results support the view that inactivity‐dependent modulation of cortical circuits produces an increase in their responsiveness. Among the implications of our findings, the silencing of cortical circuits can boost linearly the cortical responsiveness but with negative impact on their frequency transfer and with a loss of the information content of the sensory signal.

  August 13, 2016   doi: 10.1113/JP272215   open full text
• Differential impact of acute and prolonged cAMP agonist exposure on protein kinase A activation and human myometrium contractile activity.
  Pei F. Lai, Rachel M. Tribe, Mark R. Johnson.
  The Journal of Physiology. August 08, 2016
  Key points Over 15 million babies are born prematurely each year with approximately 1 million of these babies dying as a direct result of preterm delivery. β2‐Adrenoreceptor agonists that act via cAMP can reduce uterine contractions to delay preterm labour, but their ability to repress uterine contractions lasts ≤ 48 h and their use does not improve neonatal outcomes. Previous research has suggested that cAMP inhibits myometrial contractions via protein kinase A (PKA) activation, but this has yet to be demonstrated with PKA‐specific agonists. We investigated the role of PKA in mediating cAMP‐induced human myometrial relaxation, and the impact of prolonged cAMP elevation on myometrial contractility. Our findings suggest that PKA is not the sole mediator of cAMP‐induced myometrial relaxation and that prolonged prophylactic elevation of cAMP alone is unlikely to prevent preterm labour (PTL). Abstract Acute cAMP elevation inhibits myometrial contractility, but the mechanisms responsible are not fully elucidated and the long‐term effects are uncertain. Both need to be defined in pregnant human myometrium before the therapeutic potential of cAMP‐elevating agents in the prevention of preterm labour can be realised. In the present study, we tested the hypotheses that PKA activity is necessary for cAMP‐induced myometrial relaxation, and that prolonged cAMP elevation can prevent myometrial contractions. Myometrial tissues obtained from term, pre‐labour elective Caesarean sections were exposed to receptor‐independent cAMP agonists to determine the relationship between myometrial contractility (spontaneous and oxytocin‐induced), PKA activity, HSP20 phosphorylation and expression of contraction‐associated and cAMP signalling proteins. Acute (1 h) application of cAMP agonists promoted myometrial relaxation, but this was weakly related to PKA activation. A PKA‐specific activator, 6‐Bnz‐cAMP, increased PKA activity (6.8 ± 2.0 mean fold versus vehicle; P = 0.0313) without inducing myometrial relaxation. Spontaneous myometrial contractility declined after 24 h but was less marked when tissues were constantly exposed to cAMP agonists, especially for 8‐bromo‐cAMP (4.3 ± 1.2 mean fold versus vehicle; P = 0.0043); this was associated with changes to calponin, cofilin and HSP20 phosphorylated/total protein levels. Oxytocin‐induced contractions were unaffected by pre‐incubation with cAMP agonists despite treatments being able to enhance PKA activity and HSP20 phosphorylation. These data suggest that cAMP‐induced myometrial relaxation is not solely dependent on PKA activity and the ability of cAMP agonists to repress myometrial contractility is lost with prolonged exposure. We conclude that cAMP agonist treatment alone may not prevent preterm labour.

  August 08, 2016   doi: 10.1113/JP272320   open full text
• Integrative properties and transfer function of cortical neurons initiating absence seizures in a rat genetic model.
  Mark S. Williams, Tristan Altwegg‐Boussac, Mario Chavez, Sarah Lecas, Séverine Mahon, Stéphane Charpier.
  The Journal of Physiology. August 08, 2016
  Key points Absence seizures are accompanied by spike‐and‐wave discharges in cortical electroencephalograms. These complex paroxysmal activities, affecting the thalamocortical networks, profoundly alter cognitive performances and preclude conscious perception. Here, using a well‐recognized genetic model of absence epilepsy, we investigated in vivo how information processing was impaired in the ictogenic neurons, i.e. the population of cortical neurons responsible for seizure initiation. In between seizures, ictogenic neurons were more prone to generate bursting activity and their firing response to weak depolarizing events was considerably facilitated compared to control neurons. In the course of seizures, information processing became unstable in ictogenic cells, alternating between an increased and a decreased responsiveness to excitatory inputs, depending on the spike and wave patterns. The state‐dependent modulation in the excitability of ictogenic neurons affects their inter‐seizure transfer function and their time‐to‐time responsiveness to incoming inputs during absences. Abstract Epileptic seizures result from aberrant cellular and/or synaptic properties that can alter the capacity of neurons to integrate and relay information. During absence seizures, spike‐and‐wave discharges (SWDs) interfere with incoming sensory inputs and preclude conscious experience. The Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a well‐established animal model of absence epilepsy, allows exploration of the cellular basis of this impaired information processing. Here, by combining in vivo electrocorticographic and intracellular recordings from GAERS and control animals, we investigated how the pro‐ictogenic properties of seizure‐initiating cortical neurons modify their integrative properties and input–output operation during inter‐ictal periods and during the spike (S‐) and wave (W‐) cortical patterns alternating during seizures. In addition to a sustained depolarization and an excessive firing rate in between seizures, ictogenic neurons exhibited a pronounced hyperpolarization‐activated depolarization compared to homotypic control neurons. Firing frequency versus injected current relations indicated an increased sensitivity of GAERS cells to weak excitatory inputs, without modifications in the trial‐to‐trial variability of current‐induced firing. During SWDs, the W‐component resulted in paradoxical effects in ictogenic neurons, associating an increased membrane input resistance with a reduction in the current‐evoked firing responses. Conversely, the collapse of cell membrane resistance during the S‐component was accompanied by an elevated current‐evoked firing relative to W‐sequences, which remained, however, lower compared to inter‐ictal periods. These findings show a dynamic modulation of ictogenic neurons’ intrinsic properties that may alter inter‐seizure cortical function and participate in compromising information processing in cortical networks during absences.

  August 08, 2016   doi: 10.1113/JP272162   open full text
• Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake.
  Pawel E. Ferdek, Monika A. Jakubowska, Julia V. Gerasimenko, Oleg V. Gerasimenko, Ole H. Petersen.
  The Journal of Physiology. August 08, 2016
  Key points Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas. Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored. Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells. Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells. Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Abstract Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na+‐dependent bile acid uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca2+ and significantly reduced in the absence of Na+, showing that bile‐dependent cell death was a downstream event of Ca2+ signals. Finally, combined application of TLC‐S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC‐S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells.

  August 08, 2016   doi: 10.1113/JP272774   open full text
• Serotonin controls initiation of locomotion and afferent modulation of coordination via 5‐HT7 receptors in adult rats.
  Anna M. Cabaj, Henryk Majczyński, Erika Couto, Phillip F. Gardiner, Katinka Stecina, Urszula Sławińska, Larry M. Jordan.
  The Journal of Physiology. August 08, 2016
  Key points Experiments on neonatal rodent spinal cord showed that serotonin (5‐HT), acting via 5‐HT7 receptors, is required for initiation of locomotion and for controlling the action of interneurons responsible for inter‐ and intralimb coordination, but the importance of the 5‐HT system in adult locomotion is not clear. Blockade of spinal 5‐HT7 receptors interfered with voluntary locomotion in adult rats and fictive locomotion in paralysed decerebrate rats with no afferent feedback, consistent with a requirement for activation of descending 5‐HT neurons for production of locomotion. The direct control of coordinating interneurons by 5‐HT7 receptors observed in neonatal animals was not found during fictive locomotion, revealing a developmental shift from direct control of locomotor interneurons in neonates to control of afferent input from the moving limb in adults. An understanding of the afferents controlled by 5‐HT during locomotion is required for optimal use of rehabilitation therapies involving the use of serotonergic drugs. Abstract Serotonergic pathways to the spinal cord are implicated in the control of locomotion based on studies using serotonin type 7 (5‐HT7) receptor agonists and antagonists and 5‐HT7 receptor knockout mice. Blockade of these receptors is thought to interfere with the activity of coordinating interneurons, a conclusion derived primarily from in vitro studies on isolated spinal cord of neonatal rats and mice. Developmental changes in the effects of serotonin (5‐HT) on spinal neurons have recently been described, and there is increasing data on control of sensory input by 5‐HT7 receptors on dorsal root ganglion cells and/or dorsal horn neurons, leading us to determine the effects of 5‐HT7 receptor blockade on voluntary overground locomotion and on locomotion without afferent input from the moving limb (fictive locomotion) in adult animals. Intrathecal injections of the selective 5‐HT7 antagonist SB269970 in adult intact rats suppressed locomotion by partial paralysis of hindlimbs. This occurred without a direct effect on motoneurons as revealed by an investigation of reflex activity. The antagonist disrupted intra‐ and interlimb coordination during locomotion in all intact animals but not during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR). MLR‐evoked fictive locomotion was transiently blocked, then the amplitude and frequency of rhythmic activity were reduced by SB269970, consistent with the notion that the MLR activates 5‐HT neurons, leading to excitation of central pattern generator neurons with 5‐HT7 receptors. Effects on coordination in adults required the presence of afferent input, suggesting a switch to 5‐HT7 receptor‐mediated control of sensory pathways during development.

  August 08, 2016   doi: 10.1113/JP272271   open full text
• The role of the store‐operated calcium entry channel Orai1 in cultured rat hippocampal synapse formation and plasticity.
  Eduard Korkotian, Efrat Oni‐Biton, Menahem Segal.
  The Journal of Physiology. August 08, 2016
  Key points The role of non‐synaptic calcium entry in the formation and functions of dendritic spines was studied in dissociated cultured rat hippocampal neurons. Orai1, a store‐operated calcium channel, is found in dendritic spines. Orai1 co‐localizes in dendritic spines with STIM2 under conditions of lower [Ca2+]o. Orai1 channels are associated with the formation of new dendritic spines in response to elevated [Ca2+]o. Lack of Orai1, either by transfection with a dominant negative construct or with small interfering RNA to Orai1, results in retarded dendritic spines, an increase in density of filopodia, lower synaptic connectivity and the ability to undergo plastic changes. These results highlight a novel role for Orai1 in synapse formation, maturation and plasticity. Abstract The possible role of store operated calcium entry (SOCE) through the Orai1 channel in the formation and functions of dendritic spines was studied in cultured hippocampal neurons. In calcium store‐depleted neurons, a transient elevation of extracellular calcium concentration ([Ca2+]o) caused a rise in [Ca2+]i that was mediated by activation of the SOCE. The store depletion resulted in an increase in stromal interacting molecule 2 (an endoplasmic calcium sensor) association with Orai1 in dendritic spines. The response to the rise in [Ca2+]o was larger in spines endowed with a cluster of Orai1 molecules than in spines devoid of Orai1. Transfection of neurons with a dominant negative Orai1 resulted in retarded maturation of dendritic spines, a reduction in synaptic connectivity with afferent neurons and a reduction in the ability to undergo morphological changes following induction of chemical long‐term potentiation. Similarly, small interfering RNA (siRNA)‐treated neurons had fewer mature dendritic spines, and lower rates of mEPSCs compared to scrambled control siRNA‐treated neurons. Thus, influx of calcium through Orai1 channels facilitates the maturation of dendritic spines and the formation of functional synapses in central neurons.

  August 08, 2016   doi: 10.1113/JP272645   open full text
• Electrolyte transport pathways induced in the midgut epithelium of Drosophila melanogaster larvae by commensal gut microbiota and pathogens.
  Shubha R. Shanbhag, Abraham T. Vazhappilly, Abhay Sane, Natalie M. D'Silva, Subrata Tripathi.
  The Journal of Physiology. August 04, 2016
  Key points The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions. Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non‐sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes. Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly. We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier. Abstract Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post‐embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non‐sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non‐specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non‐sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter‐enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.

  August 04, 2016   doi: 10.1113/JP272617   open full text
• Agonist‐induced sensitisation of the irritant receptor ion channel TRPA1.
  Jannis E. Meents, Michael J. M. Fischer, Peter A. McNaughton.
  The Journal of Physiology. August 04, 2016
  Key points The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium‐free environment and this sensitisation is very long lasting following agonist removal. We show that agonist‐induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist‐induced sensitisation may provide an explanation for sensitisation following long‐term exposure to harmful irritants and pollutants, particularly in the airways. Abstract The TRPA1 ion channel is expressed in nociceptive (pain‐sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non‐covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N‐terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non‐covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1.

  August 04, 2016   doi: 10.1113/JP272237   open full text
• Nicotinic receptor activation contrasts pathophysiological bursting and neurodegeneration evoked by glutamate uptake block on rat hypoglossal motoneurons.
  Silvia Corsini, Maria Tortora, Andrea Nistri.
  The Journal of Physiology. August 03, 2016
  Key points Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death. Abstract Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl‐threo‐β‐benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS‐vulnerable hypoglossal motoneurons (HMs). On 50% of patch‐clamped HMs, TBOA induced intense network bursts that were inhibited by 1–10 μm nicotine, whereas nAChR antagonists facilitated burst emergence in non‐burster cells. Furthermore, nicotine inhibited excitatory transmission and enhanced synaptic inhibition. Strong neuroprotection by nicotine prevented the HM loss observed after 4 h of TBOA exposure. This neuroprotective action was due to suppression of downstream effectors of neurotoxicity such as increased intracellular levels of reactive oxygen species, impaired energy metabolism and upregulated genes involved in endoplasmic reticulum (ER) stress. In addition, HMs surviving TBOA toxicity often expressed UDP‐glucose glycoprotein glucosyltransferase, a key element in repair of misfolded proteins: this phenomenon was absent after nicotine application, indicative of ER stress prevention. Our results suggest nAChRs to be potential targets for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.

  August 03, 2016   doi: 10.1113/JP272591   open full text
• Xanthine oxidoreductase mediates membrane docking of milk‐fat droplets but is not essential for apocrine lipid secretion.
  Jenifer Monks, Monika Dzieciatkowska, Elise S. Bales, David J. Orlicky, Richard M. Wright, James L. McManaman.
  The Journal of Physiology. August 03, 2016
  Key points Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Abstract Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane‐bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary‐specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild‐type gland but not in the knockout, suggesting that XOR mediates ‘docking’ to this membrane. Secreted milk fat globules were isolated from mouse milk of wild‐type and XOR MGKO dams, and subjected to LC‐MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

  August 03, 2016   doi: 10.1113/JP272390   open full text
• Superior mitochondrial adaptations in human skeletal muscle after interval compared to continuous single‐leg cycling matched for total work.
  Martin J. MacInnis, Evelyn Zacharewicz, Brian J. Martin, Maria E. Haikalis, Lauren E. Skelly, Mark A. Tarnopolsky, Robyn M. Murphy, Martin J. Gibala.
  The Journal of Physiology. August 03, 2016
  Key points A classic unresolved issue in human integrative physiology involves the role of exercise intensity, duration and volume in regulating skeletal muscle adaptations to training. We employed counterweighted single‐leg cycling as a unique within‐subject model to investigate the role of exercise intensity in promoting training‐induced increases in skeletal muscle mitochondrial content. Six sessions of high‐intensity interval training performed over 2 weeks elicited greater increases in citrate synthase maximal activity and mitochondrial respiration compared to moderate‐intensity continuous training matched for total work and session duration. These data suggest that exercise intensity, and/or the pattern of contraction, is an important determinant of exercise‐induced skeletal muscle remodelling in humans. Abstract We employed counterweighted single‐leg cycling as a unique model to investigate the role of exercise intensity in human skeletal muscle remodelling. Ten young active men performed unilateral graded‐exercise tests to measure single‐leg V̇O2, peak and peak power (Wpeak). Each leg was randomly assigned to complete six sessions of high‐intensity interval training (HIIT) [4 × (5 min at 65% Wpeak and 2.5 min at 20% Wpeak)] or moderate‐intensity continuous training (MICT) (30 min at 50% Wpeak), which were performed 10 min apart on each day, in an alternating order. The work performed per session was matched for MICT (143 ± 8.4 kJ) and HIIT (144 ± 8.5 kJ, P > 0.05). Post‐training, citrate synthase (CS) maximal activity (10.2 ± 0.8 vs. 8.4 ± 0.9 mmol kg protein−1 min−1) and mass‐specific [pmol O2•(s•mg wet weight)−1] oxidative phosphorylation capacities (complex I: 23.4 ± 3.2 vs. 17.1 ± 2.8; complexes I and II: 58.2 ± 7.5 vs. 42.2 ± 5.3) were greater in HIIT relative to MICT (interaction effects, P < 0.05); however, mitochondrial function [i.e. pmol O2•(s•CS maximal activity)−1] measured under various conditions was unaffected by training (P > 0.05). In whole muscle, the protein content of COXIV (24%), NDUFA9 (11%) and mitofusin 2 (MFN2) (16%) increased similarly across groups (training effects, P < 0.05). Cytochrome c oxidase subunit IV (COXIV) and NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9) were more abundant in type I than type II fibres (P < 0.05) but training did not increase the content of COXIV, NDUFA9 or MFN2 in either fibre type (P > 0.05). Single‐leg V̇O2, peak was also unaffected by training (P > 0.05). In summary, single‐leg cycling performed in an interval compared to a continuous manner elicited superior mitochondrial adaptations in human skeletal muscle despite equal total work.

  August 03, 2016   doi: 10.1113/JP272570   open full text
• A novel mutant Na+/HCO3− cotransporter NBCe1 in a case of compound‐heterozygous inheritance of proximal renal tubular acidosis.
  Evan J. Myers, Lu Yuan, Melanie A. Felmlee, Yuan‐Yuan Lin, Yan Jiang, Yu Pei, Ou Wang, Mei Li, Xiao‐Ping Xing, Aniko Marshall, Wei‐Bo Xia, Mark D. Parker.
  The Journal of Physiology. August 02, 2016
  Key points The inheritance of two defective alleles of SLC4A4, the gene that encodes the widely‐expressed electrogenic sodium bicarbonate cotransporter NBCe1, results in the bicarbonate‐wasting disease proximal renal tubular acidosis (pRTA). In the present study, we report the first case of compound‐heterozygous inheritance of pRTA (p.Arg510His/p.Gln913Arg) in an individual with low blood pH, blindness and neurological signs that resemble transient ischaemic attacks. We employ fluorescence microscopy on non‐polarized (human embryonic kidney) and polarized (Madin–Darby canine kidney) renal cell lines and electrophysiology on Xenopus oocytes to characterize the mutant transporters (R510H and Q913R). Both mutant transporters exhibit enhanced intracellular retention in renal cells, an observation that probably explains the HCO3− transport deficit in the individual. Both mutants retain a close‐to‐normal per molecule Na+/HCO3− cotransport activity in Xenopus oocytes, suggesting that they are suitable candidates for folding‐correction therapy. However, Q913R expression is uniquely associated with a depolarizing, HCO3− independent, Cl−‐conductance in oocytes that could have pathological consequences if expressed in the cells of patients. Abstract Proximal renal tubular acidosis (pRTA) is a rare, recessively‐inherited disease characterized by abnormally acidic blood, blindness, as well as below average height and weight. pRTA is typically associated with homozygous mutation of the solute carrier 4 family gene SLC4A4. SLC4A4 encodes the electrogenic sodium bicarbonate cotransporter NBCe1, a membrane protein that acts to maintain intracellular and plasma pH. We present the first description of a case of compound‐heterozygous inheritance of pRTA. The individual has inherited two mutations in NBCe1: p.Arg510His (R510H) and p.Gln913Arg (Q913R), one from each parent. In addition to the usual features of pRTA, the patient exhibits unusual signs, such as muscle spasms and fever. We have recreated these mutant transporters for expression in model systems. We find that both of the mutant proteins exhibit substantial intracellular retention when expressed in mammalian renal cell lines. When expressed in Xenopus oocytes, we find that the R510H and Q913R‐mutant NBCe1 molecules exhibit apparently normal Na+/HCO3− cotransport activity but that Q913R is associated with an unusual HCO3− independent anion‐leak. We conclude that a reduced accumulation of NBCe1 protein in the basolateral membrane of proximal‐tubule epithelia is the most probable cause of pRTA in this case. We further note that the Q913R‐associated anion‐leak could itself be pathogenic if expressed in the plasma membrane of mammalian cells, compromising the benefit of strategies aiming to enhance mutant NBCe1 accumulation in the plasma membrane.

  August 02, 2016   doi: 10.1113/JP272252   open full text
• Inhibitory masking controls the threshold sensitivity of retinal ganglion cells.
  Feng Pan, Abduqodir Toychiev, Yi Zhang, Tamas Atlasz, Hariharasubramanian Ramakrishnan, Kaushambi Roy, Béla Völgyi, Abram Akopian, Stewart A. Bloomfield.
  The Journal of Physiology. August 02, 2016
  Key points Retinal ganglion cells (RGCs) in dark‐adapted retinas show a range of threshold sensitivities spanning ∼3 log units of illuminance. Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways. The masking inhibition is subserved by GABAC receptors, probably on bipolar cell axon terminals. The GABAergic masking inhibition appears independent of dopaminergic circuitry that has been shown also to affect RGC sensitivity. The results indicate a novel mechanism whereby inhibition controls the sensitivity of different cohorts of RGCs. This can limit and thereby ensure that appropriate signals are carried centrally in scotopic conditions when sensitivity rather than acuity is crucial. Abstract The responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark‐adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions.

  August 02, 2016   doi: 10.1113/JP272267   open full text
• Secondary hyperalgesia is mediated by heat‐insensitive A‐fibre nociceptors.
  Emanuel N. den Broeke, Cédric Lenoir, André Mouraux.
  The Journal of Physiology. August 02, 2016
  Key points It is believed that secondary hyperalgesia (the increased sensitivity to mechanical nociceptive stimuli that develops after cutaneous tissue injury in the surrounding uninjured skin) is mediated by a subclass of nociceptors: the slowly adapting A‐fibre mechano‐heat nociceptors (AMH‐type I). Here we tested this hypothesis. By using intense long‐lasting heat stimuli, which are known to activate these slowly adapting AMH‐type I nociceptors, we show that the perceived intensity elicited by these stimuli is not increased in the area of secondary hyperalgesia. Moreover, we show that during an A‐fibre nerve conduction block the perception elicited by the long‐lasting heat stimuli is significantly reduced in a time window that matches the response profile of the AMH‐type I nociceptors. AMH‐type I nociceptors contribute to the perception of sustained heat, but they do not mediate secondary hyperalgesia. Therefore, we propose that secondary hyperalgesia is mediated by high threshold mechanoreceptors. Abstract Secondary hyperalgesia refers to the increase in sensitivity to mechanical nociceptive stimuli delivered outside the area of tissue injury. Previous studies have suggested that secondary hyperalgesia is mediated by a specific class of myelinated nociceptors: slowly adapting A‐fibre mechano‐ and heat‐sensitive (AMH) type I nociceptors. Here, we tested this hypothesis by examining whether long‐lasting heat stimuli, which are known to activate AMH‐type I nociceptors, elicit enhanced responses when delivered to the area of secondary hyperalgesia induced by high frequency electrical stimulation of the skin (HFS). Before and 20 min after HFS, sustained 30 s radiant heat stimuli were delivered to the area of increased mechanical pinprick sensitivity while participants continuously rated intensity of perception using an online visual analog scale (0–100 mm). After HFS, no significant enhancement of heat perception was observed in the area of increased pinprick sensitivity. To establish that myelinated nociceptors actually contribute to the perception of sustained heat, we conducted a second experiment in which sustained heat stimuli were presented before and during an A‐fibre nerve conduction block, achieved by applying a rubber band with weights which compresses the superficial radial nerve against the radius. During the block, heat perception was significantly reduced 17–33 s after the onset of the heat stimulus (before: mean = 53 mm, during: mean = 31 mm; P = 0.03), matching the response profile of AMH‐type I nociceptors. These results support the notion that AMH‐type I nociceptors contribute to the perception of sustained heat, but also show that these afferents do not mediate secondary hyperalgesia.

  August 02, 2016   doi: 10.1113/JP272599   open full text
• Parallel processing of afferent olfactory sensory information.
  Christopher E. Vaaga, Gary L. Westbrook.
  The Journal of Physiology. August 02, 2016
  Key points The functional synaptic connectivity between olfactory receptor neurons and principal cells within the olfactory bulb is not well understood. One view suggests that mitral cells, the primary output neuron of the olfactory bulb, are solely activated by feedforward excitation. Using focal, single glomerular stimulation, we demonstrate that mitral cells receive direct, monosynaptic input from olfactory receptor neurons. Compared to external tufted cells, mitral cells have a prolonged afferent‐evoked EPSC, which serves to amplify the synaptic input. The properties of presynaptic glutamate release from olfactory receptor neurons are similar between mitral and external tufted cells. Our data suggest that afferent input enters the olfactory bulb in a parallel fashion. Abstract Primary olfactory receptor neurons terminate in anatomically and functionally discrete cortical modules known as olfactory bulb glomeruli. The synaptic connectivity and postsynaptic responses of mitral and external tufted cells within the glomerulus may involve both direct and indirect components. For example, it has been suggested that sensory input to mitral cells is indirect through feedforward excitation from external tufted cells. We also observed feedforward excitation of mitral cells with weak stimulation of the olfactory nerve layer; however, focal stimulation of an axon bundle entering an individual glomerulus revealed that mitral cells receive monosynaptic afferent inputs. Although external tufted cells had a 4.1‐fold larger peak EPSC amplitude, integration of the evoked currents showed that the synaptic charge was 5‐fold larger in mitral cells, reflecting the prolonged response in mitral cells. Presynaptic afferents onto mitral and external tufted cells had similar quantal amplitude and release probability, suggesting that the larger peak EPSC in external tufted cells was the result of more synaptic contacts. The results of the present study indicate that the monosynaptic afferent input to mitral cells depends on the strength of odorant stimulation. The enhanced spiking that we observed in response to brief afferent input provides a mechanism for amplifying sensory information and contrasts with the transient response in external tufted cells. These parallel input paths may have discrete functions in processing olfactory sensory input.

  August 02, 2016   doi: 10.1113/JP272755   open full text
• N‐linked glycosylation of Kv1.2 voltage‐gated potassium channel facilitates cell surface expression and enhances the stability of internalized channels.
  Desiree A. Thayer, Shi‐Bing Yang, Yuh Nung Jan, Lily Y. Jan.
  The Journal of Physiology. August 02, 2016
  Key points Kv1.2 and related voltage‐gated potassium channels have a highly conserved N‐linked glycosylation site in the first extracellular loop, with complex glycosylation in COS‐7 cells similar to endogenous Kv1.2 glycosylation in hippocampal neurons. COS‐7 cells expressing Kv1.2 show a crucial role of this N‐linked glycosylation in the forward trafficking of Kv1.2 to the cell membrane. Although both wild‐type and non‐glycosylated mutant Kv1.2 channels that have reached the cell membrane are internalized at a comparable rate, mutant channels are degraded at a faster rate. Treatment of wild‐type Kv1.2 channels on the cell surface with glycosidase to remove sialic acids also results in the faster degradation of internalized channels. Glycosylation of Kv1.2 is important with respect to facilitating trafficking to the cell membrane and enhancing the stability of channels that have reached the cell membrane. Abstract Studies in cultured hippocampal neurons and the COS‐7 cell line demonstrate important roles for N‐linked glycosylation of Kv1.2 channels in forward trafficking and protein degradation. Kv1.2 channels can contain complex N‐linked glycans, which facilitate cell surface expression of the channels. Additionally, the protein stability of cell surface‐expressed Kv1.2 channels is affected by glycosylation via differences in the degradation of internalized channels. The present study reveals the importance of N‐linked complex glycosylation in boosting Kv1.2 channel density. Notably, sialic acids at the terminal sugar branches play an important role in dampening the degradation of Kv1.2 internalized from the cell membrane to promote its stability.

  August 02, 2016   doi: 10.1113/JP272394   open full text
• cis Retinol oxidation regulates photoreceptor access to the retina visual cycle and cone pigment regeneration.
  Shinya Sato, Vladimir J. Kefalov.
  The Journal of Physiology. August 02, 2016
  Key points This study explores the nature of the cis retinol that Müller cells in the retina provide to cones for the regeneration of their visual pigment. We report that the retina visual cycle provides cones exclusively with 11‐cis chromophore in both salamander and mouse and show that this selectivity is dependent on the 11‐cis‐specific cellular retinaldehyde binding protein (CRALBP) present in Müller cells. Even though salamander blue cones and green rods share the same visual pigment, only blue cones but not green rods are able to dark‐adapt in the retina following a bleach and to use exogenous 9‐cis retinol for pigment regeneration, suggesting that access to the retina visual cycle is cone‐specific and pigment‐independent. Our results show that the retina produces 11‐cis retinol that can be oxidized and used for pigment regeneration and dark adaptation selectively in cones and not in rods. Abstract Chromophore supply by the retinal Müller cells (retina visual cycle) supports the efficient pigment regeneration required for cone photoreceptor function in bright light. Surprisingly, a large fraction of the chromophore produced by dihydroceramide desaturase‐1, the putative all‐trans retinol isomerase in Müller cells, appears to be 9‐cis retinol. In contrast, the canonical retinal pigment epithelium (RPE) visual cycle produces exclusively 11‐cis retinal. Here, we used the different absorption spectra of 9‐cis and 11‐cis pigments to identify the isoform of the chromophore produced by the visual cycle of the intact retina. We found that the spectral sensitivity of salamander and mouse cones dark‐adapted in the isolated retina (with only the retina visual cycle) was similar to that of cones dark‐adapted in the intact eye (with both the RPE and retina visual cycles) and consistent with pure 11‐cis pigment composition. However, in mice lacking the cellular retinaldehyde binding protein (CRALBP), cone spectral sensitivity contained a substantial 9‐cis component. Thus, the retina visual cycle provides cones exclusively with 11‐cis chromophore and this process is mediated by the 11‐cis selective CRALBP in Müller cells. Finally, despite sharing the same pigment, salamander blue cones, but not green rods, recovered their sensitivity in the isolated retina. Exogenous 9‐cis retinol produced robust sensitivity recovery in bleached red and blue cones but not in red and green rods, suggesting that cis retinol oxidation restricts access to the retina visual cycle to cones.

  August 02, 2016   doi: 10.1113/JP272831   open full text
• Store‐operated calcium entry is required for sustained contraction and Ca2+ oscillations of airway smooth muscle.
  Jun Chen, Michael J. Sanderson.
  The Journal of Physiology. August 02, 2016
  Key points Airway hyper‐responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (ASMCs). Agonist‐induced Ca2+ oscillations underlie this contraction of ASMCs and the magnitude of this contraction is proportional to the Ca2+ oscillation frequency. Sustained contraction and Ca2+ oscillations require an influx of extracellular Ca2+, although the mechanisms and pathways mediating this Ca2+ influx during agonist‐induced ASMC contraction are not well defined. By inhibiting store‐operated calcium entry (SOCE) or voltage‐gated Ca2+ channels (VGCCs), we show that SOCE, rather than Ca2+ influx via VGCCs, provides the major Ca2+ entry pathway into ASMCs to sustain ASMCs contraction and Ca2+ oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper‐responsiveness in asthma. Abstract Asthma is characterized by airway hyper‐responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca2+ oscillations within airway smooth muscle cells (ASMCs). Sustained Ca2+ oscillations require a Ca2+ influx to replenish Ca2+ losses across the plasma membrane. Our previous studies implied store‐operated calcium entry (SOCE) as the major pathway for this Ca2+ influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975A and GSK5498A) as well as L‐type voltage‐gated Ca2+ channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca2+ oscillations and SOCE‐mediated Ca2+ influx in ASMCs within mouse precision‐cut lung slices. We found that both GSK7975A and GSK5498A were able to fully relax methacholine‐induced airway contraction by abolishing the Ca2+ oscillations, in a manner similar to that observed in zero extracellular Ca2+ ([Ca2+]e). In addition, GSK7975A and GSK5498A inhibited increases in intracellular Ca2+ ([Ca2+]i) in ASMCs with depleted Ca2+‐stores in response to increased [Ca2+]e, demonstrating a response consistent with the inhibition of SOCE. However, GSK7975A and GSK5498A did not reduce Ca2+ release via IP3 receptors stimulated with IP3 released from caged‐IP3. By contrast, nifedipine and nimodipine only partially reduced airway contraction, Ca2+ oscillation frequency and SOCE‐mediated Ca2+ influx. These data suggest that SOCE is the major Ca2+ influx pathway for ASMCs with respect to sustaining agonist‐induced airway contraction and the underlying Ca2+ oscillations. The mechanisms of SOCE may therefore form novel targets for new bronchodilators.

  August 02, 2016   doi: 10.1113/JP272694   open full text
• Hepatic mitochondrial oxidative phosphorylation is normal in obese patients with and without type 2 diabetes.
  Michael Taulo Lund, Marianne Kristensen, Merethe Hansen, Louise Tveskov, Andrea Karen Floyd, Mikael Støckel, Ben Vainer, Steen Seier Poulsen, Jørn Wulff Helge, Clara Prats, Flemming Dela.
  The Journal of Physiology. August 01, 2016
  Key points Hepatic insulin resistance in patients with obesity or type 2 diabetes has been suggested to result from hepatic mitochondrial dysfunction. High‐resolution respirometry (HRR) can be used to assess oxidative phosphorylation by measuring the mitochondrial oxygen consumption rate in the individual complexes of the mitochondria. By using HRR, the present study demonstrates no difference in hepatic mitochondrial oxidative phosphorylation among subjects with obesity with or without type 2 diabetes and non‐obese controls. Furthermore, the amount of mitochondria, assessed by the citrate synthase activity, is not different between the three groups. Together the present findings indicate that hepatic mitochondrial oxidative phosphorylation capacity is not impaired in patients with obesity or type 2 diabetes. Abstract Obese patients with type 2 diabetes (T2DM) and without type 2 diabetes (OB) are characterized by high hepatic lipid content and hepatic insulin resistance. This may be linked to impaired hepatic mitochondrial oxidative phosphorylation (OXPHOS) capacity. The aim of the present study was to investigate and compare hepatic mitochondrial OXPHOS capacity in T2DM, OB and non‐obese controls (CON). Seventeen obese patients (nine OB and eight T2DM) and six CON patients had perioperative liver biopsies taken. Samples were divided into three parts to measure (1) complex I, II and IV linked respiration, (2) citrate synthase (CS) activity and (3) lipid droplet (LD) size and area (% of total tissue area filled by LDs). State 3 respiration of complex I, II and IV and the CS activity did not differ in OB, T2DM and CON. LD size was significantly higher in T2DM compared with CON, and LD area tended (P = 0.10) to be higher in T2DM and OB compared with CON. The present findings indicate that hepatic OXPHOS capacity is not different in patients with markedly different weight and glycaemic control. Furthermore, the results do not support impaired hepatic mitochondrial respiratory capacity playing a major role in the development of obesity‐induced type 2 diabetes.

  August 01, 2016   doi: 10.1113/JP272105   open full text
• Respiratory modulation of human autonomic function on Earth.
  Dwain L. Eckberg, William H. Cooke, André Diedrich, Italo Biaggioni, Jay C. Buckey, James A. Pawelczyk, Andrew C. Ertl, James F. Cox, Tom A. Kuusela, Kari U. O. Tahvanainen, Tadaaki Mano, Satoshi Iwase, Friedhelm J. Baisch, Benjamin D. Levine, Beverley Adams‐Huet, David Robertson, C. Gunnar Blomqvist.
  The Journal of Physiology. July 26, 2016
  Key points We studied healthy supine astronauts on Earth with electrocardiogram, non‐invasive arterial pressure, respiratory carbon dioxide concentrations, breathing depth and sympathetic nerve recordings. The null hypotheses were that heart beat interval fluctuations at usual breathing frequencies are baroreflex mediated, that they persist during apnoea, and that autonomic responses to apnoea result from changes of chemoreceptor, baroreceptor or lung stretch receptor inputs. R‐R interval fluctuations at usual breathing frequencies are unlikely to be baroreflex mediated, and disappear during apnoea. The subjects’ responses to apnoea could not be attributed to changes of central chemoreceptor activity (hypocapnia prevailed); altered arterial baroreceptor input (vagal baroreflex gain declined and muscle sympathetic nerve burst areas, frequencies and probabilities increased, even as arterial pressure climbed to new levels); or altered pulmonary stretch receptor activity (major breathing frequency and tidal volume changes did not alter vagal tone or sympathetic activity). Apnoea responses of healthy subjects may result from changes of central respiratory motoneurone activity. Abstract We studied eight healthy, supine astronauts on Earth, who followed a simple protocol: they breathed at fixed or random frequencies, hyperventilated and then stopped breathing, as a means to modulate and expose to view important, but obscure central neurophysiological mechanisms. Our recordings included the electrocardiogram, finger photoplethysmographic arterial pressure, tidal volume, respiratory carbon dioxide concentrations and peroneal nerve muscle sympathetic activity. Arterial pressure, vagal tone and muscle sympathetic outflow were comparable during spontaneous and controlled‐frequency breathing. Compared with spontaneous, 0.1 and 0.05 Hz breathing, however, breathing at usual frequencies (∼0.25 Hz) lowered arterial baroreflex gain, and provoked smaller arterial pressure and R‐R interval fluctuations, which were separated by intervals that were likely to be too short and variable to be attributed to baroreflex physiology. R‐R interval fluctuations at usual breathing frequencies disappear during apnoea, and thus cannot provide evidence for the existence of a central respiratory oscillation. Apnoea sets in motion a continuous and ever changing reorganization of the relations among stimulatory and inhibitory inputs and autonomic outputs, which, in our study, could not be attributed to altered chemoreceptor, baroreceptor, or pulmonary stretch receptor activity. We suggest that responses of healthy subjects to apnoea are driven importantly, and possibly prepotently, by changes of central respiratory motoneurone activity. The companion article extends these observations and asks the question, Might terrestrial responses to our 20 min breathing protocol find expression as long‐term neuroplasticity in serial measurements made over 20 days during and following space travel?

  July 26, 2016   doi: 10.1113/JP271654   open full text
• Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea‐pigs.
  Frank Fabris, Yuankun Yue, Yongxia Qu, Mohamed Chahine, Eric Sobie, Peng Lee, Rosemary Wieczorek, Xian‐Cheng Jiang, Pier‐Leopoldo Capecchi, Franco Laghi‐Pasini, Pietro‐Enea Lazzerini, Mohamed Boutjdir.
  The Journal of Physiology. July 26, 2016
  Key points Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias. We have recently demonstrated that anti‐SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether‐à‐go‐go‐related gene (HERG) K+ channel by inhibiting the corresponding current, IKr, at the pore region. Immunization of guinea‐pigs with a peptide (E‐pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit IKr, lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti‐SSA/Ro‐positive sera from patients with connective tissue diseases showed high reactivity to the E‐pore peptide. The translational impact is the development of a peptide‐based approach for the diagnosis and treatment of autoimmune‐associated long QT syndrome. Abstract We recently demonstrated that anti‐SSA/52 kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether‐à‐go‐go‐related gene (HERG) K+ channel at the extracellular pore (E‐pore) region, where homology with SSA/52 kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea‐pigs with a peptide corresponding to the E‐pore region (E‐pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea‐pigs were immunized with a 31‐amino‐acid peptide corresponding to the E‐pore region of HERG. On days 10–62 after immunization, ECGs were recorded and blood was sampled for the detection of E‐pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E‐pore peptide by enzyme‐linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E‐pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E‐pore peptide was found using anti‐SSA/Ro Ab‐positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti‐SSA/Ro Ab‐positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E‐pore region induce QTc prolongation in immunized guinea‐pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti‐SSA/Ro Ab‐positive sera from patients with connective tissue diseases with the E‐pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune‐associated QTc prolongation.

  July 26, 2016   doi: 10.1113/JP272151   open full text
• Respiratory modulation of human autonomic function: long‐term neuroplasticity in space.
  Dwain L. Eckberg, André Diedrich, William H. Cooke, Italo Biaggioni, Jay C. Buckey, James A. Pawelczyk, Andrew C. Ertl, James F. Cox, Tom A. Kuusela, Kari U.O. Tahvanainen, Tadaaki Mano, Satoshi Iwase, Friedhelm J. Baisch, Benjamin D. Levine, Beverley Adams‐Huet, David Robertson, C. Gunnar Blomqvist.
  The Journal of Physiology. July 26, 2016
  Key points We studied healthy astronauts before, during and after the Neurolab Space Shuttle mission with controlled breathing and apnoea, to identify autonomic changes that might contribute to postflight orthostatic intolerance. Measurements included the electrocardiogram, finger photoplethysmographic arterial pressure, respiratory carbon dioxide levels, tidal volume and peroneal nerve muscle sympathetic activity. Arterial pressure fell and then rose in space, and drifted back to preflight levels after return to Earth. Vagal metrics changed in opposite directions: vagal baroreflex gain and two indices of vagal fluctuations rose and then fell in space, and descended to preflight levels upon return to Earth. Sympathetic burst frequencies (but not areas) were greater than preflight in space and on landing day, and astronauts’ abilities to modulate both burst areas and frequencies during apnoea were sharply diminished. Spaceflight triggers long‐term neuroplastic changes reflected by reciptocal sympathetic and vagal motoneurone responsiveness to breathing changes. Abstract We studied six healthy astronauts five times, on Earth, in space on the first and 12th or 13th day of the 16 day Neurolab Space Shuttle mission, on landing day, and 5–6 days later. Astronauts followed a fixed protocol comprising controlled and random frequency breathing and apnoea, conceived to perturb their autonomic function and identify changes, if any, provoked by microgravity exposure. We recorded the electrocardiogram, finger photoplethysmographic arterial pressure, tidal carbon dioxide concentrations and volumes, and peroneal nerve muscle sympathetic activity on Earth (in the supine position) and in space. (Sympathetic nerve recordings were made during three sessions: preflight, late mission and landing day.) Arterial pressure changed systematically from preflight levels: pressure fell during early microgravity exposure, rose as microgravity exposure continued, and drifted back to preflight levels after return to Earth. Vagal metrics changed in opposite directions: vagal baroreflex gain and two indices of vagal fluctuations (root mean square of successive normal R‐R intervals; and proportion of successive normal R‐R intervals greater than 50 ms, divided by the total number of normal R‐R intervals) rose significantly during early microgravity exposure, fell as microgravity exposure continued, and descended to preflight levels upon return to Earth. Sympathetic mechanisms also changed. Burst frequencies (but not areas) during fixed frequency breathing were greater than preflight in space and on landing day, but their control during apnoea was sharply altered: astronauts increased their burst frequencies from already high levels, but they could not modulate either burst areas or frequencies appropriately. Space travel provokes long‐lasting sympathetic and vagal neuroplastic changes in healthy humans.

  July 26, 2016   doi: 10.1113/JP271656   open full text
• Formation of mitochondrial‐derived vesicles is an active and physiologically relevant mitochondrial quality control process in the cardiac system.
  Virgilio J. J. Cadete, Sonia Deschênes, Alexanne Cuillerier, François Brisebois, Ayumu Sugiura, Amy Vincent, Doug Turnbull, Martin Picard, Heidi M. McBride, Yan Burelle.
  The Journal of Physiology. July 24, 2016
  Key points Mitochondrial‐derived vesicle (MDV) formation occurs under baseline conditions and is rapidly upregulated in response to stress‐inducing conditions in H9c2 cardiac myoblasts. In mice formation of MDVs occurs readily in the heart under normal healthy conditions while mitophagy is comparatively less prevalent. In response to acute stress induced by doxorubicin, mitochondrial dysfunction develops in the heart, triggering MDV formation and mitophagy. MDV formation is thus active in the cardiac system, where it probably constitutes a baseline housekeeping mechanism and a first line of defence against stress. Abstract The formation of mitochondrial‐derived vesicles (MDVs), a process inherited from bacteria, has emerged as a potentially important mitochondrial quality control (QC) mechanism to selectively deliver damaged material to lysosomes for degradation. However, the existence of this mechanism in various cell types, and its physiological relevance, remains unknown. Our aim was to investigate the dynamics of MDV formation in the cardiac system in vitro and in vivo. Immunofluorescence in cell culture, quantitative transmission electron microscopy and electron tomography in vivo were used to study MDV production in the cardiac system. We show that in cardiac cells MDV production occurs at baseline, is commensurate with the dependence of cells on oxidative metabolism, is more frequent than mitophagy and is up‐regulated on the time scale of minutes to hours in response to prototypical mitochondrial stressors (antimycin‐A, xanthine/xanthine oxidase). We further show that MDV production is up‐regulated together with mitophagy in response to doxorubicin‐induced mitochondrial and cardiac dysfunction. Here we provide the first quantitative data demonstrating that MDV formation is a mitochondrial QC operating in the heart.

  July 24, 2016   doi: 10.1113/JP272703   open full text
• Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr) in a length‐dependent fashion.
  Christopher N. Toepfer, Timothy G. West, Michael A. Ferenczi.
  The Journal of Physiology. July 24, 2016
  Key points Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (ktr) at submaximal [Ca2+]. Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca2+]‐activated cardiac trabecula accelerates ktr. Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows ktr and reduces force generation. ktr is dependent on sarcomere length in the physiological range 1.85–1.94 μm and RLC phosphorylation modulates this response. We demonstrate that Frank–Starling is evident at maximal [Ca2+] activation and therefore does not necessarily require length‐dependent change in [Ca2+]‐sensitivity of thin filament activation. The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank–Starling law in the heart. These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. Abstract Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin‐associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (ktr) in maximally activating [Ca2+]. Activation was achieved by rapidly increasing the temperature (temperature‐jump of 0.5–20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85–1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC‐exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two‐state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross‐bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+] and are not explained by changes in the Ca2+‐sensitivity of acto‐myosin interactions. The length‐dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank–Starling law of the heart.

  July 24, 2016   doi: 10.1113/JP272441   open full text
• Sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in Ambystoma tigrinum retina.
  Ted J. Warren, Matthew J. Hook, Claudiu T. Supuran, Wallace B. Thoreson.
  The Journal of Physiology. July 21, 2016
  Key points In the vertebrate retina, photoreceptors influence the signalling of neighbouring photoreceptors through lateral‐inhibitory interactions mediated by horizontal cells (HCs). These interactions create antagonistic centre‐surround receptive fields important for detecting edges and generating chromatically opponent responses in colour vision. The mechanisms responsible for inhibitory feedback from HCs involve changes in synaptic cleft pH that modulate photoreceptor calcium currents. However, the sources of synaptic protons involved in feedback and the mechanisms for their removal from the cleft when HCs hyperpolarize to light remain unknown. Our results indicate that Na+–H+ exchangers are the principal source of synaptic cleft protons involved in HC feedback but that synaptic cleft alkalization during light‐evoked hyperpolarization of HCs also involves changes in bicarbonate transport across the HC membrane. In addition to delineating processes that establish lateral inhibition in the retina, these results contribute to other evidence showing the key role for pH in regulating synaptic signalling throughout the nervous system. Abstract Lateral‐inhibitory feedback from horizontal cells (HCs) to photoreceptors involves changes in synaptic cleft pH accompanying light‐evoked changes in HC membrane potential. We analysed HC to cone feedback by studying surround‐evoked light responses of cones and by obtaining paired whole cell recordings from cones and HCs in salamander retina. We tested three potential sources for synaptic cleft protons: (1) generation by extracellular carbonic anhydrase (CA), (2) release from acidic synaptic vesicles and (3) Na+/H+ exchangers (NHEs). Neither antagonizing extracellular CA nor blocking loading of protons into synaptic vesicles eliminated feedback. However, feedback was eliminated when extracellular Na+ was replaced with choline and significantly reduced by an NHE inhibitor, cariporide. Depriving NHEs of intracellular protons by buffering HC cytosol with a pH 9.2 pipette solution eliminated feedback, whereas alkalinizing the cone cytosol did not, suggesting that HCs are a major source for protons in feedback. We also examined mechanisms for changing synaptic cleft pH in response to changes in HC membrane potential. Increasing the trans‐membrane proton gradient by lowering the extracellular pH from 7.8 to 7.4 to 7.1 strengthened feedback. While maintaining constant extracellular pH with 1 mm HEPES, removal of bicarbonate abolished feedback. Elevating intracellular bicarbonate levels within HCs prevented this loss of feedback. A bicarbonate transport inhibitor, 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS), also blocked feedback. Together, these results suggest that NHEs are the primary source of extracellular protons in HC feedback but that changes in cleft pH accompanying changes in HC membrane voltage also require bicarbonate flux across the HC membrane.

  July 21, 2016   doi: 10.1113/JP272533   open full text
• Organization of flexor–extensor interactions in the mammalian spinal cord: insights from computational modelling.
  Natalia A. Shevtsova, Ilya A. Rybak.
  The Journal of Physiology. July 21, 2016
  Key points Alternation of flexor and extensor activity in the mammalian spinal cord is mediated by two classes of genetically identified inhibitory interneurons, V1 and V2b. The V1 interneurons are essential for high‐speed locomotor activity. They secure flexor–extensor alternations in the intact cord but lose this function after hemisection, suggesting that they are activated by inputs from the contralateral side of the cord. The V2b interneurons are involved in flexor–extensor alternations in both intact cord and hemicords. We used a computational model of the spinal network, simulating the left and right rhythm‐generating circuits interacting via several commissural pathways, and extended this model by incorporating V1 and V2b neuron populations involved in flexor–extensor interactions on each cord side. The model reproduces multiple experimental data on selective silencing and activation of V1 and/or V2b neurons and proposes the organization of their connectivity providing flexor–extensor alternation in the spinal cord. Abstract Alternating flexor and extensor activity represents the fundamental property underlying many motor behaviours including locomotion. During locomotion this alternation appears to arise in rhythm‐generating circuits and transpires at all levels of the spinal cord including motoneurons. Recent studies in vitro and in vivo have shown that flexor–extensor alternation during locomotion involves two classes of genetically identified, inhibitory interneurons: V1 and V2b. Particularly, in the isolated mouse spinal cord, abrogation of neurotransmission derived by both V1 and V2b interneurons resulted in flexor–extensor synchronization, whereas selective inactivation of only one of these neuron types did not abolish flexor–extensor alternation. After hemisection, inactivation of only V2b interneurons led to the flexor–extensor synchronization, while inactivation of V1 interneurons did not affect flexor–extensor alternation. Moreover, optogenetic activation of V2b interneurons suppressed extensor‐related activity, while similar activation of V1 interneurons suppressed both flexor and extensor oscillations. Here, we address these issues using the previously published computational model of spinal circuitry simulating bilateral interactions between left and right rhythm‐generating circuits. In the present study, we incorporate V1 and V2b neuron populations on both sides of the cord to make them critically involved in flexor–extensor interactions. The model reproduces multiple experimental data on the effects of hemisection and selective silencing or activation of V1 and V2b neurons and suggests connectivity profiles of these neurons and their specific roles in left–right (V1) and flexor–extensor (both V2b and V1) interactions in the spinal cord that can be tested experimentally.

  July 21, 2016   doi: 10.1113/JP272437   open full text
• Sex differences in response to miRNA‐34a therapy in mouse models of cardiac disease: identification of sex‐, disease‐ and treatment‐regulated miRNAs.
  Bianca C. Bernardo, Jenny Y. Y. Ooi, Aya Matsumoto, Yow Keat Tham, Saloni Singla, Helen Kiriazis, Natalie L. Patterson, Junichi Sadoshima, Susanna Obad, Ruby C. Y. Lin, Julie R. McMullen.
  The Journal of Physiology. July 20, 2016
  Key points MicroRNA (miRNA)‐based therapies are in development for numerous diseases, including heart disease. Currently, very limited basic information is available on the regulation of specific miRNAs in male and female hearts in settings of disease. The identification of sex‐specific miRNA signatures has implications for translation into the clinic and suggests the need for customised therapy. In the present study, we found that a miRNA‐based treatment inhibiting miRNA‐34a (miR‐34a) was more effective in females in a setting of moderate dilated cardiomyopathy than in males. Furthermore, the treatment showed little benefit for either sex in a setting of more severe dilated cardiomyopathy associated with atrial fibrillation. The results highlight the importance of understanding the effect of miRNA‐based therapies in cardiac disease settings in males and females. Abstract MicroRNA (miRNA)‐34a (miR‐34a) is elevated in the diseased heart in mice and humans. Previous studies have shown that inhibiting miR‐34a in male mice in settings of pathological cardiac hypertrophy or ischaemia protects the heart against progression to heart failure. Whether inhibition of miR‐34a protects the female heart is unknown. Furthermore, the therapeutic potential of silencing miR‐34a in settings of dilated cardiomyopathy (DCM) and atrial fibrillation (AF) has not been assessed previously. In the present study, we examined the effect of silencing miR‐34a in males and females in (1) a model of moderate DCM and (2) a model of severe DCM with AF. The cardiac disease models were administered with a locked nucleic acid‐modified oligonucleotide (LNA‐antimiR‐34a) at 6–7 weeks of age when the models display cardiac dysfunction and conduction abnormalities. Cardiac function and morphology were measured 6 weeks after treatment. In the present study, we show that inhibition of miR‐34a provides more protection in the DCM model in females than males. Disease prevention in LNA‐antimiR‐34a treated DCM female mice was characterized by attenuated heart enlargement and lung congestion, lower expression of cardiac stress genes (B‐type natriuretic peptide, collagen gene expression), less cardiac fibrosis and better cardiac function. There was no evidence of significant protection in the severe DCM and AF model in either sex. Sex‐ and treatment‐dependent regulation of miRNAs was also identified in the diseased heart, and may explain the differential response of males and females. These studies highlight the importance of examining the impact of miRNA‐based drugs in both sexes and under different disease conditions.

  July 20, 2016   doi: 10.1113/JP272512   open full text
• Functional and structural properties of ion channels at the nerve terminal depends on compact myelin.
  Emmanuelle Berret, Sei Eun Kim, Seul Yi Lee, Christopher Kushmerick, Jun Hee Kim.
  The Journal of Physiology. July 18, 2016
  Key points In the present study, we document the role of compact myelin in regulating the structural and functional properties of ion channels at the nerve terminals, using electrophysiology, dynamic Na+ imaging and immunohistochemistry. The subcellular segregation of Na+ channel expression and intracellular Na+ dynamics at the heminode and terminal was lost in the dysmyelinated axon from Long–Evans shaker rats, which lack compact myelin. In Long–Evans shaker rats, loss of the Navβ4 subunit specifically at the heminode reduced resurgent and persistent Na+ currents, whereas K+ channel expression and currents were increased. The results of the present study suggest that there is a specific role for compact myelin in dictating protein expression and function at the axon heminode and in regulating excitability of the nerve terminal. Abstract Axon myelination increases the conduction velocity and precision of action potential propagation. Although the negative effects of demyelination are generally attributed to conduction failure, accumulating evidence suggests that myelination also regulates the structural properties and molecular composition of the axonal membrane. In the present study, we investigated how myelination affects ion channel expression and function, particularly at the last axon heminode before the nerve terminal, which regulates the presynaptic excitability of the nerve terminal. We compared the structure and physiology of normal axons and those of the Long–Evans shaker (LES) rat, which lacks compact myelin. The normal segregation of Na+ channel expression and dynamics at the heminode and terminal was lost in the LES rat. Specifically, NaV‐α subunits were dispersed and NaVβ4 subunit was absent, whereas the density of K+ channels was increased at the heminode. Correspondingly, resurgent and persistent Na+ currents were reduced and K+ current was increased. Taken together, these data suggest a specific role for compact myelin in the orchestration of ion channel expression and function at the axon heminode and in regulating excitability of the nerve terminal.

  July 18, 2016   doi: 10.1113/JP272205   open full text
• E74‐like factor 3 and nuclear factor‐κB regulate lipocalin‐2 expression in chondrocytes.
  Javier Conde, Miguel Otero, Morena Scotece, Vanessa Abella, Verónica López, Jesús Pino, Rodolfo Gómez, Francisca Lago, Mary B. Goldring, Oreste Gualillo.
  The Journal of Physiology. July 18, 2016
  Key points E74‐like factor 3 (ELF3) is a transcription factor regulated by inflammation in different physio‐pathological situations. Lipocalin‐2 (LCN2) emerged as a relevant adipokine involved in the regulation of inflammation. In this study we showed for the first time the involvement of ELF3 in the control of LCN2 expression and its cooperation with nuclear factor‐κB (NFκB). Our results will help to better understand of the role of ELF3, NFκB and LCN2 in the pathophysiology of articular cartilage. Abstract E74‐like factor 3 (ELF3) is a transcription factor induced by inflammatory cytokines in chondrocytes that increases gene expression of catabolic and inflammatory mediators. Lipocalin 2 (LCN2) is a novel adipokine that negatively impacts articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated the control of LCN2 gene expression by ELF3 in the context of interleukin 1 (IL‐1)‐driven inflammatory responses in chondrocytes. The interaction of ELF3 and nuclear factor‐κB (NFκB) in modulating LCN2 levels was also explored. LCN2 mRNA and protein levels, as well those of several other ELF3 target genes, were determined by RT‐qPCR and Western blotting. Human primary chondrocytes, primary chondrocytes from wild‐type and Elf3 knockout mice, and immortalized human T/C‐28a2 and murine ATDC5 cell lines were used in in vitro assays. The activities of various gene reporter constructs were evaluated by luciferase assays. Gene overexpression and knockdown were performed using specific expression vectors and siRNA technology, respectively. ELF3 overexpression transactivated the LCN2 promoter and increased the IL‐1‐induced mRNA and protein levels of LCN2, as well as the mRNA expression of other pro‐inflammatory mediators, in human and mouse chondrocytes. We also identified a collaborative loop between ELF3 and NFκB that amplifies the induction of LCN2. Our findings show a novel role for ELF3 and NFκB in the induction of the pro‐inflammatory adipokine LCN2, providing additional evidence of the interaction between ELF3 and NFκB in modulating inflammatory responses, and a better understanding of the mechanisms of action of ELF3 in chondrocytes.

  July 18, 2016   doi: 10.1113/JP272240   open full text
• Functional adaptations of the coronary microcirculation to anaemia in fetal sheep.
  Sonnet S. Jonker, Lowell Davis, Divya Soman, J. Todd Belcik, Brian P. Davidson, Tamara M. Atkinson, Adrienne Wilburn, Samantha Louey, George D. Giraud, Jonathan R. Lindner.
  The Journal of Physiology. July 18, 2016
  Key points In fetuses, chronic anaemia stimulates cardiac growth; simultaneously, blood flow to the heart muscle itself is increased, and reserve blood flow capacity of the coronary vascular bed is preserved. Here we examined functional adaptations of the capillaries and small blood vessels responsible for delivering oxygen to the anaemic fetal heart muscle using contrast‐enhanced echocardiography. We demonstrate that coronary microvascular flux rate doubled in anaemic fetuses compared to control fetuses, both at rest and during maximal flow, suggesting reduced microvascular resistance consistent with capillary widening. Cardiac fractional microvascular blood volume was not greater in anaemic fetuses, suggesting that growth of new microvascular vessels does not contribute to the increased flow per volume of myocardium. These unusual changes in microvascular function during anaemia may indicate novel adaptive strategies in the fetal heart. Abstract Fetal anaemia causes cardiac adaptations that have immediate and life‐long repercussions on heart function and health. It is known that resting and maximal coronary conductance both increase during chronic fetal anaemia, but the coronary microvascular changes responsible for the adaptive response are unknown. Until recently, technical limitations have prevented quantifying functional capillary‐level adaptations in the in vivo fetal heart. Our objective was to characterise functional microvascular adaptations in chronically anaemic fetal sheep. Chronically instrumented fetuses were randomized to a control group (n = 11) or were made anaemic by isovolumetric haemorrhage (n = 12) for 1 week prior to myocardial contrast echocardiography at 85% of gestation. Anaemia augmented cardiac mass by 23% without changing body weight. In anaemic fetuses, microvascular blood flow per volume of myocardium was twice that of control fetuses at rest, during vasodilatory hyperaemia, and during hyperaemia plus increased aortic pressure. The elevated blood flow was attributable almost entirely to an increase in microvascular blood flux rate whereas microvascular blood volumes were not different between groups at baseline, during hyperaemia, or with hyperaemia plus increased aortic pressure. Increased coronary microvascular flux rate in response to chronic fetal anaemia is consistent with expected reductions in capillary resistance from capillary diameter widening detected in earlier histological studies.

  July 18, 2016   doi: 10.1113/JP272696   open full text
• Satellite cell activation and apoptosis in skeletal muscle from severely burned children.
  Christopher S. Fry, Craig Porter, Labros S. Sidossis, Christopher Nieten, Paul T. Reidy, Gabriel Hundeshagen, Ronald Mlcak, Blake B. Rasmussen, Jong O. Lee, Oscar E. Suman, David N. Herndon, Celeste C. Finnerty.
  The Journal of Physiology. July 15, 2016
  Key points Severe burns result in profound skeletal muscle atrophy that hampers recovery. The activity of skeletal muscle stem cells, satellite cells, acutely following a severe burn is unknown and may contribute to the recovery of lean muscle. Severe burn injury induces skeletal muscle regeneration and myonuclear apoptosis. Satellite cells undergo concurrent apoptosis and activation acutely following a burn, with a net reduction in satellite cell content compared to healthy controls. The activation and apoptosis of satellite cells probably impacts the recovery of lean tissue following a severe burn, contributing to prolonged frailty in burn survivors. Abstract Severe burns result in profound skeletal muscle atrophy; persistent muscle loss and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma and we propose that an impaired muscle satellite cell response is key in the aetiology of burn‐induced cachexia. Muscle biopsies from the m. vastus lateralis were obtained from 12 male pediatric burn patients (>30% total body surface area burn) and 12 young, healthy male subjects. Satellite cell content, activation and apoptosis were determined via immunohistochemistry, as were muscle fibre regeneration and myonuclear apoptosis. Embryonic myosin heavy chain expression and central nucleation, indices of skeletal muscle regeneration, were elevated in burn patients (P < 0.05). Myonuclear apoptosis, quantified by TUNEL positive myonuclei and cleaved caspase‐3 positive myonuclei, was also elevated in burn patients (P < 0.05). Satellite cell content was reduced in burn patients, with approximately 20% of satellite cells positive for TUNEL staining, indicating DNA damage associated with apoptosis (P < 0.05). Additionally, a significant percentage of satellite cells in burn patients expressed Ki67, a marker for cellular proliferation (P < 0.05). Satellite cell activation was also observed in burn patients with increased expression of MyoD compared to healthy controls (P < 0.05). Robust skeletal muscle atrophy occurs after burn injury, even in muscles located distally to the site of injury. The activation and apoptosis of satellite cells probably impacts the recovery of lean tissue following a severe burn, contributing to prolonged frailty in burn survivors.

  July 15, 2016   doi: 10.1113/JP272520   open full text
• Cerebral oxidative metabolism is decreased with extreme apnoea in humans; impact of hypercapnia.
  Anthony R. Bain, Philip N. Ainslie, Ryan L. Hoiland, Otto F. Barak, Marija Cavar, Ivan Drvis, Mike Stembridge, Douglas M. MacLeod, Damian M. Bailey, Zeljko Dujic, David B. MacLeod.
  The Journal of Physiology. July 09, 2016
  Key points The present study describes the cerebral oxidative and non‐oxidative metabolism in man during a prolonged apnoea (ranging from 3 min 36 s to 7 min 26 s) that generates extremely low levels of blood oxygen and high levels of carbon dioxide. The cerebral oxidative metabolism, measured from the product of cerebral blood flow and the radial artery‐jugular venous oxygen content difference, was reduced by ∼29% at the termination of apnoea, although there was no change in the non‐oxidative metabolism. A subset study with mild and severe hypercapnic breathing at the same level of hypoxia suggests that hypercapnia can partly explain the cerebral metabolic reduction near the apnoea breakpoint. A hypercapnia‐induced oxygen‐conserving response may protect the brain against severe oxygen deprivation associated with prolonged apnoea. Abstract Prolonged apnoea in humans is reflected in progressive hypoxaemia and hypercapnia. In the present study, we explore the cerebral metabolic responses under extreme hypoxia and hypercapnia associated with prolonged apnoea. We hypothesized that the cerebral metabolic rate for oxygen (CMRO2) will be reduced near the termination of apnoea, attributed in part to the hypercapnia. Fourteen elite apnoea‐divers performed a maximal apnoea (range 3 min 36 s to 7 min 26 s) under dry laboratory conditions. In a subset study with the same divers, the impact of hypercapnia on cerebral metabolism was determined using varying levels of hypercapnic breathing, against the background of similar hypoxia. In both studies, the CMRO2 was calculated from the product of cerebral blood flow (ultrasound) and the radial artery‐internal jugular venous oxygen content difference. Non‐oxidative cerebral metabolism was calculated from the ratio of oxygen and carbohydrate (lactate and glucose) metabolism. The CMRO2 was reduced by ∼29% (P < 0.01, Cohen's d = 1.18) near the termination of apnoea compared to baseline, although non‐oxidative metabolism remained unaltered. In the subset study, in similar backgrounds of hypoxia (arterial O2 tension: ∼38.4 mmHg), severe hypercapnia (arterial CO2 tension: ∼58.7 mmHg), but not mild‐hypercapnia (arterial CO2 tension: ∼46.3 mmHg), depressed the CMRO2 (∼17%, P = 0.04, Cohen's d = 0.87). Similarly to the apnoea, there was no change in the non‐oxidative metabolism. These data indicate that hypercapnia can partly explain the reduction in CMRO2 near the apnoea breakpoint. This hypercapnic‐induced oxygen conservation may protect the brain against severe hypoxaemia associated with prolonged apnoea.

  July 09, 2016   doi: 10.1113/JP272404   open full text
• Resistance training‐induced changes in integrated myofibrillar protein synthesis are related to hypertrophy only after attenuation of muscle damage.
  Felipe Damas, Stuart M. Phillips, Cleiton A. Libardi, Felipe C. Vechin, Manoel E. Lixandrão, Paulo R. Jannig, Luiz A. R. Costa, Aline V. Bacurau, Tim Snijders, Gianni Parise, Valmor Tricoli, Hamilton Roschel, Carlos Ugrinowitsch.
  The Journal of Physiology. July 09, 2016
  Key points Skeletal muscle hypertrophy is one of the main outcomes from resistance training (RT), but how it is modulated throughout training is still unknown. We show that changes in myofibrillar protein synthesis (MyoPS) after an initial resistance exercise (RE) bout in the first week of RT (T1) were greater than those seen post‐RE at the third (T2) and tenth week (T3) of RT, with values being similar at T2 and T3. Muscle damage (Z‐band streaming) was the highest during post‐RE recovery at T1, lower at T2 and minimal at T3. When muscle damage was the highest, so was the integrated MyoPS (at T1), but neither were related to hypertrophy; however, integrated MyoPS at T2 and T3 were correlated with hypertrophy. We conclude that muscle hypertrophy is the result of accumulated intermittent increases in MyoPS mainly after a progressive attenuation of muscle damage. Abstract Skeletal muscle hypertrophy is one of the main outcomes of resistance training (RT), but how hypertrophy is modulated and the mechanisms regulating it are still unknown. To investigate how muscle hypertrophy is modulated through RT, we measured day‐to‐day integrated myofibrillar protein synthesis (MyoPS) using deuterium oxide and assessed muscle damage at the beginning (T1), at 3 weeks (T2) and at 10 weeks of RT (T3). Ten young men (27 (1) years, mean (SEM)) had muscle biopsies (vastus lateralis) taken to measure integrated MyoPS and muscle damage (Z‐band streaming and indirect parameters) before, and 24 h and 48 h post resistance exercise (post‐RE) at T1, T2 and T3. Fibre cross‐sectional area (fCSA) was evaluated using biopsies at T1, T2 and T3. Increases in fCSA were observed only at T3 (P = 0.017). Changes in MyoPS post‐RE at T1, T2 and T3 were greater at T1 (P < 0.03) than at T2 and T3 (similar values between T2 and T3). Muscle damage was the highest during post‐RE recovery at T1, attenuated at T2 and further attenuated at T3. The change in MyoPS post‐RE at both T2 and T3, but not at T1, was strongly correlated (r ≈ 0.9, P < 0.04) with muscle hypertrophy. Initial MyoPS response post‐RE in an RT programme is not directed to support muscle hypertrophy, coinciding with the greatest muscle damage. However, integrated MyoPS is quickly ‘refined’ by 3 weeks of RT, and is related to muscle hypertrophy. We conclude that muscle hypertrophy is the result of accumulated intermittent changes in MyoPS post‐RE in RT, which coincides with progressive attenuation of muscle damage.

  July 09, 2016   doi: 10.1113/JP272472   open full text
• The effect of lung deformation on the spatial distribution of pulmonary blood flow.
  Tatsuya J. Arai, Rebecca J. Theilmann, Rui Carlos Sá, Michael T. Villongco, Susan R. Hopkins.
  The Journal of Physiology. July 08, 2016
  Key points Pulmonary perfusion measurement using magnetic resonance imaging combined with deformable image registration enabled us to quantify the change in the spatial distribution of pulmonary perfusion at different lung volumes. The current study elucidated the effects of tidal volume lung inflation [functional residual capacity (FRC) + 500 ml and FRC + 1 litre] on the change in pulmonary perfusion distribution. Changes in hydrostatic pressure distribution as well as transmural pressure distribution due to the change in lung height with tidal volume inflation are probably bigger contributors to the redistribution of pulmonary perfusion than the changes in pulmonary vasculature resistance caused by lung tissue stretch. Abstract Tidal volume lung inflation results in structural changes in the pulmonary circulation, potentially affecting pulmonary perfusion. We hypothesized that perfusion is recruited to regions receiving the greatest deformation from a tidal breath, thus ensuring ventilation–perfusion matching. Density‐normalized perfusion (DNP) magnetic resonance imaging data were obtained in healthy subjects (n = 7) in the right lung at functional residual capacity (FRC), FRC+500 ml, and FRC+1.0 l. Using deformable image registration, the displacement of a sagittal lung slice acquired at FRC to the larger volumes was calculated. Registered DNP images were normalized by the mean to estimate perfusion redistribution (nDNP). Data were evaluated across gravitational regions (dependent, middle, non‐dependent) and by lobes (upper, RUL; middle, RML; lower, RLL). Lung inflation did not alter mean DNP within the slice (P = 0.10). The greatest expansion was seen in the dependent region (P < 0.0001: dependent vs non‐dependent, P < 0.0001: dependent vs middle) and RLL (P = 0.0015: RLL vs RUL, P < 0.0001: RLL vs RML). Neither nDNP recruitment to RLL [+500 ml = −0.047(0.145), +1 litre = 0.018(0.096)] nor to dependent lung [+500 ml = −0.058(0.126), +1 litre = −0.023(0.106)] were found. Instead, redistribution was seen in decreased nDNP in the non‐dependent [+500 ml = −0.075(0.152), +1 litre = −0.137(0.167)) and increased nDNP in the gravitational middle lung [+500 ml = 0.098(0.058), +1 litre = 0.093(0.081)] (P = 0.01). However, there was no significant lobar redistribution (P < 0.89). Contrary to our hypothesis, based on the comparison between gravitational and lobar perfusion data, perfusion was not redistributed to the regions of the most inflation. This suggests that either changes in hydrostatic pressure or transmural pressure distribution in the gravitational direction are implicated in the redistribution of perfusion away from the non‐dependent lung.

  July 08, 2016   doi: 10.1113/JP272030   open full text
• Systemic leukotriene B4 receptor antagonism lowers arterial blood pressure and improves autonomic function in the spontaneously hypertensive rat.
  Paul J. Marvar, Emma B. Hendy, Thomas D. Cruise, Dawid Walas, Danielle DeCicco, Rajanikanth Vadigepalli, James S. Schwaber, Hidefumi Waki, David Murphy, Julian F. R. Paton.
  The Journal of Physiology. July 08, 2016
  Key points Evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4), a potent chemoattractant involved in the inflammatory response, but its mode of action is poorly understood. In the SHR, we observed an increase in T cells and macrophages in the brainstem; in addition, gene expression profiling data showed that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. When LTB4 receptor 1 (BLT1) receptors were blocked with CP‐105,696, arterial pressure was reduced in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in systolic blood pressure (BP) indicators. These data provide new evidence for the role of LTB4 as an important neuro‐immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension. Abstract Accumulating evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4), a potent chemoattractant involved in the inflammatory response. However, the mechanism for LTB4‐mediated inflammation in hypertension is poorly understood. Here we report in the SHR, increased brainstem infiltration of T cells and macrophages plus gene expression profiling data showing that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. Chronic blockade of the LTB4 receptor 1 (BLT1) receptor with CP‐105,696, reduced arterial pressure in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in low and high frequency spectra of systolic blood pressure, and an increase in spontaneous baroreceptor reflex gain (sBRG). These data provide new evidence for the role of LTB4 as an important neuro‐immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension.

  July 08, 2016   doi: 10.1113/JP272065   open full text
• Group III/IV muscle afferents limit the intramuscular metabolic perturbation during whole body exercise in humans.
  Gregory M. Blain, Tyler S. Mangum, Simranjit K. Sidhu, Joshua C. Weavil, Thomas J. Hureau, Jacob E. Jessop, Amber D. Bledsoe, Russell S. Richardson, Markus Amann.
  The Journal of Physiology. July 08, 2016
  Key points The purpose of this study was to determine the role of group III/IV muscle afferents in limiting the endurance exercise‐induced metabolic perturbation assayed in muscle biopsy samples taken from locomotor muscle. Lumbar intrathecal fentanyl was used to attenuate the central projection of μ‐opioid receptor‐sensitive locomotor muscle afferents during a 5 km cycling time trial. The findings suggest that the central projection of group III/IV muscle afferent feedback constrains voluntary neural ‘drive’ to working locomotor muscle and limits the exercise‐induced intramuscular metabolic perturbation. Therefore, the CNS might regulate the degree of metabolic perturbation within locomotor muscle and thereby limit peripheral fatigue. It appears that the group III/IV muscle afferents are an important neural link in this regulatory mechanism, which probably serves to protect locomotor muscle from the potentially severe functional impairment as a consequence of severe intramuscular metabolic disturbance. Abstract To investigate the role of metabo‐ and mechanosensitive group III/IV muscle afferents in limiting the intramuscular metabolic perturbation during whole body endurance exercise, eight subjects performed 5 km cycling time trials under control conditions (CTRL) and with lumbar intrathecal fentanyl impairing lower limb muscle afferent feedback (FENT). Vastus lateralis muscle biopsies were obtained before and immediately after exercise. Motoneuronal output was estimated through vastus lateralis surface electromyography (EMG). Exercise‐induced changes in intramuscular metabolites were determined using liquid and gas chromatography‐mass spectrometry. Quadriceps fatigue was quantified by pre‐ to post‐exercise changes in potentiated quadriceps twitch torque (ΔQTsingle) evoked by electrical femoral nerve stimulation. Although motoneuronal output was 21 ± 12% higher during FENT compared to CTRL (P < 0.05), time to complete the time trial was similar (∼8.8 min). Compared to CTRL, power output during FENT was 10 ± 4% higher in the first half of the time trial, but 11 ± 5% lower in the second half (both P < 0.01). The exercise‐induced increase in intramuscular inorganic phosphate, H+, adenosine diphosphate, lactate and phosphocreatine depletion was 55 ± 30, 62 ± 18, 129 ± 63, 47 ± 14 (P < 0.001) and 27 ± 14% (P < 0.01) greater in FENT than CTRL. ΔQTsingle was greater following FENT than CTRL (−52 ± 2 vs −31 ± 1%, P < 0.001) and this difference was positively correlated with the difference in inorganic phosphate (r2 = 0.79; P < 0.01) and H+ (r2 = 0.92; P < 0.01). In conclusion, during whole body exercise, group III/IV muscle afferents provide feedback to the CNS which, in turn, constrains motoneuronal output to the active skeletal muscle. This regulatory mechanism limits the exercise‐induced intramuscular metabolic perturbation, preventing an abnormal homeostatic challenge and excessive peripheral fatigue.

  July 08, 2016   doi: 10.1113/JP272283   open full text
• The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.
  Amelia Burroughs, Andrew K. Wise, Jianqiang Xiao, Conor Houghton, Tianyu Tang, Colleen Y. Suh, Eric J. Lang, Richard Apps, Nadia L. Cerminara.
  The Journal of Physiology. July 07, 2016
  Key points Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Abstract Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition, complex spikes with a greater number of spikelets were associated with a subsequent reduction in simple spike firing rate. We therefore suggest that one important function of spikelets is the modulation of Purkinje cell simple spike firing frequency, which has implications for controlling cerebellar cortical output and motor learning.

  July 07, 2016   doi: 10.1113/JP272259   open full text
• A physiological increase in maternal cortisol alters uteroplacental metabolism in the pregnant ewe.
  O. R. Vaughan, K. L. Davies, J. W. Ward, M. J. Blasio, A. L. Fowden.
  The Journal of Physiology. July 06, 2016
  Key points Fetal nutrient supply is dependent, in part, upon the transport capacity and metabolism of the placenta. The stress hormone, cortisol, alters metabolism in the adult and fetus but it is not known whether cortisol in the pregnant mother affects metabolism of the placenta. In this study, when cortisol concentrations were raised in pregnant sheep by infusion, proportionately more of the glucose taken up by the uterus was consumed by the uteroplacental tissues while less was transferred to the fetus, despite an increased placental glucose transport capacity. Concomitantly, the uteroplacental tissues produced lactate at a greater rate. The results show that maternal cortisol concentrations regulate uteroplacental glycolytic metabolism, producing lactate for use in utero. Prolonged increases in placental lactate production induced by cortisol overexposure may contribute to the adverse effects of maternal stress on fetal wellbeing. Abstract Fetal nutrition is determined by maternal availability, placental transport and uteroplacental metabolism of carbohydrates. Cortisol affects maternal and fetal metabolism, but whether maternal cortisol concentrations within the physiological range regulate uteroplacental carbohydrate metabolism remains unknown. This study determined the effect of maternal cortisol infusion (1.2 mg kg−1 day−1 i.v. for 5 days, n = 20) on fetal glucose, lactate and oxygen supplies in pregnant ewes on day ∼130 of pregnancy (term = 145 days). Compared to saline infusion (n = 21), cortisol infusion increased maternal, but not fetal, plasma cortisol (P < 0.05). Cortisol infusion also raised maternal insulin, glucose and lactate concentrations, and blood pH, PCO2 and HCO3− concentration. Although total uterine glucose uptake determined by Fick's principle was unaffected, a greater proportion was consumed by the uteroplacental tissues, so net fetal glucose uptake was 29% lower in cortisol‐infused than control ewes (P < 0.05). Concomitantly, uteroplacental lactate production was > 2‐fold greater in cortisol‐ than saline‐treated ewes (P < 0.05), although uteroplacental O2 consumption was unaffected by maternal treatment. Materno‐fetal clearance of non‐metabolizable [3H]methyl‐d‐glucose and placental SLC2A8 (glucose transporter 8) gene expression were also greater with cortisol treatment. Fetal plasma glucose, lactate or α‐amino nitrogen concentrations were unaffected by treatment although fetal plasma fructose and hepatic lactate dehydrogenase activity were greater in cortisol‐ than saline‐treated ewes (P < 0.05). Fetal plasma insulin levels and body weight were also unaffected by maternal treatment. During stress, cortisol‐dependent regulation of uteroplacental glycolysis may allow increased maternal control over fetal nutrition and metabolism. However, when maternal cortisol concentrations are raised chronically, prolonged elevation of uteroplacental lactate production may compromise fetal wellbeing.

  July 06, 2016   doi: 10.1113/JP272301   open full text
• Using physiologically based models for clinical translation: predictive modelling, data interpretation or something in‐between?
  Steven A. Niederer, Nic P. Smith.
  The Journal of Physiology. July 03, 2016
  Heart disease continues to be a significant clinical problem in Western society. Predictive models and simulations that integrate physiological understanding with patient information derived from clinical data have huge potential to contribute to improving our understanding of both the progression and treatment of heart disease. In particular they provide the potential to improve patient selection and optimisation of cardiovascular interventions across a range of pathologies. Currently a significant proportion of this potential is still to be realised. In this paper we discuss the opportunities and challenges associated with this realisation. Reviewing the successful elements of model translation for biophysically based models and the emerging supporting technologies, we propose three distinct modes of clinical translation. Finally we outline the challenges ahead that will be fundamental to overcome if the ultimate goal of fully personalised clinical cardiac care is to be achieved. A schematic diagram of the relationship between the success factors driving clinical translation of models and the related enabling technologies and tools. These inputs are currently driving model exemplars through three channels. CRT, cardiac resynchronisation therapy; FFR, fractional flow reserve. LVAD, left ventricular assist device.

  July 03, 2016   doi: 10.1113/JP272003   open full text
• Calcium/calmodulin‐dependent kinase II and nitric oxide synthase 1‐dependent modulation of ryanodine receptors during β‐adrenergic stimulation is restricted to the dyadic cleft.
  Eef Dries, Demetrio J. Santiago, Daniel M. Johnson, Guillaume Gilbert, Patricia Holemans, Sanne M. Korte, H. Llewelyn Roderick, Karin R. Sipido.
  The Journal of Physiology. July 03, 2016
  Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation. Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. Abstract In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.

  July 03, 2016   doi: 10.1113/JP271965   open full text
• Cav1.2 and Cav1.3 L‐type calcium channels independently control short‐ and long‐term sensitization to pain.
  Houda Radwani, Maria José Lopez‐Gonzalez, Daniel Cattaert, Olivier Roca‐Lapirot, Eric Dobremez, Rabia Bouali‐Benazzouz, Emelía Eiríksdóttir, Ülo Langel, Alexandre Favereaux, Mohammed Errami, Marc Landry, Pascal Fossat.
  The Journal of Physiology. July 03, 2016
  Key points L‐type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short‐ and long‐term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind‐up. These results identify Cav1.3 as a key conductance responsible for short‐term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long‐term plasticity associated with neuropathic pain. Up‐regulation of Cav1.2 and down‐regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind‐up may be not relevant in the treatment of neuropathy. Abstract Short‐term central sensitization to pain temporarily increases the responsiveness of nociceptive pathways after peripheral injury. In dorsal horn neurons (DHNs), short‐term sensitization can be monitored through the study of wind‐up. Wind‐up, a progressive increase in DHNs response following repetitive peripheral stimulations, depends on the post‐synaptic L‐type calcium channels. In the dorsal horn of the spinal cord, two L‐type calcium channels are present, Cav1.2 and Cav1.3, each displaying specific kinetics and spatial distribution. In the present study, we used a mathematical model of DHNs in which we integrated the specific patterns of expression of each Cav subunits. This mathematical approach reveals that Cav1.3 is necessary for the onset of wind‐up, whereas Cav1.2 is not and that synaptically triggered wind‐up requires NMDA receptor activation. We then switched to a biological preparation in which we knocked down Cav subunits and confirmed the prominent role of Cav1.3 in both naive and spinal nerve ligation model of neuropathy (SNL). Interestingly, although a clear mechanical allodynia dependent on Cav1.2 expression was observed after SNL, the amplitude of wind‐up was decreased. These results were confirmed with our model when adapting Cav1.3 conductance to the changes observed after SNL. Finally, our mathematical approach predicts that, although wind‐up amplitude is decreased in SNL, plateau potentials are not altered, suggesting that plateau and wind‐up are not fully equivalent. Wind‐up and long‐term hyperexcitability of DHNs are differentially controlled by Cav1.2 and Cav1.3, therefore confirming that short‐ and long‐term sensitization are two different phenomena triggered by distinct mechanisms.

  July 03, 2016   doi: 10.1113/JP272725   open full text
• The transcription factor NeuroD2 coordinates synaptic innervation and cell intrinsic properties to control excitability of cortical pyramidal neurons.
  Fading Chen, Jacqueline T. Moran, Yihui Zhang, Kristin M. Ates, Diankun Yu, Laura A. Schrader, Partha M. Das, Frank E. Jones, Benjamin J. Hall.
  The Journal of Physiology. July 01, 2016
  Key points Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks to allow proper brain function. Disrupting this balance may lead to autism spectral disorders and epilepsy. We show the basic helix–loop–helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell‐intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo. We identify two genes potentially downstream of NeuroD2‐mediated transcription that regulate these parameters: gastrin‐releasing peptide and the small conductance, calcium‐activated potassium channel, SK2. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability. Our results offer insight into how synaptic innervation and intrinsic excitability are coordinated during cortical development. Abstract Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks for proper brain function. Disruption of this balance during development may lead to autism spectral disorders and epilepsy. Synaptic excitation is counterbalanced by synaptic inhibition but also by attenuation of cell‐intrinsic neuronal excitability. To maintain proper excitation levels during development, neurons must sense activity over time and regulate the expression of genes that control these parameters. While this is a critical process, little is known about the transcription factors involved in coordinating gene expression to control excitatory/inhibitory synaptic balance. We show here that the basic helix–loop–helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell‐intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo as shown by ex vivo analysis of a NeuroD2 knockout mouse. Using microarray analysis and comparing wild‐type and NeuroD2 knockout cortical networks, we identified two potential gene targets of NeuroD2 that contribute to these processes: gastrin‐releasing peptide (GRP) and the small conductance, calcium‐activated potassium channel, SK2. We found that the GRP receptor antagonist RC‐3059 and the SK2 specific blocker apamin partially reversed the effects of increased NeuroD2 expression on inhibitory synaptic drive and action potential repolarization, respectively. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability and offer insight into how these processes are coordinated during cortical development.

  July 01, 2016   doi: 10.1113/JP271953   open full text
• AMP‐activated protein kinase inhibits Kv1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation.
  Javier Moral‐Sanz, Amira D. Mahmoud, Fiona A. Ross, Jodene Eldstrom, David Fedida, D. Grahame Hardie, A. Mark Evans.
  The Journal of Physiology. June 30, 2016
  Key points Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (Kv) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP‐activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv1.5 channels in pulmonary arterial myocytes. AMPK activation by 5‐aminoimidazole‐4‐carboxamide riboside, A769662 or C13 attenuated Kv1.5 currents in pulmonary arterial myocytes, and this effect was non‐additive with respect to Kv1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv1.5 channels in cell‐free assays, and inhibited K+ currents when introduced into HEK 293 cells stably expressing Kv1.5. These results suggest that AMPK is the primary mediator of reductions in Kv1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. Abstract Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (Kv) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP‐activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1‐dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5‐aminoimidazole‐4‐carboxamide riboside and C13 and by intracellular dialysis from a patch‐pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2β2γ1 or α1β1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK‐sensitive K+ currents, which were also blocked by the selective Kv1.5 channel inhibitor diphenyl phosphine oxide‐1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv1.5 channels were phosphorylated by AMPK in cell‐free assays, and K+ currents carried by Kv1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv1.5 and/or an associated protein.

  June 30, 2016   doi: 10.1113/JP272032   open full text
• Passive heat therapy improves endothelial function, arterial stiffness and blood pressure in sedentary humans.
  Vienna E. Brunt, Matthew J. Howard, Michael A. Francisco, Brett R. Ely, Christopher T. Minson.
  The Journal of Physiology. June 30, 2016
  Key points A recent 30 year prospective study showed that lifelong sauna use reduces cardiovascular‐related and all‐cause mortality; however, the specific cardiovascular adaptations that cause this chronic protection are currently unknown. We investigated the effects of 8 weeks of repeated hot water immersion (‘heat therapy’) on various biomarkers of cardiovascular health in young, sedentary humans. We showed that, relative to a sham group which participated in thermoneutral water immersion, heat therapy increased flow‐mediated dilatation, reduced arterial stiffness, reduced mean arterial and diastolic blood pressure, and reduced carotid intima media thickness, with changes all on par or greater than what is typically observed in sedentary subjects with exercise training. Our results show for the first time that heat therapy has widespread and robust effects on vascular function, and as such, could be a viable treatment option for improving cardiovascular health in a variety of patient populations, particularly those with limited exercise tolerance and/or capabilities. Abstract The majority of cardiovascular diseases are characterized by disorders of the arteries, predominantly caused by endothelial dysfunction and arterial stiffening. Intermittent hot water immersion (‘heat therapy’) results in elevations in core temperature and changes in cardiovascular haemodynamics, such as cardiac output and vascular shear stress, that are similar to exercise, and thus may provide an alternative means of improving health which could be utilized by patients with low exercise tolerance and/or capabilities. We sought to comprehensively assess the effects of 8 weeks of heat therapy on biomarkers of vascular function in young, sedentary subjects. Twenty young, sedentary subjects were assigned to participate in 8 weeks (4–5 times per week) of heat therapy (n = 10; immersion in a 40.5°C bath sufficient to maintain rectal temperature ≥ 38.5°C for 60 min per session) or thermoneutral water immersion (n = 10; sham). Eight weeks of heat therapy increased flow‐mediated dilatation from 5.6 ± 0.3 to 10.9 ± 1.0% (P < 0.01) and superficial femoral dynamic arterial compliance from 0.06 ± 0.01 to 0.09 ±0.01 mm2 mmHg−1 (P = 0.03), and reduced (i.e. improved) aortic pulse wave velocity from 7.1 ± 0.3 to 6.1 ± 0.3 m s−1 (P = 0.03), carotid intima media thickness from 0.43 ± 0.01 to 0.37 ± 0.01 mm (P < 0.001), and mean arterial blood pressure from 83 ± 1 to 78 ± 2 mmHg (P = 0.02). No changes were observed in the sham group or for carotid arterial compliance, superficial femoral intima media thickness or endothelium‐independent dilatation. Heat therapy improved endothelium‐dependent dilatation, arterial stiffness, intima media thickness and blood pressure, indicating improved cardiovascular health. These data suggest heat therapy may provide a simple and effective tool for improving cardiovascular health in various populations.

  June 30, 2016   doi: 10.1113/JP272453   open full text
• Diacylglycerol‐mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C.
  Raymond M. Sturgeon, Neil S. Magoski.
  The Journal of Physiology. June 30, 2016
  Key points In Aplysia, reproduction is initiated by the bag cell neurons and a prolonged period of enhanced excitability known as the afterdischarge. Phosphoinositide turnover is upregulated during the afterdischarge resulting in the hydrolysis of phosphatidylinositol‐4,5‐bisphosphate by phospholipase C (PLC) and the release of diacylglycerol (DAG) and inositol trisphosphate (IP3). In whole‐cell voltage‐clamped cultured bag cell neurons, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), a synthetic DAG analogue, activates a dose‐dependent, transient, inward current (IOAG) that is enhanced by IP3, mimicked by PLC activation and dependent on basal protein kinase C (PKC) activity. OAG depolarizes bag cell neurons and triggers action potential firing in culture, and prolongs electrically stimulated afterdischarges in intact bag cell neuron clusters ex vivo. Although PKC alone cannot activate the current, it is required for IOAG; this is the first description of required obligate PKC activity working in concert with PLC, DAG and IP3 to maintain the depolarization required for prolonged excitability in Aplysia reproduction. Abstract Following synaptic input, the bag cell neurons of Aplysia undergo a long‐term afterdischarge of action potentials to secrete egg‐laying hormone and initiate reproduction. Early in the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol‐4,5‐bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). In Aplysia, little is known about the action of DAG, or any interaction with IP3; thus, we examined the effects of a synthetic DAG analogue, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), on whole‐cell voltage‐clamped cultured bag cell neurons. OAG induced a large, prolonged, Ca2+‐permeable, concentration‐dependent inward current (IOAG) that reversed at ∼−20 mV and was enhanced by intracellular IP3. A similar current was evoked by either another DAG analogue, 1,2‐dioctanoyl‐sn‐glycerol (DOG), or activating PLC with N‐(3‐trifluoromethylphenyl)‐2,4,6‐trimethylbenzenesulfonamide (m‐3M3FBS). IOAG was reduced by the general cation channel blockers Gd3+ or flufenamic acid. Work in other systems indicated that OAG activates channels independently of protein kinase C (PKC); however, we found pretreating bag cell neurons with any of the PKC inhibitors bisindolylmaleimide, sphinganine, or H7, attenuated IOAG. However, stimulating PKC with phorbol 12‐myristate 13‐acetate (PMA) did not evoke current or enhance IOAG; moreover, unlike PMA, OAG failed to trigger PKC, as confirmed by an independent bioassay. Finally, OAG or m‐3M3FBS depolarized cultured neurons, and while OAG did not provoke afterdischarges from bag cell neurons in the nervous system, it did double the duration of synaptically elicited afterdischarges. To our knowledge, this is the first report of obligate PKC activity for IOAG gating. An interaction between phosphoinositol metabolites and PKC could control the cation channel to influence afterdischarge duration.

  June 30, 2016   doi: 10.1113/JP272152   open full text
• Time course of EPSCs in ON‐type starburst amacrine cells is independent of dendritic location.
  Todd Stincic, Robert G. Smith, W. Rowland Taylor.
  The Journal of Physiology. June 29, 2016
  Key points Direction selectivity has been widely studied as an example of a complex neural computation. Directional GABA release from starburst amacrine cells (SBACs) is critical for generating directional signals in direction‐selective ganglion cells. The mechanisms producing the directional release remain unclear. For SBACs, ordered distribution of sustained and transient bipolar cell inputs along the dendrites is proposed to generate directional GABA release. This study tests whether this hypothesis applies to ON‐type SBACs. EPSCs activated at proximal and distal dendritic locations have the same time course. Therefore, the ordered arrangement of inputs from bipolar cells with different kinetic properties cannot be responsible for generating directional GABA release from ON‐type SBACs. Abstract Direction selectivity in the retina relies critically on directionally asymmetric GABA release from the dendritic tips of starburst amacrine cells (SBACs). GABA release from each radially directed dendrite is larger for motion outward from the soma toward the dendritic tips than for motion inwards toward the soma. The biophysical mechanisms generating these directional signals remain controversial. A model based on electron‐microscopic reconstructions of the mouse retina proposed that an ordered arrangement of kinetically distinct bipolar cell inputs to ON‐ and OFF‐type SBACs could produce directional GABA release. We tested this prediction by measuring the time course of EPSCs in ON‐type SBACs in the mouse retina, activated by proximal and distal light stimulation. Contrary to the prediction, the kinetics of the excitatory inputs were independent of dendritic location. Computer simulations based on 3D reconstructions of SBAC dendrites demonstrated that the response kinetics of distal inputs were not significantly altered by dendritic filtering. These direct physiological measurements, do not support the hypothesis that directional signals in SBACs arise from the ordered arrangement of kinetically distinct bipolar cell inputs.

  June 29, 2016   doi: 10.1113/JP272384   open full text
• Glutamate transporter activity promotes enhanced Na+/K+‐ATPase‐mediated extracellular K+ management during neuronal activity.
  Brian Roland Larsen, Rikke Holm, Bente Vilsen, Nanna MacAulay.
  The Journal of Physiology. June 29, 2016
  Key points Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na+/K+‐ATPase isoform combination is activated by the K+ transients occurring during neuronal activity. In the present study, we report that glutamate transporter‐mediated astrocytic Na+ transients stimulate the Na+/K+‐ATPase and thus the clearance of extracellular K+. Specifically, the astrocytic α2β1 Na+/K+‐ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+. Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+/K+‐ATPase β‐subunits. Abstract Neuronal activity is associated with transient [K+]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+/K+‐ATPase of which several subunit isoform combinations exist. The astrocytic Na+/K+‐ATPase α2β2 isoform constellation responds directly to increased [K+]o but, in addition, Na+/K+‐ATPase‐mediated K+ clearance could be governed by astrocytic [Na+]i. During most neuronal activity, glutamate is released in the synaptic cleft and is re‐absorbed by astrocytic Na+‐coupled glutamate transporters, thereby elevating [Na+]i. It thus remains unresolved whether the different Na+/K+‐ATPase isoforms are controlled by [K+]o or [Na+]i during neuronal activity. Hippocampal slice recordings of stimulus‐induced [K+]o transients with ion‐sensitive microelectrodes revealed reduced Na+/K+‐ATPase‐mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+/K+‐ATPase was not fully saturated at basal astrocytic [Na+]i, irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+/K+‐ATPase‐dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+/K+‐ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+]i transients associated with activity‐induced glutamate transporter activity.

  June 29, 2016   doi: 10.1113/JP272531   open full text
• A role for loop G in the β1 strand in GABAA receptor activation.
  Daniel T. Baptista‐Hon, Alexander Krah, Ulrich Zachariae, Tim G. Hales.
  The Journal of Physiology. June 27, 2016
  Key points The role of the β1 strand in GABAA receptor function is unclear. It lies anti‐parallel to the β2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABAA β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration–response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2‐aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the β2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of β1 strand loop G residues during agonist activation of the GABAA receptor. Abstract The GABAA receptor α subunit β1 strand runs anti‐parallel to the β2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the β1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of β1 strand residues in the GABAA β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a β strand structure. We investigated the functional role of these β1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1β2γ2 receptors by 50‐fold and eight‐fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration–response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2‐aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA‐evoked currents mediated by α1(F45C)β2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)β2γ2 and α1(T47C)β2γ2 receptors, indicating movement of the β1 strand even during allosteric activation. This is in contrast to α1(F64C)β2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the β1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.

  June 27, 2016   doi: 10.1113/JP272463   open full text
• Dynamin‐1 deletion enhances post‐tetanic potentiation and quantal size after tetanic stimulation at the calyx of Held.
  Satyajit Mahapatra, Xuelin Lou.
  The Journal of Physiology. June 27, 2016
  Key points Post‐tetanic potentiation (PTP) is attributed mainly to an increase in release probability (Pr) and/or readily‐releasable pool (RRP) in many synapses, but the role of endocytosis in PTP is unknown. Using the calyx of Held synapse from tissue‐specific dynamin‐1 knockout (cKO) mice (P16–20), we report that cKO synapses show enhanced PTP compared to control. We found significant increases in both spontaneous excitatory postsynaptic current (spEPSC) amplitude and RRP size (estimated by a train of 30 APs at 100 Hz) in cKO over control during PTP. Actin depolymerization blocks the increase in spEPSC amplitude in both control and cKO, and it abolishes the enhancement of PTP in cKO. PTP is sensitive to the PKC inhibitor GF109203X in both control and cKO. We conclude that an activity‐dependent quantal size increase contributes to the enhancement of PTP in cKO over control and an altered endocytosis affects short‐term plasticity through quantal size changes. Abstract High‐frequency stimulation leads to post‐tetanic potentiation (PTP) at many types of synapses. Previous studies suggest that PTP results primarily from a protein kinase C (PKC)‐dependent increase in release probability (Pr) and/or readily‐releasable pool (RRP) of synaptic vesicles (SVs), but the role of SV endocytosis in PTP is unknown. Using the mature calyx of Held (P16–20), we report that tissue‐specific ablation of dynamin‐1 (cKO), an endocytic protein crucial for SV regeneration, enhances PTP in cKO over control. To explore the mechanism of this enhancement, we estimated the changes in paired‐pulse ratios (PPRs) and RRP size during PTP. RRP was estimated by the back‐extrapolation of cumulative EPSC amplitudes during a train of 30 action potentials at 100 Hz (termed RRPtrain). We found an increase in RRPtrain during PTP in both control and cKO, but no significant changes in the PPR. Moreover, the amplitude and frequency of spontaneous excitatory postsynaptic currents (spEPSCs) increased during PTP in both control and cKO; however, the spEPSC amplitude in cKO during PTP was significantly larger than in control. Actin depolymerization reagent latrunculin‐B (Lat‐B) abolished the activity‐dependent increase in spEPSC amplitude in both control and cKO, but selectively blocked the enhancement of PTP in cKO, without affecting PTP in control. PKC inhibitor GF109203X nearly abolished PTP in both control and cKO. These data suggest that the quantal size increase contributes to the enhancement of PTP in dynamin‐1 cKO, and this change depends on strong synaptic activity and actin polymerization.

  June 27, 2016   doi: 10.1113/JP271937   open full text
• Intensity‐dependent timing and precision of startle response latency in larval zebrafish.
  Eileen L. Troconis, Alexander J. Ordoobadi, Thomas F. Sommers, Razina Aziz‐Bose, Ashley R. Carter, Josef G. Trapani.
  The Journal of Physiology. June 27, 2016
  Key points Using high‐speed videos time‐locked with whole‐animal electrical recordings, simultaneous measurement of behavioural kinematics and field potential parameters of C‐start startle responses allowed for discrimination between short‐latency and long‐latency C‐starts (SLCs vs. LLCs) in larval zebrafish. Apart from their latencies, SLC kinematics and SLC field potential parameters were intensity independent. Increasing stimulus intensity increased the probability of evoking an SLC and decreased mean SLC latencies while increasing their precision; subtraction of field potential latencies from SLC latencies revealed a fixed time delay between the two measurements that was intensity independent. The latency and the precision in the latency of the SLC field potentials were linearly correlated to the latencies and precision of the first evoked action potentials (spikes) in hair‐cell afferent neurons of the lateral line. Together, these findings indicate that first spike latency (FSL) is a fast encoding mechanism that can serve to precisely initiate startle responses when speed is critical for survival. Abstract Vertebrates rely on fast sensory encoding for rapid and precise initiation of startle responses. In afferent sensory neurons, trains of action potentials (spikes) encode stimulus intensity within the onset time of the first evoked spike (first spike latency; FSL) and the number of evoked spikes. For speed of initiation of startle responses, FSL would be the more advantageous mechanism to encode the intensity of a threat. However, the intensity dependence of FSL and spike number and whether either determines the precision of startle response initiation is not known. Here, we examined short‐latency startle responses (SLCs) in larval zebrafish and tested the hypothesis that first spike latencies and their precision (jitter) determine the onset time and precision of SLCs. We evoked startle responses via activation of Channelrhodopsin (ChR2) expressed in ear and lateral line hair cells and acquired high‐speed videos of head‐fixed larvae while simultaneously recording underlying field potentials. This method allowed for discrimination between primary SLCs and less frequent, long‐latency startle responses (LLCs). Quantification of SLC kinematics and field potential parameters revealed that, apart from their latencies, they were intensity independent. We found that increasing stimulus intensity decreased SLC latencies while increasing their precision, which was significantly correlated with corresponding changes in field potential latencies and their precision. Single afferent neuron recordings from the lateral line revealed a similar intensity‐dependent decrease in first spike latencies and their jitter, which could account for the intensity‐dependent changes in timing and precision of startle response latencies.

  June 27, 2016   doi: 10.1113/JP272466   open full text
• The calcium–frequency response in the rat ventricular myocyte: an experimental and modelling study.
  Sara Gattoni, Åsmund Treu Røe, Michael Frisk, William E. Louch, Steven A. Niederer, Nicolas P. Smith.
  The Journal of Physiology. June 26, 2016
  Key points In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force–frequency response (FFR). The majority of mammals have a positive force–frequency relationship (FFR). In rat the FFR is controversial. We derive a species‐ and temperature‐specific data‐driven model of the rat ventricular myocyte. As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium–frequency response (CFR) in our model and three altered models. The results show a biphasic peak calcium–frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency‐dependent increase in diastolic calcium. Alterations to the model reveal that inclusion of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated L‐type channel and transient outward K+ current activity enhances the positive magnitude calcium–frequency response, and the absence of CAMKII‐mediated increase in activity of the sarco/endoplasmic reticulum Ca2+‐ATPase induces a negative magnitude calcium–frequency response. Abstract An increase in heart rate affects the strength of cardiac contraction by altering the Ca2+ transient as a response to physiological demands. This is described by the force–frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat‐based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca2+ transient characteristics and showed a biphasic peak calcium–frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated increase in sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L‐type channel and transient outward K+ current activity and (3) Na+/K+ pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca2+–frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca2+.

  June 26, 2016   doi: 10.1113/JP272011   open full text
• The human motor neuron pools receive a dominant slow‐varying common synaptic input.
  Francesco Negro, Utku Şükrü Yavuz, Dario Farina.
  The Journal of Physiology. June 21, 2016
  Key points Motor neurons in a pool receive both common and independent synaptic inputs, although the proportion and role of their common synaptic input is debated. Classic correlation techniques between motor unit spike trains do not measure the absolute proportion of common input and have limitations as a result of the non‐linearity of motor neurons. We propose a method that for the first time allows an accurate quantification of the absolute proportion of low frequency common synaptic input (<5 Hz) to motor neurons in humans. We applied the proposed method to three human muscles and determined experimentally that they receive a similar large amount (>60%) of common input, irrespective of their different functional and control properties. These results increase our knowledge about the role of common and independent input to motor neurons in force control. Abstract Motor neurons receive both common and independent synaptic inputs. This observation is classically based on the presence of a significant correlation between pairs of motor unit spike trains. The functional significance of different relative proportions of common input across muscles, individuals and conditions is still debated. One of the limitations in our understanding of correlated input to motor neurons is that it has not been possible so far to quantify the absolute proportion of common input with respect to the total synaptic input received by the motor neurons. Indeed, correlation measures of pairs of output spike trains only allow for relative comparisons. In the present study, we report for the first time an approach for measuring the proportion of common input in the low frequency bandwidth (<5 Hz) to a motor neuron pool in humans. This estimate is based on a phenomenological model and the theoretical fitting of the experimental values of coherence between the permutations of groups of motor unit spike trains. We demonstrate the validity of this theoretical estimate with several simulations. Moreover, we applied this method to three human muscles: the abductor digiti minimi, tibialis anterior and vastus medialis. Despite these muscles having different functional roles and control properties, as confirmed by the results of the present study, we estimate that their motor pools receive a similar and large (>60%) proportion of common low frequency oscillations with respect to their total synaptic input. These results suggest that the central nervous system provides a large amount of common input to motor neuron pools, in a similar way to that for muscles with different functional and control properties.

  June 21, 2016   doi: 10.1113/JP271748   open full text
• Molecular and cellular neurocardiology: development, and cellular and molecular adaptations to heart disease.
  Beth A. Habecker, Mark E. Anderson, Susan J. Birren, Keiichi Fukuda, Neil Herring, Donald B. Hoover, Hideaki Kanazawa, David J. Paterson, Crystal M. Ripplinger.
  The Journal of Physiology. June 17, 2016
  Abstract The nervous system and cardiovascular system develop in concert and are functionally interconnected in both health and disease. This white paper focuses on the cellular and molecular mechanisms that underlie neural–cardiac interactions during development, during normal physiological function in the mature system, and during pathological remodelling in cardiovascular disease. The content on each subject was contributed by experts, and we hope that this will provide a useful resource for newcomers to neurocardiology as well as aficionados.

  June 17, 2016   doi: 10.1113/JP271840   open full text
• Translational neurocardiology: preclinical models and cardioneural integrative aspects.
  J. L. Ardell, M. C. Andresen, J. A. Armour, G. E. Billman, P.‐S. Chen, R. D. Foreman, N. Herring, D. S. O'Leary, H. N. Sabbah, H. D. Schultz, K. Sunagawa, I. H. Zucker.
  The Journal of Physiology. June 17, 2016
  Abstract Neuronal elements distributed throughout the cardiac nervous system, from the level of the insular cortex to the intrinsic cardiac nervous system, are in constant communication with one another to ensure that cardiac output matches the dynamic process of regional blood flow demand. Neural elements in their various ‘levels’ become differentially recruited in the transduction of sensory inputs arising from the heart, major vessels, other visceral organs and somatic structures to optimize neuronal coordination of regional cardiac function. This White Paper will review the relevant aspects of the structural and functional organization for autonomic control of the heart in normal conditions, how these systems remodel/adapt during cardiac disease, and finally how such knowledge can be leveraged in the evolving realm of autonomic regulation therapy for cardiac therapeutics.

  June 17, 2016   doi: 10.1113/JP271869   open full text
• Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut.
  Ching‐Ying Huang, Wei‐Ting Kuo, Chung‐Yen Huang, Tsung‐Chun Lee, Chin‐Tin Chen, Wei‐Hao Peng, Kuo‐Shyan Lu, Chung‐Yi Yang, Linda Chia‐Hui Yu.
  The Journal of Physiology. June 17, 2016
  Key points Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria‐derived septic complications. Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive. A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes. Pyruvate suppressed epithelial cell death in an ATP‐independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut. Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti‐necroptotic role of glycolytic pyruvate under ischaemic stress. Abstract Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor‐interacting protein kinase (RIP)‐dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium‐glucose transporter 1 ameliorated ischaemia/reperfusion‐induced epithelial injury, partly via anti‐apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1‐dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell‐permeable pyruvate suppressed epithelial cell death in an ATP‐independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal‐to‐serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP.

  June 17, 2016   doi: 10.1113/JP272208   open full text
• Immature endothelial cells initiate endothelin‐mediated constriction of newborn arteries.
  Fumin Chang, Sheila Flavahan, Nicholas A. Flavahan.
  The Journal of Physiology. June 16, 2016
  Key points Endothelial expression and the release of endothelin‐1 (ET‐1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET‐1 peptides and, in response to endothelial stimulation, rapidly release ET‐1 and initiate powerful ET‐1‐mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET‐1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re‐emergence of this phenotype may contribute to the development of vascular disease. Abstract Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin‐1 (ET‐1) and initiates endothelium‐dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial‐denudation, by ET‐1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin‐converting enzyme (phosphoramidon or SM19712). ET‐1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET‐1 caused similar concentration‐dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET‐1 from P1 but not P21 aortas. Endothelial expression of ET‐1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET‐1 peptides, rapidly release ET‐1 in response to endothelial stimulation, and initiate ET‐1‐mediated endothelium‐dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET‐1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury.

  June 16, 2016   doi: 10.1113/JP272176   open full text
• Attenuated flow‐induced dilatation of middle cerebral arteries is related to increased vascular oxidative stress in rats on a short‐term high salt diet.
  Anita Cosic, Ivana Jukic, Ana Stupin, Martina Mihalj, Zrinka Mihaljevic, Sanja Novak, Rosemary Vukovic, Ines Drenjancevic.
  The Journal of Physiology. June 16, 2016
  Key points Recent studies have shown that high salt (HS) intake leads to endothelial dysfunction and impaired vascular reactivity in different vascular beds in both animal and human models, due to increased oxidative stress. The objective of this study was to assess vascular response to flow‐induced dilatation (FID) and to elucidate the role of vascular oxidative stress/antioxidative capacity in middle cerebral arteries (MCAs) of HS‐fed rats in vitro. The novelty of this study is in demonstrating impaired flow‐induced dilatation of MCAs and down‐regulation of vascular antioxidant genes with HS intake, leading to increased levels of oxidative stress in blood vessels and peripheral lymph organs, which together contribute to impaired FID. In addition, results show increased oxidative stress in leukocytes of peripheral lymph organs, suggesting the occurrence of inflammatory processes due to HS intake. Recirculation of leukocytes might additionally increase vascular oxidative stress in vivo. Abstract The aim of this study was to determine flow‐induced dilatation (FID) and the role of oxidative stress/antioxidative capacity in isolated, pressurized middle cerebral arteries (MCAs) of high salt (HS)‐fed rats. Healthy male Sprague‐Dawley rats (11 weeks old) were fed low salt (0.4% NaCl; LS group) or high salt (4% NaCl; HS group) diets for 1 week. Reactivity of MCAs in response to stepwise increases in pressure gradient (Δ10–Δ100 mmHg) was determined in the absence or presence of the superoxide dismutase (SOD) mimetic TEMPOL and/or the nitric oxide synthases (NOS) inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME). mRNA levels of antioxidative enzymes, NAPDH‐oxidase components, inducible (iNOS) and endothelial nitric oxide synthases (eNOS) were determined by quantitative real‐time PCR. Blood pressure (BP), antioxidant enzymes activity, oxidative stress in peripheral leukocytes, lipid peroxidation products and the antioxidant capacity of plasma were measured for both groups. FID was reduced in the HS group compared to the LS group. The presence of TEMPOL restored dilatation in the HS group, with no effect in the LS group. Expression of glutathione peroxidase 4 (GPx4) and iNOS in the HS group was significantly decreased; oxidative stress was significantly higher in the HS group compared to the LS group. HS intake significantly induced basal reactive oxygen species production in the leukocytes of mesenteric lymph nodes and splenocytes, and intracellular production after stimulation in peripheral lymph nodes. Antioxidant enzyme activity and BP were not affected by HS diet. Low GPx4 expression, increased superoxide production in leukocytes, and decreased iNOS expression are likely to underlie increased oxidative stress and reduced nitric oxide bioavailability, leading to impairment of FID in the HS group without changes in BP values.

  June 16, 2016   doi: 10.1113/JP272297   open full text
• Rac1 governs exercise‐stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice.
  Lykke Sylow, Ida L. Nielsen, Maximilian Kleinert, Lisbeth L. V. Møller, Thorkil Ploug, Peter Schjerling, Philip J. Bilan, Amira Klip, Thomas E. Jensen, Erik A. Richter.
  The Journal of Physiology. June 16, 2016
  Key point Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. The GTPase Rac1 can be activated by muscle contraction and has been found to be necessary for insulin‐stimulated glucose uptake, although its role in exercise‐stimulated glucose uptake is unknown. We show that Rac1 regulates the translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle during exercise. We find that Rac1 knockout mice display significantly reduced glucose uptake in skeletal muscle during exercise. Abstract Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. Despite extensive efforts, the signalling mechanisms vital for glucose uptake during exercise are not yet fully understood, although the GTPase Rac1 is a candidate molecule. The present study investigated the role of Rac1 in muscle glucose uptake and substrate utilization during treadmill exercise in mice in vivo. Exercise‐induced uptake of radiolabelled 2‐deoxyglucose at 65% of maximum running capacity was blocked in soleus muscle and decreased by 80% and 60% in gastrocnemius and tibialis anterior muscles, respectively, in muscle‐specific inducible Rac1 knockout (mKO) mice compared to wild‐type littermates. By developing an assay to quantify endogenous GLUT4 translocation, we observed that GLUT4 content at the sarcolemma in response to exercise was reduced in Rac1 mKO muscle. Our findings implicate Rac1 as a regulatory element critical for controlling glucose uptake during exercise via regulation of GLUT4 translocation.

  June 16, 2016   doi: 10.1113/JP272039   open full text
• Cerebrovascular disorders caused by hyperfibrinogenaemia.
  Nino Muradashvili, Reeta Tyagi, Neetu Tyagi, Suresh C. Tyagi, David Lominadze.
  The Journal of Physiology. June 16, 2016
  Key points Hyperfibrinogenaemia (HFg) results in vascular remodelling, and fibrinogen (Fg) and amyloid β (Aβ) complex formation is a hallmark of Alzheimer's disease. However, the interconnection of these effects, their mechanisms and implications in cerebrovascular diseases are not known. Using a mouse model of HFg, we showed that at an elevated blood level, Fg increases cerebrovascular permeability via mainly caveolar protein transcytosis. This enhances deposition of Fg in subendothelial matrix and interstitium making the immobilized Fg a readily accessible substrate for binding Aβ and cellular prion protein (PrPC), the protein that is thought to have a greater effect on memory than Aβ. We showed that enhanced formation of Fg–Aβ and Fg–PrPC complexes are associated with reduction in short‐term memory. The present study delineates a new mechanistic pathway for vasculo‐neuronal dysfunctions found in inflammatory cardiovascular and cerebrovascular diseases associated with an elevated blood level of Fg. Abstract Many cardiovascular diseases are associated with inflammation and as such are accompanied by an increased blood level of fibrinogen (Fg). Besides its well‐known prothrombotic effects Fg seems to have other destructive roles in developing microvascular dysfunction that include changes in vascular reactivity and permeability. Increased permeability of brain microvessels has the most profound effects as it may lead to cerebrovascular remodelling and result in memory reduction. The goal of the present study was to define mechanisms of cerebrovascular permeability and associated reduction in memory induced by elevated blood content of Fg. Genetically modified, transgenic hyperfibrinogenic (HFg) mice were used to study cerebrovascular transcellular and paracellular permeability in vivo. The extent of caveolar formation and the role of caveolin‐1 signalling were evaluated by immunohistochemistry (IHC) and Western blot (WB) analysis in brain samples from experimental animals. Formation of Fg complexes with amyloid β (Aβ) and with cellular prion protein (PrPC) were also assessed with IHC and WB analysis. Short‐term memory of mice was assessed by novel object recognition and Y‐maze tests. Caveolar protein transcytosis was found to have a prevailing role in overall increased cerebrovascular permeability in HFg mice. These results were associated with enhanced formation of caveolae. Increased formation of Fg–PrPC and Fg–Aβ complexes were correlated with reduction in short‐term memory in HFg mice. Using the model of hyperfibrinogenaemia, the present study shows a novel mechanistic pathway of inflammation‐induced and Fg‐mediated reduction in short‐term memory.

  June 16, 2016   doi: 10.1113/JP272558   open full text
• Short‐term depression of gap junctional coupling in reticular thalamic neurons of absence epileptic rats.
  Denise Kohmann, Annika Lüttjohann, Thomas Seidenbecher, Philippe Coulon, Hans‐Christian Pape.
  The Journal of Physiology. June 16, 2016
  Key points Gap junctional electrical coupling between neurons of the reticular thalamic nucleus (RTN) is critical for hypersynchrony in the thalamo‐cortical network. This study investigates the role of electrical coupling in pathological rhythmogenesis in RTN neurons in a rat model of absence epilepsy. Rhythmic activation resulted in a Ca2+‐dependent short‐term depression (STD) of electrical coupling between pairs of RTN neurons in epileptic rats, but not in RTN of a non‐epileptic control strain. Pharmacological blockade of gap junctions in RTN in vivo induced a depression of seizure activity. The STD of electrical coupling represents a mechanism of Ca2+ homeostasis in RTN aimed to counteract excessive synchronization. Abstract Neurons in the reticular thalamic nucleus (RTN) are coupled by electrical synapses, which play a major role in regulating synchronous activity. This study investigates electrical coupling in RTN neurons from a rat model of childhood absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS), compared with a non‐epileptic control (NEC) strain, to assess the impact on pathophysiological rhythmogenesis. Whole‐cell recordings were obtained from pairs of RTN neurons of GAERS and NEC in vitro. Coupling was determined by injection of hyperpolarizing current steps in one cell and monitoring evoked voltage responses in both activated and coupled cell. The coupling coefficient (cc) was compared under resting condition, during pharmacological interventions and repeated activation using a series of current injections. The effect of gap junctional coupling on seizure expression was investigated by application of gap junctional blockers into RTN of GAERS in vivo. At resting conditions, cc did not differ between GAERS and NEC. During repeated activation, cc declined in GAERS but not in NEC. This depression in cc was restored within 25 s and was prevented by intracellular presence of BAPTA in the activated but not in the coupled cell. Local application of gap junctional blockers into RTN of GAERS in vivo resulted in a decrease of spike wave discharge (SWD) activity. Repeated activation results in a short‐term depression (STD) of gap junctional coupling in RTN neurons of GAERS, depending on intracellular Ca2+ mechanisms in the activated cell. As blockage of gap junctions in vivo results in a decrease of SWD activity, the STD observed in GAERS is considered a compensatory mechanism, aimed to dampen SWD activity.

  June 16, 2016   doi: 10.1113/JP271811   open full text
• Clinical neurocardiology defining the value of neuroscience‐based cardiovascular therapeutics.
  Kalyanam Shivkumar, Olujimi A. Ajijola, Inder Anand, J. Andrew Armour, Peng‐Sheng Chen, Murray Esler, Gaetano M. Ferrari, Michael C. Fishbein, Jeffrey J. Goldberger, Ronald M. Harper, Michael J. Joyner, Sahib S. Khalsa, Rajesh Kumar, Richard Lane, Aman Mahajan, Sunny Po, Peter J. Schwartz, Virend K. Somers, Miguel Valderrabano, Marmar Vaseghi, Douglas P. Zipes.
  The Journal of Physiology. June 14, 2016
  Abstract The autonomic nervous system regulates all aspects of normal cardiac function, and is recognized to play a critical role in the pathophysiology of many cardiovascular diseases. As such, the value of neuroscience‐based cardiovascular therapeutics is increasingly evident. This White Paper reviews the current state of understanding of human cardiac neuroanatomy, neurophysiology, pathophysiology in specific disease conditions, autonomic testing, risk stratification, and neuromodulatory strategies to mitigate the progression of cardiovascular diseases.

  June 14, 2016   doi: 10.1113/JP271870   open full text
• GABAB receptors enhance excitatory responses in isolated rat retinal ganglion cells.
  Jay Garaycochea, Malcolm M. Slaughter.
  The Journal of Physiology. June 14, 2016
  Key points GABA is an inhibitory transmitter but can sometimes produce paradoxical excitatory effects through synaptic networks. We found a novel GABA‐mediated excitation within a single retinal cell. It involves a chain of events from receptor stimulation to the sequential modulation of two associated channels, resulting in enhanced neuroexcitability. GABAB receptor activation selectively suppresses N‐type calcium channels. The BK‐type potassium channels are exclusively linked to the N‐type calcium channel. Thus, stimulation of GABAB receptors suppresses an outward current, increasing the excitatory range of single neurons. Abstract GABAB receptors (GABABRs) suppress voltage‐gated calcium channels and activate G‐protein coupled potassium channels (GIRK and TREK channels), both mechanisms serving to inhibit neurons. In isolated rat retinal spiking neurons, GABABRs produce both actions but the net effect is to enhance excitatory signals. This is because GABABRs selectively suppress N‐type calcium channels, which in turn are specifically linked to BK channels. Consequently, when GABABRs are stimulated there is a reduction in outward current, allowing neurons to extend their level of depolarization. Whereas many retinal neurons use L‐type channels to stimulate vesicle fusion, the suppression of N‐type channels augments dynamic range without affecting transmitter release.

  June 14, 2016   doi: 10.1113/JP272374   open full text
• Ventilatory responses to muscle metaboreflex activation in chronic obstructive pulmonary disease.
  Richard M. Bruce, Alice Turner, Michael J. White.
  The Journal of Physiology. June 14, 2016
  Key points Recent evidence indicates a role for group III/IV muscle afferents in reflex control of the human ventilatory response to exercise. Dyspnoea in chronic obstructive pulmonary disease (COPD) may be linked to this reflex response. This study shows that activation of the muscle metaboreflex causes a ventilatory response in COPD patients but not in healthy controls. This indicates abnormal involvement of muscle afferents in the control of ventilation in COPD which may be a contributing factor to exercise dyspnoea. Abstract Blockade of thin fibre muscle afferent feedback during dynamic exercise reduces exercise hyperpnoea in health and chronic obstructive pulmonary disease (COPD). Therefore, we hypothesised that activation of the muscle metaboreflex at rest would cause hyperpnoea. We evaluated the effect of muscle metaboreflex activation on ventilation, in resting COPD patients and healthy participants. Following a bout of rhythmic hand grip exercise, post exercise circulatory occlusion (PECO) was applied to the resting forearm to sustain activation of the muscle metaboreflex, in 18 COPD patients (FEV1/FVC ratio < 70%), 9 also classified as chronically hypercapnic, and 9 age‐ and gender‐matched controls. The cardiovascular response to exercise and the sustained blood pressure elevation during PECO was similar in patients and controls. During exercise ventilation increased by 6.64 ± 0.84 in controls and significantly (P < 0.05) more, 8.38 ± 0.81 l min−1, in patients. During PECO it fell to baseline levels in controls but remained significantly (P < 0.05) elevated by 2.78 ± 0.51 l min−1 in patients until release of circulatory occlusion, with no significant difference in responses between patient groups. Muscle metaboreflex activation causes increased ventilation in COPD patients but not in healthy participants. Chronic hypercapnia in COPD patients does not exaggerate this response. The muscle metaboreflex appears to be abnormally involved in the control of ventilation in COPD and may be a contributing factor to exercise dyspnoea.

  June 14, 2016   doi: 10.1113/JP272329   open full text
• Error signals driving locomotor adaptation: cutaneous feedback from the foot is used to adapt movement during perturbed walking.
  Julia T. Choi, Peter Jensen, Jens Bo Nielsen, Laurent J. Bouyer.
  The Journal of Physiology. June 14, 2016
  Key points Sensory input from peripheral receptors are important for the regulation of walking patterns. Cutaneous input mediates muscle responses to deal with immediate external perturbations. In this study we focused on the role of cutaneous feedback in locomotor adaptation that takes place over minutes of training. We show that interfering with cutaneous feedback reduced adaptation to ankle perturbations during walking. These results help us understand the neural mechanisms underlying walking adaptation, and have clinical implications for treating walking impairments after neurological injuries. Abstract Locomotor patterns must be adapted to external forces encountered during daily activities. The contribution of different sensory inputs to detecting perturbations and adapting movements during walking is unclear. In the present study, we examined the role of cutaneous feedback in adapting walking patterns to force perturbations. Forces were applied to the ankle joint during the early swing phase using an electrohydraulic ankle–foot orthosis. Repetitive 80 Hz electrical stimulation was applied to disrupt cutaneous feedback from the superficial peroneal nerve (foot dorsum) and medial plantar nerve (foot sole) during walking (Choi et al. 2013). Sensory tests were performed to measure the cutaneous touch threshold and perceptual threshold of force perturbations. Ankle movement were measured when the subjects walked on the treadmill over three periods: baseline (1 min), adaptation (1 min) and post‐adaptation (3 min). Subjects (n = 10) showed increased touch thresholds measured with Von Frey monofilaments and increased force perception thresholds with stimulation. Stimulation reduced the magnitude of walking adaptation to force perturbation. In addition, we compared the effects of interrupting cutaneous feedback using anaesthesia (n = 5) instead of repetitive nerve stimulation. Foot anaesthesia reduced ankle adaptation to external force perturbations during walking. The results of the present study suggest that cutaneous input plays a role in force perception, and may contribute to the ‘error’ signal involved in driving walking adaptation when there is a mismatch between expected and actual force.

  June 14, 2016   doi: 10.1113/JP271996   open full text
• The L‐type Ca2+ channel facilitates abnormal metabolic activity in the cTnI‐G203S mouse model of hypertrophic cardiomyopathy.
  Helena Viola, Victoria Johnstone, Henrietta Cserne Szappanos, Tara Richman, Tatiana Tsoutsman, Aleksandra Filipovska, Christopher Semsarian, Livia Hool.
  The Journal of Physiology. June 12, 2016
  Key points Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism. The L‐type Ca2+ channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network. We find that L‐type Ca2+ channel kinetics are altered in cTnI‐G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI‐G203S cardiac myocytes. These responses occur as a result of impaired communication between the L‐type Ca2+ channel and cytoskeletal protein F‐actin, involving decreased movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel, resulting in a ‘hypermetabolic’ mitochondrial state. We propose that L‐type Ca2+ channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity. Abstract Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L‐type Ca2+ channel (ICa‐L) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of ICa‐L in regulating mitochondrial function in 25‐ to 30‐week‐old cardiomyopathic mice expressing the human disease‐causing mutation Gly203Ser in cTnI (cTnI‐G203S). The inactivation rate of ICa‐L is significantly faster in cTnI‐G203S myocytes [cTnI‐G203S: τ1 = 40.68 ± 3.22, n = 10 vs. wild‐type (wt): τ1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of ICa‐L caused a greater increase in mitochondrial membrane potential (Ψm, 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between ICa‐L and F‐actin, involving lack of dynamic movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel. Similar responses were observed in precardiomyopathic mice. ICa‐L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa‐L and mitochondria, resulting in a ‘hypermetabolic’ state. This might contribute to development of cTnI‐G203S cardiomyopathy because the response is present in young precardiomyopathic mice. ICa‐L antagonists might be effective in reducing the cardiomyopathy by altering mitochondrial function.

  June 12, 2016   doi: 10.1113/JP271681   open full text
• Use dependence of peripheral nociceptive conduction in the absence of tetrodotoxin‐resistant sodium channel subtypes.
  Tal Hoffmann, Katrin Kistner, Mohammed Nassar, Peter W. Reeh, Christian Weidner.
  The Journal of Physiology. June 12, 2016
  Key points This study examines conduction in peripheral nerves and its use dependence in tetrodotoxin‐resistant (TTXr) sodium channel (Nav1.8, Nav1.9) knockout and wildtype animals. We observed use‐dependent decreases of single fibre and compound action potential amplitude in peripheral mouse C‐fibres (wildtype). This matches the previously published hypothesis that increased Na/K‐pump activity is not the underlying mechanism for use‐dependent changes of neural conduction. Knocking out TTXr sodium channels influences use‐dependent changes of conductive properties (action potential amplitude, latency, conduction safety) in the order Nav1.8 KO > Nav1.9KO > wildtype. This is most likely explained by different subsets of presumably (relatively) Nav1.7‐rich conducting fibres in knockout animals as compared to wildtypes, in combination with reduced per‐pulse sodium influx. Abstract Use dependency of peripheral nerves, especially of nociceptors, correlates with receptive properties. Slow inactivation of voltage‐gated sodium channels has been discussed to be the underlying mechanism – pointing to a receptive class‐related difference of sodium channel equipment. Using electrophysiological recordings of single unmyelinated cutaneous fibres and their compound action potential (AP), we evaluated use‐dependent changes in mouse peripheral nerves, and the contribution of the tetrodotoxin‐resistant (TTXr) sodium channels Nav1.8 and Nav1.9 to these changes. Nerve fibres were electrically stimulated using single or double pulses at 2 Hz. Use‐dependent changes of latency, AP amplitude, and duration as well as the fibres’ ability to follow the stimulus were evaluated. AP amplitudes substantially diminished in used fibres from C57BL/6 but increased in Nav1.8 knockout (KO) mice, with Nav1.9 KO in between. Activity‐induced latency slowing was in contrast the most pronounced in Nav1.8 KOs and the least in wildtype mice. The genotype was also predictive of how long fibres could follow the double pulsed stimulus with wildtype fibres blocking first and Nav1.8 KO fibres enduring the longest. In contrast, changes in spike duration were less pronounced and displayed no significant tendency. Thus, all major measures of peripheral nerve accommodation (amplitude, latency and durability) depended on genotype. All use‐dependent changes appeared in the order NaV1.8 KO > NaV1.9 KO > wildtype, which is most likely explained by the relative contribution of Nav1.7 varying in the same order and the amounts of per‐pulse sodium influx expected in the opposite order.

  June 12, 2016   doi: 10.1113/JP272082   open full text
• Cellular and circuit mechanisms underlying spinocerebellar ataxias.
  Pratap Meera, Stefan M. Pulst, Thomas S. Otis.
  The Journal of Physiology. June 12, 2016
  Abstract Degenerative ataxias are a common form of neurodegenerative disease that affect about 20 individuals per 100,000. The autosomal dominant spinocerebellar ataxias (SCAs) are caused by a variety of protein coding mutations (single nucleotide changes, deletions and expansions) in single genes. Affected genes encode plasma membrane and intracellular ion channels, membrane receptors, protein kinases, protein phosphatases and proteins of unknown function. Although SCA‐linked genes are quite diverse they share two key features: first, they are highly, although not exclusively, expressed in cerebellar Purkinje neurons (PNs), and second, when mutated they lead ultimately to the degeneration of PNs. In this review we summarize ataxia‐related changes in PN neurophysiology that have been observed in various mouse knockout lines and in transgenic models of human SCA. We also highlight emerging evidence that altered metabotropic glutamate receptor signalling and disrupted calcium homeostasis in PNs form a common, early pathophysiological mechanism in SCAs. Together these findings indicate that aberrant calcium signalling and profound changes in PN neurophysiology precede PN cell loss and are likely to lead to cerebellar circuit dysfunction that explains behavioural signs of ataxia characteristic of the disease.

  June 12, 2016   doi: 10.1113/JP271897   open full text
• Adrenal, metabolic and cardio‐renal dysfunction develops after pregnancy in rats born small or stressed by physiological measurements during pregnancy.
  Jean N. Cheong, James S. M. Cuffe, Andrew J. Jefferies, Karen M. Moritz, Mary E. Wlodek.
  The Journal of Physiology. June 12, 2016
  Key points Women born small are at an increased risk of developing pregnancy complications. Stress may further increase a woman's likelihood for an adverse pregnancy. Adverse pregnancy adaptations can lead to long‐term diseases even after her pregnancy. The current study investigated the effects of stress during pregnancy on the long‐term adrenal, metabolic and cardio‐renal health of female rats that were born small. Stress programmed increased adrenal Mc2r gene expression, a higher insulin secretory response to glucose during intraperitoneal glucose tolerance test (+36%) and elevated renal creatinine clearance after pregnancy. Females that were born small had increased homeostatic model assessment‐insulin resistance and elevated systolic blood pressure after pregnancy, regardless of stress exposure. These findings suggest that being born small or being stressed during pregnancy programs long‐term adverse health outcomes after pregnancy. However, stress in pregnancy does not exacerbate the long‐term adverse health outcomes for females that were born small. Abstract Females born small are more likely to experience complications during their pregnancy, including pregnancy‐induced hypertension, pre‐eclampsia and gestational diabetes. The risk of developing complications is increased by stress exposure during pregnancy. In addition, pregnancy complications may predispose the mother to diseases after pregnancy. We determined whether stress during pregnancy would exacerbate the adrenal, metabolic and cardio‐renal dysfunction of growth‐restricted females in later life. Late gestation bilateral uterine vessel ligation was performed in Wistar Kyoto rats to induce growth restriction. At 4 months, growth‐restricted and control female offspring were mated with normal males. Those allocated to the stressed group had physiological measurements [metabolic cage, tail cuff blood pressure, intraperitoneal glucose tolerance test (IPGTT)] conducted during pregnancy whilst the unstressed groups were unhandled. After the completion of pregnancy, dams were aged to 12 months and blood pressure, and metabolic and renal function were assessed. At 13 months, adrenal glands, pancreases and plasma were collected at post‐mortem. Females stressed during pregnancy had increased adrenal Mc2r gene expression (+22%), higher insulin secretory response to glucose during IPGTT (+36%) and higher creatinine clearance (+29%, indicating increased estimated glomerular filtration rate). In contrast, females that were born small had increased homeostatic model assessment‐insulin resistance (+54%), increased water intake (+23%), urine output (+44%) and elevated systolic blood pressure (+7%) regardless of exposure to stress. Our findings suggest that low maternal birth weight and maternal stress exposure during pregnancy are both independently detrimental for long‐term adrenal, metabolic and cardio‐renal health of the mother, although their effects were not exacerbated.

  June 12, 2016   doi: 10.1113/JP272212   open full text
• Decreased arterial PO2, not O2 content, increases blood flow through intrapulmonary arteriovenous anastomoses at rest.
  Joseph W. Duke, James T. Davis, Benjamin J. Ryan, Jonathan E. Elliott, Kara M. Beasley, Jerold A. Hawn, William C. Byrnes, Andrew T. Lovering.
  The Journal of Physiology. June 09, 2016
  Key points The mechanism(s) that regulate hypoxia‐induced blood flow through intrapulmonary arteriovenous anastomoses (QIPAVA) are currently unknown. Our previous work has demonstrated that the mechanism of hypoxia‐induced QIPAVA is not simply increased cardiac output, pulmonary artery systolic pressure or sympathetic nervous system activity and, instead, it may be a result of hypoxaemia directly. To determine whether it is reduced arterial PO2 (PaO2) or O2 content (CaO2) that causes hypoxia‐induced QIPAVA, individuals were instructed to breathe room air and three levels of hypoxic gas at rest before (control) and after CaO2 was reduced by 10% by lowering the haemoglobin concentration (isovolaemic haemodilution; Low [Hb]). QIPAVA, assessed by transthoracic saline contrast echocardiography, significantly increased as PaO2 decreased and, despite reduced CaO2 (via isovolaemic haemodilution), was similar at iso‐PaO2. These data suggest that, with alveolar hypoxia, low PaO2 causes the hypoxia‐induced increase in QIPAVA, although where and how this is detected remains unknown. Abstract Alveolar hypoxia causes increased blood flow through intrapulmonary arteriovenous anastomoses (QIPAVA) in healthy humans at rest. However, it is unknown whether the stimulus regulating hypoxia‐induced QIPAVA is decreased arterial PO2 (PaO2) or O2 content (CaO2). CaO2 is known to regulate blood flow in the systemic circulation and it is suggested that IPAVA may be regulated similar to the systemic vasculature. Thus, we hypothesized that reduced CaO2 would be the stimulus for hypoxia‐induced QIPAVA. Blood volume (BV) was measured using the optimized carbon monoxide rebreathing method in 10 individuals. Less than 5 days later, subjects breathed room air, as well as 18%, 14% and 12.5% O2, for 30 min each, in a randomized order, before (CON) and after isovolaemic haemodilution (10% of BV withdrawn and replaced with an equal volume of 5% human serum albumin–saline mixture) to reduce [Hb] (Low [Hb]). PaO2 was measured at the end of each condition and QIPAVA was assessed using transthoracic saline contrast echocardiography. [Hb] was reduced from 14.2 ± 0.8 to 12.8 ± 0.7 g dl−1 (10 ± 2% reduction) from CON to Low [Hb] conditions. PaO2 was no different between CON and Low [Hb], although CaO2 was 10.4%, 9.2% and 9.8% lower at 18%, 14% and 12.5% O2, respectively. QIPAVA significantly increased as PaO2 decreased and, despite reduced CaO2, was similar at iso‐PaO2. These data suggest that, with alveolar hypoxia, low PaO2 causes the hypoxia‐induced increase in QIPAVA. Whether the low PO2 is detected at the carotid body, airway and/or the vasculature remains unknown.

  June 09, 2016   doi: 10.1113/JP272211   open full text
• Computational cardiology and risk stratification for sudden cardiac death: one of the grand challenges for cardiology in the 21st century.
  Adam P. Hill, Matthew D. Perry, Najah Abi‐Gerges, Jean‐Philippe Couderc, Bernard Fermini, Jules C. Hancox, Bjorn C. Knollmann, Gary R. Mirams, Jon Skinner, Wojciech Zareba, Jamie I. Vandenberg.
  The Journal of Physiology. June 09, 2016
  Risk stratification in the context of sudden cardiac death has been acknowledged as one of the major challenges facing cardiology for the past four decades. In recent years, the advent of high performance computing has facilitated organ‐level simulation of the heart, meaning we can now examine the causes, mechanisms and impact of cardiac dysfunction in silico. As a result, computational cardiology, largely driven by the Physiome project, now stands at the threshold of clinical utility in regards to risk stratification and treatment of patients at risk of sudden cardiac death. In this white paper, we outline a roadmap of what needs to be done to make this translational step, using the relatively well‐developed case of acquired or drug‐induced long QT syndrome as an exemplar case. Proposed roadmap for computational cardiology. A, general approach for clinical application of computational cardiology outlining three stages of development: (i) development of baseline models; (ii) disease specific simulations; and (iii) translation. At each state black arrows represents the iterative process of in silico simulation and in vitro/in vivo validation that is critical for successful implementation. The human ventricular mesh was provided by Dr Mikael Wallman and Professor Blanca Rodriguez, and developed as described in Wallman et al. 2014. B, specific priority goals for convergence of computational and clinical cardiology. Expanded discussion of specific goals is presented in the section headed The future: moving computational cardiology into the clinic.

  June 09, 2016   doi: 10.1113/JP272015   open full text
• Acoustic trauma slows AMPA receptor‐mediated EPSCs in the auditory brainstem, reducing GluA4 subunit expression as a mechanism to rescue binaural function.
  Nadia Pilati, Deborah M. Linley, Haresh Selvaskandan, Osvaldo Uchitel, Matthias H. Hennig, Cornelia Kopp‐Scheinpflug, Ian D. Forsythe.
  The Journal of Physiology. June 09, 2016
  Key points Lateral superior olive (LSO) principal neurons receive AMPA receptor (AMPAR) ‐ and NMDA receptor (NMDAR)‐mediated EPSCs and glycinergic IPSCs. Both EPSCs and IPSCs have slow kinetics in prehearing animals, which during developmental maturation accelerate to sub‐millisecond decay time‐constants. This correlates with a change in glutamate and glycine receptor subunit composition quantified via mRNA levels. The NMDAR‐EPSCs accelerate over development to achieve decay time‐constants of 2.5 ms. This is the fastest NMDAR‐mediated EPSC reported. Acoustic trauma (AT, loud sounds) slow AMPAR‐EPSC decay times, increasing GluA1 and decreasing GluA4 mRNA. Modelling of interaural intensity difference suggests that the increased EPSC duration after AT shifts interaural level difference to the right and compensates for hearing loss. Two months after AT the EPSC decay times recovered to control values. Synaptic transmission in the LSO matures by postnatal day 20, with EPSCs and IPSCs having fast kinetics. AT changes the AMPAR subunits expressed and slows the EPSC time‐course at synapses in the central auditory system. Abstract Damaging levels of sound (acoustic trauma, AT) diminish peripheral synapses, but what is the impact on the central auditory pathway? Developmental maturation of synaptic function and hearing were characterized in the mouse lateral superior olive (LSO) from postnatal day 7 (P7) to P96 using voltage‐clamp and auditory brainstem responses. IPSCs and EPSCs show rapid acceleration during development, so that decay kinetics converge to similar sub‐millisecond time‐constants (τ, 0.87 ± 0.11 and 0.77 ± 0.08 ms, respectively) in adult mice. This correlated with LSO mRNA levels for glycinergic and glutamatergic ionotropic receptor subunits, confirming a switch from Glyα2 to Glyα1 for IPSCs and increased expression of GluA3 and GluA4 subunits for EPSCs. The NMDA receptor (NMDAR)‐EPSC decay τ accelerated from >40 ms in prehearing animals to 2.6 ± 0.4 ms in adults, as GluN2C expression increased. In vivo induction of AT at around P20 disrupted IPSC and EPSC integration in the LSO, so that 1 week later the AMPA receptor (AMPAR)‐EPSC decay was slowed and mRNA for GluA1 increased while GluA4 decreased. In contrast, GlyR IPSC and NMDAR‐EPSC decay times were unchanged. Computational modelling confirmed that matched IPSC and EPSC kinetics are required to generate mature interaural level difference functions, and that longer‐lasting EPSCs compensate to maintain binaural function with raised auditory thresholds after AT. We conclude that LSO excitatory and inhibitory synaptic drive matures to identical time‐courses, that AT changes synaptic AMPARs by expression of subunits with slow kinetics (which recover over 2 months) and that loud sounds reversibly modify excitatory synapses in the brain, changing synaptic function for several weeks after exposure.

  June 09, 2016   doi: 10.1113/JP271929   open full text
• Influence of brain‐derived neurotrophic factor‐tyrosine receptor kinase B signalling in the nucleus tractus solitarius on baroreflex sensitivity in rats with chronic heart failure.
  Bryan K. Becker, Changhai Tian, Irving H. Zucker, Han‐Jun Wang.
  The Journal of Physiology. June 09, 2016
  Key points Impairment of baroreflex function is associated with the progression of chronic heart failure (CHF) and a poor prognosis. The baroreflex desensitization in CHF is at least partly the result of central neuronal network dysfunction. The dorsal medial nucleus tractus solitarius (dmNTS) has long been appreciated as a primary site of baroreceptor afferent termination in the central nervous system. However, the influence of neurotransmitters and neuromodulators in the dmNTS on baroreflex function both in normal and CHF states is not fully understood. The present study provides the first evidence showing a tonic sympatho‐inhibitory role for brain‐derived neurotrophic factor (BDNF) neurotransmission in the dmNTS. Most importantly, BDNF‐ tyrosine receptor kinase B (TrkB) signalling in the dmNTS is integral for normal baroreflex function as indicated by the blunting of baroreflex sensitivity (BRS) following the antagonization of TrkB, which inhibited baroreflex gain and range. Furthermore, we found that the tonic sympatho‐inhibition of BDNF was withdrawn in the CHF state, thus contributing to the increased sympathetic tone associated with CHF. Consistent with this finding, BDNF/TrkB antagonism had little effect on reducing BRS in CHF animals, which is corroborated by the observation of decreased TrkB expression in the dmNTS during CHF. Taken together, these results implicate a reduction in BDNF‐TrkB signalling in the dmNTS during CHF that contributes to sympatho‐excitation and baroreflex desensitization. The observation that the BDNF/TrkB pathway is impaired in the dmNTS during CHF provides a novel mechanism for understanding the central alterations that contribute to baroreflex desensitization during CHF. Abstract Chronic heart failure (CHF) results in blunting of arterial baroreflex sensitivity (BRS), which arises from alterations to both peripheral baroreceptors and central autonomic nuclei such as the nucleus tractus solitarius (NTS). Although glutamate is known to be an important neurotransmitter released from baroreceptor afferent synapses in the NTS, the influence of other neurotransmitters and neuromodulators remains unclear. Alterations to NTS signalling in CHF remain particularly undefined. The present study aimed to evaluate the role of brain‐derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) receptor signalling in the NTS on baroreflex control both in healthy and CHF rats. To this end, we microinjected BDNF or the highly selective TrkB receptor antagonist [N2‐2‐2‐oxoazepan‐3‐yl amino] carbonyl phenyl benzo (b)thiophene‐2‐carboxamide (ANA‐12) into the dorsal medial NTS (dmNTS) of male Sprague–Dawley rats with coronary artery ligation‐induced CHF and sham operated controls and recorded blood pressure and renal sympathetic nerve activity responses. We subsequently measured BRS before and after bilateral dmNTS microinjections of ANA‐12. In sham rats, BDNF evoked a dose‐dependent depressor and sympatho‐inhibitory effect and ANA‐12 produced the opposite response. Both of these responses were significantly blunted in CHF rats. Furthermore, bilateral microinjection of ANA‐12 into the dmNTS greatly diminished baroreflex sensitivity in sham rats, whereas it had less of an effect in CHF rats. We observed decreased levels of TrkB protein and mRNA in the dmNTS of CHF rats. These data indicate that endogenous BDNF signalling in the NTS is integral for the maintenance of BRS and that BDNF/TrkB signalling is impaired in the NTS in the CHF state.

  June 09, 2016   doi: 10.1113/JP272318   open full text
• Uncertainty and variability in computational and mathematical models of cardiac physiology.
  Gary R. Mirams, Pras Pathmanathan, Richard A. Gray, Peter Challenor, Richard H. Clayton.
  The Journal of Physiology. June 09, 2016
  Key points Mathematical and computational models of cardiac physiology have been an integral component of cardiac electrophysiology since its inception, and are collectively known as the Cardiac Physiome. We identify and classify the numerous sources of variability and uncertainty in model formulation, parameters and other inputs that arise from both natural variation in experimental data and lack of knowledge. The impact of uncertainty on the outputs of Cardiac Physiome models is not well understood, and this limits their utility as clinical tools. We argue that incorporating variability and uncertainty should be a high priority for the future of the Cardiac Physiome. We suggest investigating the adoption of approaches developed in other areas of science and engineering while recognising unique challenges for the Cardiac Physiome; it is likely that novel methods will be necessary that require engagement with the mathematics and statistics community. Abstract The Cardiac Physiome effort is one of the most mature and successful applications of mathematical and computational modelling for describing and advancing the understanding of physiology. After five decades of development, physiological cardiac models are poised to realise the promise of translational research via clinical applications such as drug development and patient‐specific approaches as well as ablation, cardiac resynchronisation and contractility modulation therapies. For models to be included as a vital component of the decision process in safety‐critical applications, rigorous assessment of model credibility will be required. This White Paper describes one aspect of this process by identifying and classifying sources of variability and uncertainty in models as well as their implications for the application and development of cardiac models. We stress the need to understand and quantify the sources of variability and uncertainty in model inputs, and the impact of model structure and complexity and their consequences for predictive model outputs. We propose that the future of the Cardiac Physiome should include a probabilistic approach to quantify the relationship of variability and uncertainty of model inputs and outputs. In the conventional approach to cardiac modelling, model inputs and parameters are assigned fixed values. These produce single outputs such as action potential time series or tissue activation sequences, as shown in the top panel of the figure. In this White Paper we argue that for models to become valuable clinical tools it will be important to treat model inputs as uncertain quantities, expressed as distributions, as shown in the lower panel of the figure.

  June 09, 2016   doi: 10.1113/JP271671   open full text
• Gluconeogenesis during endurance exercise in cyclists habituated to a long‐term low carbohydrate high‐fat diet.
  Christopher C. Webster, Timothy D. Noakes, Shaji K. Chacko, Jeroen Swart, Tertius A. Kohn, James A. H. Smith.
  The Journal of Physiology. June 08, 2016
  Key points Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis). We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization. Abstract Endogenous glucose production (EGP) occurs via hepatic glycogenolysis (GLY) and gluconeogenesis (GNG) and plays an important role in maintaining euglycaemia. Rates of GLY and GNG increase during exercise in athletes following a mixed macronutrient diet; however, these processes have not been investigated in athletes following a low carbohydrate high fat (LCHF) diet. Therefore, we studied seven well‐trained male cyclists that were habituated to either a LCHF (7% carbohydrate, 72% fat, 21% protein) or a mixed diet (51% carbohydrate, 33% fat, 16% protein) for longer than 8 months. After an overnight fast, participants performed a 2 h laboratory ride at 72% of maximal oxygen consumption. Glucose kinetics were measured at rest and during the final 30 min of exercise by infusion of [6,6‐2H2]‐glucose and the ingestion of 2H2O tracers. Rates of EGP and GLY both at rest and during exercise were significantly lower in the LCHF group than the mixed diet group (Exercise EGP: LCHF, 6.0 ± 0.9 mg kg−1 min−1, Mixed, 7.8 ± 1.1 mg kg−1 min−1, P < 0.01; Exercise GLY: LCHF, 3.2 ± 0.7 mg kg−1 min−1, Mixed, 5.3 ± 0.9 mg kg−1 min−1, P < 0.01). Conversely, no difference was detected in rates of GNG between groups at rest or during exercise (Exercise: LCHF, 2.8 ± 0.4 mg kg−1 min−1, Mixed, 2.5 ± 0.3 mg kg−1 min−1, P = 0.15). We conclude that athletes on a LCHF diet do not compensate for reduced glucose availability via higher rates of glucose synthesis compared to athletes on a mixed diet. Instead, GNG remains relatively stable, whereas glucose oxidation and GLY are influenced by dietary factors.

  June 08, 2016   doi: 10.1113/JP271934   open full text
• Phrenic motor outputs in response to bronchopulmonary C‐fibre activation following chronic cervical spinal cord injury.
  Kun‐Ze Lee.
  The Journal of Physiology. June 03, 2016
  Key points Activation of bronchopulmonary C‐fibres, the main chemosensitive afferents in the lung, can induce pulmonary chemoreflexes to modulate respiratory activity. Following chronic cervical spinal cord injury, bronchopulmonary C‐fibre activation‐induced inhibition of phrenic activity was exaggerated. Supersensitivity of phrenic motor outputs to the inhibitory effect of bronchopulmonary C‐fibre activation is due to a shift of phrenic motoneuron types and slow recovery of phrenic motoneuron discharge in cervical spinal cord‐injured animals. These data suggest that activation of bronchopulmonary C‐fibres may retard phrenic output recovery following cervical spinal cord injury. The alteration of phenotype and discharge pattern of phrenic motoneuron enables us to understand the impact of spinal cord injury on spinal respiratory activity. Abstract Cervical spinal injury interrupts bulbospinal pathways and results in cessation of phrenic bursting ipsilateral to the lesion. The ipsilateral phrenic activity can partially recover over weeks to months following injury due to the activation of latent crossed spinal pathways and exhibits a greater capacity to increase activity during respiratory challenges than the contralateral phrenic nerve. However, whether the bilateral phrenic nerves demonstrate differential responses to respiratory inhibitory inputs is unclear. Accordingly, the present study examined bilateral phrenic bursting in response to capsaicin‐induced pulmonary chemoreflexes, a robust respiratory inhibitory stimulus. Bilateral phrenic nerve activity was recorded in anaesthetized and mechanically ventilated adult rats at 8–9 weeks after C2 hemisection (C2Hx) or C2 laminectomy. Intra‐jugular capsaicin (1.5 μg kg−1) injection was performed to activate the bronchopulmonary C‐fibres to evoke pulmonary chemoreflexes. The present results indicate that capsaicin‐induced prolongation of expiratory duration was significantly attenuated in C2Hx animals. However, ipsilateral phrenic activity was robustly reduced after capsaicin treatment compared to uninjured animals. Single phrenic fibre recording experiments demonstrated that C2Hx animals had a higher proportion of late‐inspiratory phrenic motoneurons that were relatively sensitive to capsaicin treatment compared to early‐inspiratory phrenic motoneurons. Moreover, late‐inspiratory phrenic motoneurons in C2Hx animals had a weaker discharge frequency and slower recovery time than uninjured animals. These results suggest bilateral phrenic nerves differentially respond to bronchopulmonary C‐fibre activation following unilateral cervical hemisection, and the severe inhibition of phrenic bursting is due to a shift in the discharge pattern of phrenic motoneurons.

  June 03, 2016   doi: 10.1113/JP272287   open full text
• Heterogeneous firing rate response of mouse layer V pyramidal neurons in the fluctuation‐driven regime.
  Y. Zerlaut, B. Teleńczuk, C. Deleuze, T. Bal, G. Ouanounou, A. Destexhe.
  The Journal of Physiology. June 03, 2016
  Key points We recreated in vitro the fluctuation‐driven regime observed at the soma during asynchronous network activity in vivo and we studied the firing rate response as a function of the properties of the membrane potential fluctuations. We provide a simple analytical template that captures the firing response of both pyramidal neurons and various theoretical models. We found a strong heterogeneity in the firing rate response of layer V pyramidal neurons: in particular, individual neurons differ not only in their mean excitability level, but also in their sensitivity to fluctuations. Theoretical modelling suggest that this observed heterogeneity might arise from various expression levels of the following biophysical properties: sodium inactivation, density of sodium channels and spike frequency adaptation. Abstract Characterizing the input–output properties of neocortical neurons is of crucial importance for understanding the properties emerging at the network level. In the regime of low‐rate irregular firing (such as in the awake state), determining those properties for neocortical cells remains, however, both experimentally and theoretically challenging. Here, we studied this problem using a combination of theoretical modelling and in vitro experiments. We first identified, theoretically, three somatic variables that describe the dynamical state at the soma in this fluctuation‐driven regime: the mean, standard deviation and time constant of the membrane potential fluctuations. Next, we characterized the firing rate response of individual layer V pyramidal cells in this three‐dimensional space by means of perforated‐patch recordings and dynamic clamp in the visual cortex of juvenile mice in vitro. We found that individual neurons strongly differ not only in terms of their excitability, but also, and unexpectedly, in their sensitivities to fluctuations. Finally, using theoretical modelling, we attempted to reproduce these results. The model predicts that heterogeneous levels of biophysical properties such as sodium inactivation, sharpness of sodium activation and spike frequency adaptation account for the observed diversity of firing rate responses. Because the firing rate response will determine population rate dynamics during asynchronous neocortical activity, our results show that cortical populations are functionally strongly inhomogeneous in young mouse visual cortex, which should have important consequences on the strategies of cortical computation at early stages of sensory processing.

  June 03, 2016   doi: 10.1113/JP272317   open full text
• Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by β‐alanine transamination.
  Laura Blancquaert, Shahid P. Baba, Sebastian Kwiatkowski, Jan Stautemas, Sanne Stegen, Silvia Barbaresi, Weiliang Chung, Adjoa A. Boakye, J. David Hoetker, Aruni Bhatnagar, Joris Delanghe, Bert Vanheel, Maria Veiga‐da‐Cunha, Wim Derave, Inge Everaert.
  The Journal of Physiology. June 02, 2016
  Key points Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β‐alanine is an efficient substrate for the mammalian transaminating enzymes 4‐aminobutyrate‐2‐oxoglutarate transaminase and alanine‐glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β‐alanine, which is in turn controlled by degradation of β‐alanine in liver and kidney. Chronic oral β‐alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high‐intensity exercises. The present study can partly explain why the β‐alanine supplementation protocol is so inefficient, by demonstrating that exogenous β‐alanine can be effectively routed toward oxidation. Abstract The metabolic fate of orally ingested β‐alanine is largely unknown. Chronic β‐alanine supplementation is becoming increasingly popular for improving high‐intensity exercise performance because it is the rate‐limiting precursor of the dipeptide carnosine (β‐alanyl‐l‐histidine) in muscle. However, only a small fraction (3–6%) of the ingested β‐alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β‐alanine transamination enzymes, namely 4‐aminobutyrate‐2‐oxoglutarate transaminase (GABA‐T) and alanine‐glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA‐T and AGXT2 are able to transaminate β‐alanine efficiently. The reaction catalysed by GABA‐T is inhibited by vigabatrin, whereas both GABA‐T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA‐T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA‐T for vigabatrin; GABA‐T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β‐alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3‐fold increase in circulating β‐alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA‐T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β‐alanine levels, which are suppressed by hepatic and renal β‐alanine transamination upon oral β‐alanine intake.

  June 02, 2016   doi: 10.1113/JP272050   open full text
• Oral quercetin administration transiently protects respiratory function in dystrophin‐deficient mice.
  Joshua T. Selsby, Christopher G. Ballmann, Hannah R. Spaulding, Jason W. Ross, John C. Quindry.
  The Journal of Physiology. May 29, 2016
  Key point PGC‐1α pathway activation has been shown to decrease disease severity and can be driven by quercetin. Oral quercetin supplementation protected respiratory function for 4–6 months during a 12 month dosing regimen. This transient protection was probably due to a failure to sustain elevated SIRT1 activity and downstream PGC‐1α signalling. Quercetin supplementation may be a beneficial treatment as part of a cocktail provided continued SIRT1 activity elevation is achieved. Abstract Duchenne muscular dystrophy (DMD) impacts 1 : 3500 boys and leads to muscle dysfunction culminating in death due to respiratory or cardiac failure. There is an urgent need for effective therapies with the potential for immediate application for this patient population. Quercetin, a flavonoid with an outstanding safety profile, may provide therapeutic relief to DMD patients as the wait for additional therapies continues. This study evaluated the capacity of orally administered quercetin (0.2%) in 2 month old mdx mice to improve respiratory function and end‐point functional and histological outcomes in the diaphragm following 12 months of treatment. Respiratory function was protected for the first 4–6 months of treatment but appeared to become insensitive to quercetin thereafter. Consistent with this, end‐point functional measures were decreased and histopathological measures were more severe in dystrophic muscle compared to C57 and similar between control‐fed and quercetin‐fed mdx mice. To better understand the transient nature of improved respiratory function, we measured PGC‐1α pathway activity, which is suggested to be up‐regulated by quercetin supplementation. This pathway was largely suppressed in dystrophic muscle compared to healthy muscle, and at the 14 month time point dietary quercetin enrichment did not increase expression of downstream effectors. These data support the efficacy of quercetin as an intervention for DMD in skeletal muscle, and also indicate the development of age‐dependent quercetin insensitivity when continued supplementation fails to drive the PGC‐1α pathway. Continued study is needed to determine if this is related to disease severity, age or other factors.

  May 29, 2016   doi: 10.1113/JP272057   open full text
• Maternal obesity epigenetically alters visceral fat progenitor cell properties in male offspring mice.
  Xingwei Liang, Qiyuan Yang, Xing Fu, Carl J. Rogers, Bo Wang, Hong Pan, Mei‐Jun Zhu, Peter W. Nathanielsz, Min Du.
  The Journal of Physiology. May 29, 2016
  Key points Maternal obesity reduces adipogenic progenitor density in offspring adipose tissue. The ability of adipose tissue expansion in the offspring of obese mothers is limited and is associated with metabolic dysfunction of adipose tissue when challenged with a high‐fat diet. Maternal obesity induces DNA demethylation in the promoter of zinc finger protein 423, which renders progenitor cells with a high adipogenic capacity. Maternal obesity demonstrates long‐term effects on the adipogenic capacity of progenitor cells in offspring adipose tissue, demonstrating a developmental programming effect. Abstract Maternal obesity (MO) programs offspring obesity and metabolic disorders, although the underlying mechanisms remain poorly defined. Progenitor cells are the source of new adipocytes. The present study aimed to test whether MO epigenetically predisposes adipocyte progenitors in the fat of offspring to adipogenic differentiation and subsequent depletion, which leads to a failure of adipose tissue plasticity under positive energy balance, contributing to adipose tissue metabolic dysfunction. C57BL/6 female mice were fed either a control diet (10% energy from fat) or a high‐fat diet (45% energy from fat) for 8 weeks before mating. Male offspring of control (Con) and obese (OB) dams were weaned onto a regular (Reg) or obesogenic (Obe) diet until 3 months of age. At weaning, male OB offspring had a higher expression of Zinc finger protein 423 (zfp423), a key transcription factor in adipogenesis, as well as lower DNA methylation of its promoter in progenitors of epididymal fat compared to Con offspring, which was correlated with enhanced adipogenic differentiation. At 3 months of age, progenitor density was 30.9 ± 9.7% lower in OB/Obe compared to Con/Obe mice, accompanied by a limited expansion of the adipocyte number when challenged with a high‐energy diet. This difference was associated with lower DNA methylation in the zfp423 promoter in the epididymal fat of OB/Obe offspring, which was correlated with greater macrophage chemotactic protein‐1 and hypoxia‐inducible factor 1α expression. In summary, MO epigenetically limits the expansion capacity of offspring adipose tissue, providing an explanation for the adipose metabolic dysfunction of male offspring in the setting of MO.

  May 29, 2016   doi: 10.1113/JP272123   open full text
• Ventromedial hypothalamic melanocortin receptor activation: regulation of activity energy expenditure and skeletal muscle thermogenesis.
  Chaitanya K. Gavini, William C. Jones, Colleen M. Novak.
  The Journal of Physiology. May 29, 2016
  Key points The ventromedial hypothalamus (VMH) and the central melanocortin system both play vital roles in regulating energy balance by modulating energy intake and utilization. Recent evidence suggests that activation of the VMH alters skeletal muscle metabolism. We show that intra‐VMH melanocortin receptor activation increases energy expenditure and physical activity, switches fuel utilization to fats, and lowers work efficiency such that excess calories are dissipated by skeletal muscle as heat. We also show that intra‐VMH melanocortin receptor activation increases sympathetic nervous system outflow to skeletal muscle. Intra‐VMH melanocortin receptor activation also induced significant changes in the expression of mediators of energy expenditure in muscle. These results support the role of melanocortin receptors in the VMH in the modulation of skeletal muscle metabolism. Abstract The ventromedial hypothalamus (VMH) and the brain melanocortin system both play vital roles in increasing energy expenditure (EE) and physical activity, decreasing appetite and modulating sympathetic nervous system (SNS) outflow. Because of recent evidence showing that VMH activation modulates skeletal muscle metabolism, we propose the existence of an axis between the VMH and skeletal muscle, modulated by brain melanocortins, modelled on the brain control of brown adipose tissue. Activation of melanocortin receptors in the VMH of rats using a non‐specific agonist melanotan II (MTII), compared to vehicle, increased oxygen consumption and EE and decreased the respiratory exchange ratio. Intra‐VMH MTII enhanced activity‐related EE even when activity levels were held constant. MTII treatment increased gastrocnemius muscle heat dissipation during controlled activity, as well as in the home cage. Compared to vehicle‐treated rats, rats with intra‐VMH melanocortin receptor activation had higher skeletal muscle norepinephrine turnover, indicating an increased SNS drive to muscle. Lastly, intra‐VMH MTII induced mRNA expression of muscle energetic mediators, whereas short‐term changes at the protein level were primarily limited to phosphorylation events. These results support the hypothesis that melanocortin peptides act in the VMH to increase EE by lowering the economy of activity via the enhanced expression of mediators of EE in the periphery including skeletal muscle. The data are consistent with the role of melanocortins in the VMH in the modulation of skeletal muscle metabolism.

  May 29, 2016   doi: 10.1113/JP272352   open full text
• SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule.
  Mohammed Z. Ferdaus, Karl W. Barber, Karen I. López‐Cayuqueo, Andrew S. Terker, Eduardo R. Argaiz, Brandon M. Gassaway, Régine Chambrey, Gerardo Gamba, Jesse Rinehart, James A. McCormick.
  The Journal of Physiology. May 29, 2016
  Key points STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) phosphorylate and activate the renal Na+–K+–2Cl− cotransporter 2 (NKCC2) and Na+Cl− cotransporter (NCC). Mouse models suggest that OSR1 mainly activates NKCC2‐mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt‐wasting, and generated SPAK/OSR1 double knockout mice to test this. Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase. Compensatory changes in SPAK/OSR1‐independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed. Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K+]. Abstract STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) activate the renal cation cotransporters Na+–K+–2Cl− cotransporter (NKCC2) and Na+–Cl− cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross‐compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt‐wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK‐KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites was lower than in SPAK‐KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K+], with NCC activation serving as the primary effector pathway that modulates K+ secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK‐KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK‐KO and kidney‐specific OSR1 single knockout mice maintained plasma [K+] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K+].

  May 29, 2016   doi: 10.1113/JP272311   open full text
• Train stimulation of parallel fibre to Purkinje cell inputs reveals two populations of synaptic responses with different receptor signatures.
  Suma Priya Sudarsana Devi, James R. Howe, Céline Auger.
  The Journal of Physiology. May 29, 2016
  Key points Purkinje cells of the cerebellum receive ∼180,000 parallel fibre synapses, which have often been viewed as a homogeneous synaptic population and studied using single action potentials. Many parallel fibre synapses might be silent, however, and granule cells in vivo fire in bursts. Here, we used trains of stimuli to study parallel fibre inputs to Purkinje cells in rat cerebellar slices. Analysis of train EPSCs revealed two synaptic components, phase 1 and 2. Phase 1 is initially large and saturates rapidly, whereas phase 2 is initially small and facilitates throughout the train. The two components have a heterogeneous distribution at dendritic sites and different pharmacological profiles. The differential sensitivity of phase 1 and phase 2 to inhibition by pentobarbital and NBQX mirrors the differential sensitivity of AMPA receptors associated with the transmembrane AMPA receptor regulatory protein, γ‐2, gating in the low‐ and high‐open probability modes, respectively. Abstract Cerebellar granule cells fire in bursts, and their parallel fibre axons (PFs) form ∼180,000 excitatory synapses onto the dendritic tree of a Purkinje cell. As many as 85% of these synapses have been proposed to be silent, but most are labelled for AMPA receptors. Here, we studied PF to Purkinje cell synapses using trains of 100 Hz stimulation in rat cerebellar slices. The PF train EPSC consisted of two components that were present in variable proportions at different dendritic sites: one, with large initial EPSC amplitude, saturated after three stimuli and dominated the early phase of the train EPSC; and the other, with small initial amplitude, increased steadily throughout the train of 10 stimuli and dominated the late phase of the train EPSC. The two phases also displayed different pharmacological profiles. Phase 2 was less sensitive to inhibition by NBQX but more sensitive to block by pentobarbital than phase 1. Comparison of synaptic results with fast glutamate applications to recombinant receptors suggests that the high‐open‐probability gating mode of AMPA receptors containing the auxiliary subunit transmembrane AMPA receptor regulatory protein γ‐2 makes a substantial contribution to phase 2. We argue that the two synaptic components arise from AMPA receptors with different functional signatures and synaptic distributions. Comparisons of voltage‐ and current‐clamp responses obtained from the same Purkinje cells indicate that phase 1 of the EPSC arises from synapses ideally suited to transmit short bursts of action potentials, whereas phase 2 is likely to arise from low‐release‐probability or ‘silent’ synapses that are recruited during longer bursts.

  May 29, 2016   doi: 10.1113/JP272415   open full text
• Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V‐ATPase generated voltage gradient as the driving force.
  Xi Zoë Zhong, Qi Cao, Xue Sun, Xian‐Ping Dong.
  The Journal of Physiology. May 29, 2016
  Key points SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation. P2X4 receptors act as lysosomal ion channels activated by luminal ATP. SLC17A9‐mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V‐ATPase inhibitor. SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V‐ATPase as the driving force to transport ATP into the lysosome to activate P2X4. Abstract The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9‐mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V‐ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9.

  May 29, 2016   doi: 10.1113/JP271893   open full text
• Evidence of a broad histamine footprint on the human exercise transcriptome.
  Steven A. Romero, Austin D. Hocker, Joshua E. Mangum, Meredith J. Luttrell, Douglas W. Turnbull, Adam J. Struck, Matthew R. Ely, Dylan C. Sieck, Hans C. Dreyer, John R. Halliwill.
  The Journal of Physiology. May 29, 2016
  Key points Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors, in a variety of pathways that probably predate its more recent role in innate and adaptive immunity. Although histamine is normally associated with pathological conditions or allergic and anaphylactic reactions, it may contribute beneficially to the normal changes that occur within skeletal muscle during the recovery from exercise. We show that the human response to exercise includes an altered expression of thousands of protein‐coding genes, and much of this response appears to be driven by histamine. Histamine may be an important molecular transducer contributing to many of the adaptations that accompany chronic exercise training. Abstract Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors. In humans, aerobic exercise is followed by a post‐exercise activation of histamine H1 and H2 receptors localized to the previously exercised muscle. This could trigger a broad range of cellular adaptations in response to exercise. Thus, we exploited RNA sequencing to explore the effects of H1 and H2 receptor blockade on the exercise transcriptome in human skeletal muscle tissue harvested from the vastus lateralis. We found that exercise exerts a profound influence on the human transcriptome, causing the differential expression of more than 3000 protein‐coding genes. The influence of histamine blockade post‐exercise was notable for 795 genes that were differentially expressed between the control and blockade condition, which represents >25% of the number responding to exercise. The broad histamine footprint on the human exercise transcriptome crosses many cellular functions, including inflammation, vascular function, metabolism, and cellular maintenance.

  May 29, 2016   doi: 10.1113/JP272177   open full text
• Sacral nerve stimulation enhances early intestinal mucosal repair following mucosal injury in a pig model.
  Jérémy Brégeon, Emmanuel Coron, Anna Christina Cordeiro Da Silva, Julie Jaulin, Philippe Aubert, Julien Chevalier, Nathalie Vergnolle, Guillaume Meurette, Michel Neunlist.
  The Journal of Physiology. May 29, 2016
  Key points Reducing intestinal epithelial barrier (IEB) dysfunctions is recognized as being of major therapeutic interest for various intestinal disorders. Sacral nerve stimulation (SNS) is known to reduce IEB permeability. Here, we report in a pig model that SNS enhances morphological and functional recovery of IEB following mucosal injury induced via 2,4,6‐trinitrobenzenesulfonic acid. These effects are associated with an increased expression of tight junction proteins such as ZO‐1 and FAK. These results establish that SNS enhances intestinal barrier repair in acute mucosal injury. They further set the scientific basis for future use of SNS as a complementary or alternative therapeutic option for the treatment of gut disorders with IEB dysfunctions such as inflammatory bowel diseases or irritable bowel syndrome. Abstract Intestinal epithelial barrier (IEB) dysfunctions, such as increased permeability or altered healing, are central to intestinal disorders. Sacral nerve stimulation (SNS) is known to reduce IEB permeability, but its ability to modulate IEB repair remains unknown. This study aimed to characterize the impact of SNS on mucosal repair following 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced lesions. Six pigs were stimulated by SNS 3 h prior to and 3 h after TNBS enema, while sham animals (n = 8) were not stimulated. The impact of SNS on mucosal changes was evaluated by combining in vivo imaging, histological and functional methods. Biochemical and transcriptomic approaches were used to analyse the IEB and mucosal inflammatory response. We observed that SNS enhanced the recovery from TNBS‐induced increase in transcellular permeability. At 24 h, TNBS‐induced alterations of mucosal morphology were significantly less in SNS compared with sham animals. SNS reduced TNBS‐induced changes in ZO‐1 expression and its epithelial pericellular distribution, and also increased pFAK/FAK expression compared with sham. Interestingly, SNS increased the mucosal density of neutrophils, which was correlated with an increase in trypsin and TGF‐β1 levels compared with sham. Finally, SNS prevented the TNBS‐induced increases in IL‐1β and IL‐4 over time that were observed with sham treatment. In conclusion, our results show that SNS enhances mucosal repair following injury. This study highlights novel mechanisms of action of SNS and identifies SNS as a new therapy for diseases with IEB repair disorders.

  May 29, 2016   doi: 10.1113/JP271783   open full text
• Phospholipase C δ4 regulates cold sensitivity in mice.
  Yevgen Yudin, Brianna Lutz, Yuan‐Xiang Tao, Tibor Rohacs.
  The Journal of Physiology. May 29, 2016
  Key points The cold‐ and menthol‐activated transient receptor potential melastatin 8 (TRPM8) channels are thought to be regulated by phospholipase C (PLC), but neither the specific PLC isoform nor the in vivo relevance of this regulation has been established. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 channels in vivo. We show that in small PLCδ4−/− TRPM8‐positive dorsal root ganglion neurons cold, menthol and WS‐12, a selective TRPM8 agonist, evoked significantly larger currents than in wild‐type neurons, and action potential frequencies induced by menthol or by current injections were also higher in PLCδ4−/− neurons. PLCδ4−/− mice showed increased behavioural responses to evaporative cooling, and this effect was inhibited by a TRPM8 antagonist; behavioural responses to heat and mechanical stimuli were not altered. We provide evidence for the involvement of a specific PLC isoform in the regulation of cold sensitivity in mice by regulating TRPM8 activity. Abstract The transient receptor potential melastatin 8 (TRPM8) ion channel is a major sensor of environmental low temperatures. Ca2+‐induced activation of phospholipase C (PLC) has been implied in the regulation of TRPM8 channels during menthol‐ and cold‐induced desensitization in vitro. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 in sensory dorsal root ganglion (DRG) neurons. We identified two TRPM8‐positive neuronal subpopulations, based on their cell body size. Most TRPM8‐positive small neurons also responded to capsaicin, and had significantly larger menthol‐induced inward current densities than medium–large cells, most of which did not respond to capsaicin. Small, but not medium–large, PLCδ4−/− neurons showed significantly larger currents induced by cold, menthol or WS‐12, a specific TRPM8 agonist, compared to wild‐type (WT) neurons, but TRPM8 protein levels were not different between the two groups. In current‐clamp experiments small neurons had more depolarized resting membrane potentials, and required smaller current injections to generate action potentials (APs) than medium–large cells. In small PLCδ4−/− neurons, menthol application induced larger depolarizations and generation of APs with frequencies significantly higher compared to WT neurons. In behavioural experiments PLCδ4−/− mice showed greater sensitivity to evaporative cooling by acetone than control animals. Pretreatment with the TRPM8 antagonist PBMC reduced cold‐induced responses, and the effect was more pronounced in the PLCδ4−/− group. Heat and mechanical sensitivity of the PLCδ4−/− mice was not different from WT animals. Our data support the involvement of PLCδ4 in the regulation of TRPM8 channel activity in vivo.

  May 29, 2016   doi: 10.1113/JP272321   open full text
• Biphasic modulation of parallel fibre synaptic transmission by co‐activation of presynaptic GABAA and GABAB receptors in mice.
  Rebecca D. Howell, Jason R. Pugh.
  The Journal of Physiology. May 29, 2016
  Key points Many excitatory synapses co‐express presynaptic GABAA and GABAB receptors, despite their opposing actions on synaptic transmission. It is still unclear how co‐activation of these receptors modulates synapse function. We measured presynaptic GABA receptor function at parallel fibre synapses onto stellate cells in the cerebellum using whole‐cell patch‐clamp recording and photolytic uncaging of RuBi‐GABA. Activation of presynaptic GABA receptors results in a transient (∼100 ms) enhancement of synaptic transmission (mediated by GABAA receptors) followed by a long lasting (>500 ms) inhibition of transmission (mediated by GABAB receptors). When activated just prior to high‐frequency trains of stimulation, presynaptic GABAA and GABAB receptors work together to reduce short‐term facilitation/enhance depression, altering the filtering properties of synaptic transmission. Inhibition of synaptic transmission by GABAB receptors is more sensitive to GABA than enhancement by GABAA receptors, suggesting GABAB receptors may be activated by ambient GABA or release from greater distances. Abstract GABAA and GABAB receptors are co‐expressed at many presynaptic terminals in the central nervous system. Previous studies have shown that GABAA receptors typically enhance vesicle release while GABAB receptors inhibit release. However, it is not clear how the competing actions of these receptors modulate synaptic transmission when co‐activated, as is likely in vivo. We investigated this question at parallel fibre synapses in the cerebellum, which co‐express presynaptic GABAA and GABAB receptors. In acute slices from C57BL/6 mice, we find that co‐activation of presynaptic GABA receptors by photolytic uncaging of RuBi‐GABA has a biphasic effect on EPSC amplitudes recorded from stellate cells. Synchronous and asynchronous EPSCs evoked within ∼100 ms of GABA uncaging were increased, while EPSCs evoked ∼300–600 ms after GABA uncaging were reduced compared to interleaved control sweeps. We confirmed these effects are presynaptic by measuring the paired‐pulse ratio, variance of EPSC amplitudes, and response probability. During trains of high‐frequency stimulation GABAA and GABAB receptors work together (rather than oppose one another) to reduce short‐term facilitation when GABA is uncaged just prior to the onset of stimulation. We also find that GABAB receptor‐mediated inhibition can be elicited by lower GABA concentrations than GABAA receptor‐mediated enhancement of EPSCs, suggesting GABAB receptors may be selectively activated by ambient GABA or release from more distance synapses. These data suggest that GABA, acting through both presynaptic GABAA and GABAB receptors, modulate the amplitude and short‐term plasticity of excitatory synapses, a result not possible from activation of either receptor type alone.

  May 29, 2016   doi: 10.1113/JP272124   open full text
• An augmented CO2 chemoreflex and overactive orexin system are linked with hypertension in young and adult spontaneously hypertensive rats.
  Aihua Li, Sarah H. Roy, Eugene E. Nattie.
  The Journal of Physiology. May 29, 2016
  Key points Activation of central chemoreceptors by CO2 increases sympathetic nerve activity (SNA), arterial blood pressure (ABP) and breathing. These effects are exaggerated in spontaneously hypertensive rats (SHRs), resulting in an augmented CO2 chemoreflex that affects both breathing and ABP. The augmented CO2 chemoreflex and the high ABP are measureable in young SHRs (postnatal day 30–58) and become greater in adult SHRs. Blockade of orexin receptors can normalize the augmented CO2 chemoreflex and the high ABP in young SHRs and normalize the augmented CO2 chemoreflex and significantly lower the high ABP in adult SHRs. In the hypothalamus, SHRs have more orexin neurons, and a greater proportion of them increase their activity with CO2. The orexin system is overactive in SHRs and contributes to the augmented CO2 chemoreflex and hypertension. Modulation of the orexin system may be beneficial in the treatment of neurogenic hypertension. Abstract Activation of central chemoreceptors by CO2 increases arterial blood pressure (ABP), sympathetic nerve activity and breathing. In spontaneously hypertensive rats (SHRs), high ABP is associated with enhanced sympathetic nerve activity and peripheral chemoreflexes. We hypothesized that an augmented CO2 chemoreflex and overactive orexin system are linked with high ABP in both young (postnatal day 30–58) and adult SHRs (4–6 months). Our main findings are as follows. (i) An augmented CO2 chemoreflex and higher ABP in SHRs are measureable at a young age and increase in adulthood. In wakefulness, the ventilatory response to normoxic hypercapnia is higher in young SHRs (mean ± SEM: 179 ± 11% increase) than in age‐matched normotensive Wistar–Kyoto rats (114 ± 9% increase), but lower than in adult SHRs (226 ± 10% increase; P < 0.05). The resting ABP is higher in young SHRs (122 ± 5 mmHg) than in age‐matched Wistar–Kyoto rats (99 ± 5 mmHg), but lower than in adult SHRs (152 ± 4 mmHg; P < 0.05). (ii) Spontaneously hypertensive rats have more orexin neurons and more CO2‐activated orexin neurons in the hypothalamus. (iii) Antagonism of orexin receptors with a dual orexin receptor antagonist, almorexant, normalizes the augmented CO2 chemoreflex in young and adult SHRs and the high ABP in young SHRs and significantly lowers ABP in adult SHRs. (iv) Attenuation of peripheral chemoreflexes by hyperoxia does not abolish the augmented CO2 chemoreflex (breathing and ABP) in SHRs, which indicates an important role for the central chemoreflex. We suggest that an overactive orexin system may play an important role in the augmented central CO2 chemoreflex and in the development of hypertension in SHRs.

  May 29, 2016   doi: 10.1113/JP272199   open full text
• The rat suprachiasmatic nucleus: the master clock ticks at 30 Hz.
  Takahiro Tsuji, Chiharu Tsuji, Mike Ludwig, Gareth Leng.
  The Journal of Physiology. May 29, 2016
  Key points Light‐responsive neurones in the rat suprachiasmatic nucleus discharge with a harmonic distribution of interspike intervals, whereas unresponsive neurones seldom do. This harmonic patterning has a fundamental frequency of close to 30 Hz, and is the same in light‐on cells as in light‐off cells, and is unaffected by exposure to light. Light‐on cells are more active than light‐off cells in both subjective day and subjective night, and both light‐on cells and light‐off cells respond more strongly to changes in light intensity during the subjective night than during the subjective day. Paired recordings indicate that the discharge of adjacent light‐responsive cells is very tightly synchronized. The gap junction inhibitor carbenoxolone increases the spontaneous activity of suprachiasmatic nucleus neurones but does not block the harmonic discharge patterning. Abstract The suprachiasmatic nucleus (SCN) of the hypothalamus has an essential role in orchestrating circadian rhythms of behaviour and physiology. In the present study, we recorded from single SCN neurons in urethane‐anaesthetized rats, categorized them by the statistical features of their electrical activity and by their responses to light, and examined how activity in the light phase differs from activity in the dark phase. We classified cells as light‐on cells or light‐off cells according to how their firing rate changed in acute response to light, or as non‐responsive cells. In both sets of light‐responsive neurons, responses to light were stronger at subjective night than in subjective day. Neuronal firing patterns were analysed by constructing hazard functions from interspike interval data. For most light‐responsive cells, the hazard functions showed a multimodal distribution, with a harmonic sequence of modes, indicating that spike activity was driven by an oscillatory input with a fundamental frequency of close to 30 Hz; this harmonic pattern was rarely seen in non‐responsive SCN cells. The frequency of the rhythm was the same in light‐on cells as in light‐off cells, was the same in subjective day as at subjective night, and was unaffected by exposure to light. Paired recordings indicated that the discharge of adjacent light‐responsive neurons was very tightly synchronized, consistent with electrical coupling.

  May 29, 2016   doi: 10.1113/JP272331   open full text
• p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization.
  Wenwu Zhang, Youliang Huang, Susan J. Gunst.
  The Journal of Physiology. May 29, 2016
  Key points In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin. The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation. We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization. We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP). These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. Abstract The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G‐protein‐coupled receptor kinase‐interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak–GIT1–βPIX–paxillin complex was necessary for cdc42 and neuronal Wiskott–Aldrich syndrome protein (N‐WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N‐WASP activation, actin dynamics and cell contractility.

  May 29, 2016   doi: 10.1113/JP272132   open full text
• Aerobic exercise training in the treatment of non‐alcoholic fatty liver disease related fibrosis.
  Melissa A. Linden, Ryan D. Sheldon, Grace M. Meers, Laura C. Ortinau, E. Matthew Morris, Frank W. Booth, Jill A. Kanaley, Victoria J. Vieira‐Potter, James R. Sowers, Jamal A. Ibdah, John P. Thyfault, M. Harold Laughlin, R. Scott Rector.
  The Journal of Physiology. May 27, 2016
  Key points Physiologically relevant rodent models of non‐alcoholic steatohepatitis (NASH) that resemble the human condition are limited. Exercise training and energy restriction are first‐line recommendations for the treatment of NASH. Hyperphagic Otsuka Long–Evans Tokushima fatty rats fed a western diet high in fat, sucrose and cholesterol for 24 weeks developed a severe NASH with fibrosis phenotype. Moderate intensity exercise training and modest energy restriction provided some improvement in the histological features of NASH that coincided with alterations in markers of hepatic stellate cell activation and extracellular matrix remodelling. The present study highlights the importance of lifestyle modification, including exercise training and energy restriction, in the regulation of advanced liver disease. Abstract The incidence of non‐alcoholic steatohepatitis (NASH) is rising but the efficacy of lifestyle modifications to improve NASH‐related outcomes remain unclear. We hypothesized that a western diet (WD) would induce NASH in the Otsuka Long–Evans Tokushima Fatty (OLETF) rat and that lifestyle modification would improve this condition. Eight‐week‐old Long–Evans Tokushima Otsuka (L) and OLETF (O) rats consumed a control diet (10% kcal fat, 3.5% sucrose) or a WD (45% kcal fat, 17% sucrose, 1% cholesterol) for 24 weeks. At 20 weeks of age, additional WD‐fed OLETFs were randomized to sedentary (O‐SED), food restriction (O‐FR; ∼25% kcal reduction vs. O‐SED) or exercise training (O‐EX; treadmill running 20 m min–1 with a 15% incline, 60 min day–1, 5 days week–1) conditions for 12 weeks. WD induced a NASH phenotype in OLETFs characterized by hepatic fibrosis (collagen 1α1 mRNA and hydroxyproline content), as well as elevated inflammation and non‐alcoholic fatty liver disease activity scores, and hepatic stellate cell activation (α‐smooth muscle actin) compared to Long–Evans Tokushima Otsuka rats. FR and EX modestly improved NASH‐related fibrosis markers (FR: hydroxyproline content, P < 0.01; EX: collagen 1α1 mRNA, P < 0.05; both: fibrosis score, P < 0.01) and inflammation (both: inflammation score; FR: interleukin‐1β and tumor necrosis factor α) vs. O‐SED. FR reduced hepatic stellate cell activation markers (transforming growth factor‐β protein and α‐smooth muscle actin mRNA), whereas EX increased the hepatic stellate cell senescence marker CCN1 (P < 0.01 vs. O‐SED). Additionally, both FR and EX normalized extracellular matrix remodelling markers to levels similar to L‐WD (P > 0.05). Although neither EX nor FR led to complete resolution of the WD‐induced NASH phenotype, both independently benefitted liver fibrosis via altered hepatic stellate cell activation and extracellular matrix remodelling.

  May 27, 2016   doi: 10.1113/JP272235   open full text
• Extrusion versus diffusion: mechanisms for recovery from sodium loads in mouse CA1 pyramidal neurons.
  Miguel A. Mondragão, Hartmut Schmidt, Christian Kleinhans, Julia Langer, Karl W. Kafitz, Christine R. Rose.
  The Journal of Physiology. May 27, 2016
  Key points Neuronal activity causes local or global sodium signalling in neurons, depending on the pattern of synaptic activity. Recovery from global sodium loads critically relies on Na+/K+‐ATPase and an intact energy metabolism in both somata and dendrites. For recovery from local sodium loads in dendrites, Na+/K+‐ATPase activity is not required per se. Instead, recovery is predominately mediated by lateral diffusion, exhibiting rates that are 10‐fold higher than for global sodium signals. Recovery from local dendritic sodium increases is still efficient during short periods of energy deprivation, indicating that fast diffusion of sodium to non‐stimulated regions strongly reduces local energy requirements. Abstract Excitatory activity is accompanied by sodium influx into neurones as a result of the opening of voltage‐ and ligand‐activated channels. Recovery from resulting sodium transients has mainly been attributed to Na+/K+‐ATPase (NKA). Because sodium ions are highly mobile, diffusion could provide an additional pathway. We tested this in hippocampal neurones using whole‐cell patch‐clamp recordings and sodium imaging. Somatic sodium transients induced by local glutamate application recovered at a maximum rate of 8 mm min−1 (∼0.03 mm min−1 μm−2). Somatic sodium extrusion was accelerated at higher temperature and blocked by ouabain, emphasizing its dependence on NKA. Moreover, it was slowed down during inhibition of glycolysis by sodium fluoride (NaF). Local glutamate application to dendrites revealed a 10‐fold higher apparent dendritic sodium extrusion rate compared to somata. Recovery was almost unaltered by increased temperature, ouabain or NaF. We found that sodium diffused along primary dendrites with a diffusion coefficient of ∼330 μm²/s. During global glutamate application, impeding substantial net diffusion, apparent dendritic extrusion rates were reduced to somatic rates and also affected by NaF. Numerical simulations confirmed the essential role of NKA for the recovery of somatic, but not dendritic sodium loads. Our data show that sodium export upon global sodium increases is largely mediated by NKA and depends on an intact energy metabolism. For recovery from local dendritic sodium increases, diffusion dominates over extrusion, operating efficiently even during short periods of energy deprivation. Although sodium will eventually be extruded by the NKA, its diffusion‐based fast dissemination to non‐stimulated regions might reduce local energy requirements.

  May 27, 2016   doi: 10.1113/JP272431   open full text
• The acquisition of mechano‐electrical transducer current adaptation in auditory hair cells requires myosin VI.
  Walter Marcotti, Laura F. Corns, Richard J. Goodyear, Agnieszka K. Rzadzinska, Karen B. Avraham, Karen P. Steel, Guy P. Richardson, Corné J. Kros.
  The Journal of Physiology. May 27, 2016
  Key points The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano‐electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Abstract Mutations in Myo6, the gene encoding the (F‐actin) minus end‐directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano‐electrical transduction. We report that Ca2+‐dependent adaptation of the mechano‐electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca2+‐dependent adaptation.

  May 27, 2016   doi: 10.1113/JP272220   open full text
• Nuclear factor erythroid‐derived 2‐like 2 (NFE2L2, Nrf2) mediates exercise‐induced mitochondrial biogenesis and the anti‐oxidant response in mice.
  Troy L. Merry, Michael Ristow.
  The Journal of Physiology. May 27, 2016
  Key points Reactive oxygen species (ROS) and nitric oxide (NO) regulate exercise‐induced nuclear factor erythroid 2‐related factor 2 (NFE2L2) expression in skeletal muscle. NFE2L2 is required for acute exercise‐induced increases in skeletal muscle mitochondrial biogenesis genes, such as nuclear respiratory factor 1 (NRF‐1) and mitochondrial transcription factor A, and anti‐oxidant genes, such as superoxide dismutase (SOD)1, SOD2 and catalase. Following exercise training mice with impaired NFE2L2 expression have reduced exercise performance, energy expenditure, mitochondrial volume and anti‐oxidant activity. In muscle cells, ROS and NO can regulate mitochondrial biogenesis via a NFE2L2/NRF‐1‐dependent pathway. Abstract Regular exercise induces adaptations to skeletal muscle, which can include mitochondrial biogenesis and enhanced anti‐oxidant reserves. These adaptations and others are at least partly responsible for the improved health of physically active individuals. Reactive oxygen species (ROS) and nitric oxide (NO) are produced during exercise and may mediate the adaptive response to exercise in skeletal muscle. However, the mechanisms through which they act are unclear. In the present study, we aimed to determine the role of the redox‐sensitive transcription factor nuclear factor erythroid‐derived 2‐like 2 (NFE2L2) in acute exercise‐ and training‐induced mitochondrial biogenesis and the anti‐oxidant response. We report that ROS and NO regulate acute exercise‐induced expression of NFE2L2 in mouse skeletal muscle and muscle cells, and that deficiency in NFE2L2 prevents normal acute treadmill exercise‐induced increases in mRNA of the mitochondrial biogenesis markers, nuclear respiratory factor 1 (NRF‐1) and mitochondrial transcription factor A (mtTFA), and the anti‐oxidants superoxide dismutase (SOD) 1 and 2, as well as catalase, in mouse gastrocnemius muscle. Furthermore, after 5 weeks of treadmill exercise training, mice deficient in NFE2L2 had reduced exercise capacity and whole body energy expenditure, as well as skeletal muscle mitochondrial mass and SOD activity, compared to wild‐type littermates. In C2C12 myoblasts, acute treatment with exogenous H2O2 (ROS)‐ and diethylenetriamine/NO adduct (NO donor) induced increases in mtTFA, which was prevented by small interfering RNA and short hairpin RNA knockdown of either NFE2L2 or NRF‐1. Our results suggest that, during exercise, ROS and NO can act via NFE2L2 to functionally regulate skeletal muscle mitochondrial biogenesis and anti‐oxidant defence gene expression.

  May 27, 2016   doi: 10.1113/JP271957   open full text
• Altered development in GABA co‐release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1G93A mouse model of amyotrophic lateral sclerosis.
  Manuela Medelin, Vladimir Rancic, Giada Cellot, Jummi Laishram, Priyadharishini Veeraraghavan, Chiara Rossi, Luca Muzio, Lucia Sivilotti, Laura Ballerini.
  The Journal of Physiology. May 27, 2016
  Key points Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS). In vitro models allow the investigation of the genetically modified counter‐regulator of motoneuron toxicity and may help in addressing ALS therapy. Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits. This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1G93A pathogenesis. Abstract Amyotrophic lateral sclerosis (ALS) is a fatal, adult‐onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1G93A). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild‐type and SOD1G93A mice to investigate the development of glycine‐receptor‐mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co‐release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1G93A developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1G93A spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1G93A glycinergic terminals in cultured and ex vivo spinal slices.

  May 27, 2016   doi: 10.1113/JP272382   open full text
• Dual processing of visual rotation for bipedal stance control.
  Brian L. Day, Timothy Muller, Joanna Offord, Irene Di Giulio.
  The Journal of Physiology. May 27, 2016
  Key points When standing, the gain of the body‐movement response to a sinusoidally moving visual scene has been shown to get smaller with faster stimuli, possibly through changes in the apportioning of visual flow to self‐motion or environment motion. We investigated whether visual‐flow speed similarly influences the postural response to a discrete, unidirectional rotation of the visual scene in the frontal plane. Contrary to expectation, the evoked postural response consisted of two sequential components with opposite relationships to visual motion speed. With faster visual rotation the early component became smaller, not through a change in gain but by changes in its temporal structure, while the later component grew larger. We propose that the early component arises from the balance control system minimising apparent self‐motion, while the later component stems from the postural system realigning the body with gravity. Abstract The source of visual motion is inherently ambiguous such that movement of objects in the environment can evoke self‐motion illusions and postural adjustments. Theoretically, the brain can mitigate this problem by combining visual signals with other types of information. A Bayesian model that achieves this was previously proposed and predicts a decreasing gain of postural response with increasing visual motion speed. Here we test this prediction for discrete, unidirectional, full‐field visual rotations in the frontal plane of standing subjects. The speed (0.75–48 deg s–1) and direction of visual rotation was pseudo‐randomly varied and mediolateral responses were measured from displacements of the trunk and horizontal ground reaction forces. The behaviour evoked by this visual rotation was more complex than has hitherto been reported, consisting broadly of two consecutive components with respective latencies of ∼190 ms and >0.7 s. Both components were sensitive to visual rotation speed, but with diametrically opposite relationships. Thus, the early component decreased with faster visual rotation, while the later component increased. Furthermore, the decrease in size of the early component was not achieved by a simple attenuation of gain, but by a change in its temporal structure. We conclude that the two components represent expressions of different motor functions, both pertinent to the control of bipedal stance. We propose that the early response stems from the balance control system attempting to minimise unintended body motion, while the later response arises from the postural control system attempting to align the body with gravity.

  May 27, 2016   doi: 10.1113/JP271813   open full text
• Rescue of protein expression defects may not be enough to abolish the pro‐arrhythmic phenotype of long QT type 2 mutations.
  Matthew D. Perry, Chai Ann Ng, Kevin Phan, Erikka David, Kieran Steer, Mark J. Hunter, Stefan A. Mann, Mohammad Imtiaz, Adam P. Hill, Ying Ke, Jamie I. Vandenberg.
  The Journal of Physiology. May 27, 2016
  Key points Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2‐associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co‐expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. Abstract In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

  May 27, 2016   doi: 10.1113/JP271805   open full text
• Effect of aerobic fitness on capillary blood volume and diffusing membrane capacity responses to exercise.
  Vincent Tedjasaputra, Melissa M. Bouwsema, Michael K. Stickland.
  The Journal of Physiology. May 12, 2016
  Key points Endurance trained athletes exhibit enhanced cardiovascular function compared to non‐athletes, although it is considered that exercise training does not enhance lung structure and function. An increased pulmonary capillary blood volume at rest is associated with a higher V̇O2 max . In the present study, we compared the diffusion capacity, pulmonary capillary blood volume and diffusing membrane capacity responses to exercise in endurance‐trained males compared to non‐trained males. Exercise diffusion capacity was greater in athletes, secondary to an increased membrane diffusing capacity, and not pulmonary capillary blood volume. Endurance‐trained athletes appear to have differences within the pulmonary membrane that facilitate the increased O2 demand needed for high‐level exercise. Abstract Endurance‐trained athletes exhibit enhanced cardiovascular function compared to non‐athletes, allthough it is generally accepted that exercise training does not enhance lung structure and function. Recent work has shown that an increased resting pulmonary capillary blood volume (VC) is associated with a higher maximum oxygen consumption (V̇O2 max ), although there have been no studies to date examining how aerobic fitness affects the VC response to exercise. Based on previous work, we hypothesized that endurance‐trained athletes will have greater VC compared to non‐athletes during cycling exercise. Fifteen endurance‐trained athletes (HI: V̇O2 max 64.6 ± 1.8 ml kg−1 min−1) and 14 non‐endurance trained males (LO: V̇O2 max 45.0 ± 1.2 ml kg−1 min−1) were matched for age and height. Haemoglobin‐corrected diffusion capacity (DLCO), VC and diffusing membrane capacity (DM) were determined using the Roughton and Forster () multiple fraction of inspired O2 (FIO2)‐DLCO method at baseline and during incremental cycle exercise up to 90% of peak O2 consumption. During exercise, both groups exhibited increases in DLCO, DM and VC with exercise intensity. Athletes had a greater DLCO and greater DM at 80 and 90% of V̇O2 max compared to non‐athletes. However, VC was not different between groups during exercise. In contrast to our hypothesis, exercise VC was not greater in endurance‐trained subjects compared to controls; rather, the increased DLCO in athletes at peak exercise was secondary to an enhanced DM. These findings suggest that endurance‐trained athletes appear to have differences within the pulmonary membrane that facilitate the increased O2 demand needed for high‐level exercise.

  May 12, 2016   doi: 10.1113/JP272037   open full text
• A sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5‐HT3 receptor‐dependent pathway in female C57Bl/6 mice.
  Gayathri K. Balasuriya, Elisa L. Hill‐Yardin, Michael D. Gershon, Joel C. Bornstein.
  The Journal of Physiology. May 12, 2016
  Key points Cholera causes more than 100,000 deaths each year as a result of severe diarrhoea, vomiting and dehydration due to the actions of cholera toxin; more females than males are affected. Cholera toxin induces hypersecretion via release of mucosal serotonin and over‐activation of enteric neurons, but its effects on gastrointestinal motility are not well characterized. We found that cholera toxin rapidly and reversibly reduces colonic motility in female mice in oestrus, but not in males or females in prooestrus, an effect mediated by 5‐HT in the colonic mucosa and by 5‐HT3 receptors. We show that the number of mucosal enterochromaffin cells containing 5‐HT changes with the oestrous cycle in mice. These findings indicate that cholera toxin's effects on motility are rapid and depend on the oestrous cycle and therefore can help us better understand differences in responses in males and female patients. Abstract Extensive studies of the mechanisms responsible for the hypersecretion produced by cholera toxin (CT) have shown that this toxin produces a massive over‐activation of enteric neural secretomotor circuits. The effects of CT on gastrointestinal motility, however, have not been adequately characterized. We investigated effects of luminal CT on neurally mediated motor activity in ex vivo male and female mouse full length colon preparations. We used video recording and spatiotemporal maps of contractile activity to quantify colonic migrating motor complexes (CMMCs) and resting colonic diameter. We compared effects of CT in female colon from wild‐type and mice lacking tryptophan hydroxylase (TPH1KO). We also compared CMMCs in colons of female mice in oestrus with those in prooestrus. In female (but not male) colon, CT rapidly, reversibly and concentration‐dependently inhibits CMMC frequency and induces a tonic constriction. These effects were blocked by granisetron (5‐HT3 antagonist) and were absent from TPH1KO females. CT effects were prominent at oestrus but absent at prooestrus. The number of EC cells containing immunohistochemically demonstrable serotonin (5‐HT) was 30% greater in female mice during oestrus than during prooestrus or in males. We conclude that CT inhibits CMMCs via release of mucosal 5‐HT, which activates an inhibitory pathway involving 5‐HT3 receptors. This effect is sex‐ and oestrous cycle‐dependent and is probably due to an oestrous cycle‐dependent change in the number of 5‐HT‐containing EC cells in the colonic mucosa.

  May 12, 2016   doi: 10.1113/JP272071   open full text
• Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.
  Megan E. Wilkins, Alex Caley, Marc C. Gielen, Robert J. Harvey, Trevor G. Smart.
  The Journal of Physiology. May 10, 2016
  Key points Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. Abstract Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

  May 10, 2016   doi: 10.1113/JP272122   open full text
• Revisiting the physiological roles of SGLTs and GLUTs using positron emission tomography in mice.
  Monica Sala‐Rabanal, Bruce A. Hirayama, Chiara Ghezzi, Jie Liu, Sung‐Cheng Huang, Vladimir Kepe, Hermann Koepsell, Amy Yu, David R. Powell, Bernard Thorens, Ernest M. Wright, Jorge R. Barrio.
  The Journal of Physiology. May 10, 2016
  Key points Glucose transporters are central players in glucose homeostasis. There are two major classes of glucose transporters in the body, the passive facilitative glucose transporters (GLUTs) and the secondary active sodium‐coupled glucose transporters (SGLTs). In the present study, we report the use of a non‐invasive imaging technique, positron emission tomography, in mice aiming to evaluate the role of GLUTs and SGLTs in controlling glucose distribution and utilization. We show that GLUTs are most significant for glucose uptake into the brain and liver, whereas SGLTs are important in glucose recovery in the kidney. This work provides further support for the use of SGLT imaging in the investigation of the role of SGLT transporters in human physiology and diseases such as diabetes and cancer. Abstract The importance of sodium‐coupled glucose transporters (SGLTs) and facilitative glucose transporters (GLUTs) in glucose homeostasis was studied in mice using fluorine‐18 labelled glucose molecular imaging probes and non‐invasive positron emission tomography (PET) imaging. The probes were: α‐methyl‐4‐[F‐18]‐fluoro‐4‐deoxy‐d‐glucopyranoside (Me‐4FDG), a substrate for SGLTs; 4‐deoxy‐4‐[F‐18]‐fluoro‐d‐glucose (4‐FDG), a substrate for SGLTs and GLUTs; and 2‐deoxy‐2‐[F‐18]‐fluoro‐d–glucose (2‐FDG), a substrate for GLUTs. These radiolabelled imaging probes were injected i.v. into wild‐type, Sglt1–/–, Sglt2–/– and Glut2–/– mice and their dynamic whole‐body distribution was determined using microPET. The distribution of 2‐FDG was similar to that reported earlier (i.e. it accumulated in the brain, heart, liver and kidney, and was excreted into the urinary bladder). There was little change in the distribution of 2‐FDG in Glut2–/– mice, apart from a reduction in the rate of uptake into liver. The major differences between Me‐4FDG and 2‐FDG were that Me‐4FDG did not enter the brain and was not excreted into the urinary bladder. There was urinary excretion of Me‐4FDG in Sglt1–/– and Sglt2–/– mice. However, Me‐4FDG was not reabsorbed in the kidney in Glut2–/– mice. There were no differences in Me‐4FDG uptake into the heart of wild‐type, Sglt1–/– and Sglt2–/– mice. We conclude that GLUT2 is important in glucose liver transport and reabsorption of glucose in the kidney along with SGLT2 and SGLT1. Complete reabsorption of Me‐4FDG from the glomerular filtrate in wild‐type mice and the absence of reabsorption in the kidney in Glut2–/– mice confirm the importance of GLUT2 in glucose absorption across the proximal tubule.

  May 10, 2016   doi: 10.1113/JP271904   open full text
• Active subthreshold dendritic conductances shape the local field potential.
  Torbjørn V. Ness, Michiel W. H. Remme, Gaute T. Einevoll.
  The Journal of Physiology. May 10, 2016
  Key points The local field potential (LFP), the low‐frequency part of extracellular potentials recorded in neural tissue, is often used for probing neural circuit activity. Interpreting the LFP signal is difficult, however. While the cortical LFP is thought mainly to reflect synaptic inputs onto pyramidal neurons, little is known about the role of the various subthreshold active conductances in shaping the LFP. By means of biophysical modelling we obtain a comprehensive qualitative understanding of how the LFP generated by a single pyramidal neuron depends on the type and spatial distribution of active subthreshold currents. For pyramidal neurons, the h‐type channels probably play a key role and can cause a distinct resonance in the LFP power spectrum. Our results show that the LFP signal can give information about the active properties of neurons and imply that preferred frequencies in the LFP can result from those cellular properties instead of, for example, network dynamics. Abstract The main contribution to the local field potential (LFP) is thought to stem from synaptic input to neurons and the ensuing subthreshold dendritic processing. The role of active dendritic conductances in shaping the LFP has received little attention, even though such ion channels are known to affect the subthreshold neuron dynamics. Here we used a modelling approach to investigate the effects of subthreshold dendritic conductances on the LFP. Using a biophysically detailed, experimentally constrained model of a cortical pyramidal neuron, we identified conditions under which subthreshold active conductances are a major factor in shaping the LFP. We found that, in particular, the hyperpolarization‐activated inward current, Ih, can have a sizable effect and cause a resonance in the LFP power spectral density. To get a general, qualitative understanding of how any subthreshold active dendritic conductance and its cellular distribution can affect the LFP, we next performed a systematic study with a simplified model. We found that the effect on the LFP is most pronounced when (1) the synaptic drive to the cell is asymmetrically distributed (i.e. either basal or apical), (2) the active conductances are distributed non‐uniformly with the highest channel densities near the synaptic input and (3) when the LFP is measured at the opposite pole of the cell relative to the synaptic input. In summary, we show that subthreshold active conductances can be strongly reflected in LFP signals, opening up the possibility that the LFP can be used to characterize the properties and cellular distributions of active conductances.

  May 10, 2016   doi: 10.1113/JP272022   open full text
• Genetic alteration of the metal/redox modulation of Cav3.2 T‐type calcium channel reveals its role in neuronal excitability.
  Tiphaine Voisin, Emmanuel Bourinet, Philippe Lory.
  The Journal of Physiology. May 07, 2016
  Key points In this study, we describe a new knock‐in (KI) mouse model that allows the study of the H191‐dependent regulation of T‐type Cav3.2 channels. Sensitivity to zinc, nickel and ascorbate of native Cav3.2 channels is significantly impeded in the dorsal root ganglion (DRG) neurons of this KI mouse. Importantly, we describe that this H191‐dependent regulation has discrete but significant effects on the excitability properties of D‐hair (down‐hair) cells, a sub‐population of DRG neurons in which Cav3.2 currents prominently regulate excitability. Overall, this study reveals that the native H191‐dependent regulation of Cav3.2 channels plays a role in the excitability of Cav3.2‐expressing neurons. This animal model will be valuable in addressing the potential in vivo roles of the trace metal and redox modulation of Cav3.2 T‐type channels in a wide range of physiological and pathological conditions. Abstract Cav3.2 channels are T‐type voltage‐gated calcium channels that play important roles in controlling neuronal excitability, particularly in dorsal root ganglion (DRG) neurons where they are involved in touch and pain signalling. Cav3.2 channels are modulated by low concentrations of metal ions (nickel, zinc) and redox agents, which involves the histidine 191 (H191) in the channel's extracellular IS3–IS4 loop. It is hypothesized that this metal/redox modulation would contribute to the tuning of the excitability properties of DRG neurons. However, the precise role of this H191‐dependent modulation of Cav3.2 channel remains unresolved. Towards this goal, we have generated a knock‐in (KI) mouse carrying the mutation H191Q in the Cav3.2 protein. Electrophysiological studies were performed on a subpopulation of DRG neurons, the D‐hair cells, which express large Cav3.2 currents. We describe an impaired sensitivity to zinc, nickel and ascorbate of the T‐type current in D‐hair neurons from KI mice. Analysis of the action potential and low‐threshold calcium spike (LTCS) properties revealed that, contrary to that observed in WT D‐hair neurons, a low concentration of zinc and nickel is unable to modulate (1) the rheobase threshold current, (2) the afterdepolarization amplitude, (3) the threshold potential necessary to trigger an LTCS or (4) the LTCS amplitude in D‐hair neurons from KI mice. Together, our data demonstrate that this H191‐dependent metal/redox regulation of Cav3.2 channels can tune neuronal excitability. This study validates the use of this Cav3.2‐H191Q mouse model for further investigations of the physiological roles thought to rely on this Cav3.2 modulation.

  May 07, 2016   doi: 10.1113/JP271925   open full text
• Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure.
  Anju Paudyal, Sukriti Dewan, Cindy Ikie, Benjamin J Whalley, Pieter P. Tombe, Samuel Y. Boateng.
  The Journal of Physiology. May 05, 2016
  Key points The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase‐1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea‐pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. Abstract Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μm blebbistatin resulted in an ∼3‐fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO‐1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea‐pig model of angiotensin II infusion (400 ng kg–1 min–1) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO‐1 and HDAC signalling.

  May 05, 2016   doi: 10.1113/JP271809   open full text
• Properties and dynamics of inhibitory synaptic communication within the CA3 microcircuits of pyramidal cells and interneurons expressing parvalbumin or cholecystokinin.
  Z. Kohus, S. Káli, L. Rovira‐Esteban, D. Schlingloff, O. Papp, T. F. Freund, N. Hájos, A. I. Gulyás.
  The Journal of Physiology. May 05, 2016
  Key points To understand how a network operates, its elements must be identified and characterized, and the interactions of the elements need to be studied in detail. In the present study, we describe quantitatively the connectivity of two classes of inhibitory neurons in the hippocampal CA3 area (parvalbumin‐positive and cholecystokinin‐positive interneurons), a key region for the generation of behaviourally relevant synchronous activity patterns. We describe how interactions among these inhibitory cells and their local excitatory target neurons evolve over the course of physiological and pathological activity patterns. The results of the present study enable the construction of precise neuronal network models that may help us understand how network dynamics is generated and how it can underlie information processing and pathological conditions in the brain. We show how inhibitory dynamics between parvalbumin‐positive basket cells and pyramidal cells could contribute to sharp wave‐ripple generation. Abstract Different hippocampal activity patterns are determined primarily by the interaction of excitatory cells and different types of interneurons. To understand the mechanisms underlying the generation of different network dynamics, the properties of synaptic transmission need to be uncovered. Perisomatic inhibition is critical for the generation of sharp wave‐ripples, gamma oscillations and pathological epileptic activities. Therefore, we aimed to quantitatively and systematically characterize the temporal properties of the synaptic transmission between perisomatic inhibitory neurons and pyramidal cells in the CA3 area of mouse hippocampal slices, using action potential patterns recorded during physiological and pathological network states. Parvalbumin‐positive (PV+) and cholecystokinin‐positive (CCK+) interneurons showed distinct intrinsic physiological features. Interneurons of the same type formed reciprocally connected subnetworks, whereas the connectivity between interneuron classes was sparse. The characteristics of unitary interactions depended on the identity of both synaptic partners, whereas the short‐term plasticity of synaptic transmission depended mainly on the presynaptic cell type. PV+ interneurons showed frequency‐dependent depression, whereas more complex dynamics characterized the output of CCK+ interneurons. We quantitatively captured the dynamics of transmission at these different types of connection with simple mathematical models, and describe in detail the response to physiological and pathological discharge patterns. Our data suggest that the temporal propeties of PV+ interneuron transmission may contribute to sharp wave‐ripple generation. These findings support the view that intrinsic and synaptic features of PV+ cells make them ideally suited for the generation of physiological network oscillations, whereas CCK+ cells implement a more subtle, graded control in the hippocampus.

  May 05, 2016   doi: 10.1113/JP272231   open full text
• Adrenaline release evokes hyperpnoea and an increase in ventilatory CO2 sensitivity during hypoglycaemia: a role for the carotid body.
  Emma L. Thompson, Clare J. Ray, Andrew P. Holmes, Richard L. Pye, Christopher N. Wyatt, Andrew M. Coney, Prem Kumar.
  The Journal of Physiology. May 05, 2016
  Key points Hypoglycaemia is counteracted by release of hormones and an increase in ventilation and CO2 sensitivity to restore blood glucose levels and prevent a fall in blood pH. The full counter‐regulatory response and an appropriate increase in ventilation is dependent on carotid body stimulation. We show that the hypoglycaemia‐induced increase in ventilation and CO2 sensitivity is abolished by preventing adrenaline release or blocking its receptors. Physiological levels of adrenaline mimicked the effect of hypoglycaemia on ventilation and CO2 sensitivity. These results suggest that adrenaline, rather than low glucose, is an adequate stimulus for the carotid body‐mediated changes in ventilation and CO2 sensitivity during hypoglycaemia to prevent a serious acidosis in poorly controlled diabetes. Abstract Hypoglycaemia in vivo induces a counter‐regulatory response that involves the release of hormones to restore blood glucose levels. Concomitantly, hypoglycaemia evokes a carotid body‐mediated hyperpnoea that maintains arterial CO2 levels and prevents respiratory acidosis in the face of increased metabolism. It is unclear whether the carotid body is directly stimulated by low glucose or by a counter‐regulatory hormone such as adrenaline. Minute ventilation was recorded during infusion of insulin‐induced hypoglycaemia (8–17 mIU kg−1 min−1) in Alfaxan‐anaesthetised male Wistar rats. Hypoglycaemia significantly augmented minute ventilation (123 ± 4 to 143 ± 7 ml min−1) and CO2 sensitivity (3.3 ± 0.3 to 4.4 ± 0.4 ml min−1 mmHg−1). These effects were abolished by either β‐adrenoreceptor blockade with propranolol or adrenalectomy. In this hypermetabolic, hypoglycaemic state, propranolol stimulated a rise in P aC O2, suggestive of a ventilation–metabolism mismatch. Infusion of adrenaline (1 μg kg−1 min−1) increased minute ventilation (145 ± 4 to 173 ± 5 ml min−1) without altering P aC O2 or pH and enhanced ventilatory CO2 sensitivity (3.4 ± 0.4 to 5.1 ± 0.8 ml min−1 mmHg−1). These effects were attenuated by either resection of the carotid sinus nerve or propranolol. Physiological concentrations of adrenaline increased the CO2 sensitivity of freshly dissociated carotid body type I cells in vitro. These findings suggest that adrenaline release can account for the ventilatory hyperpnoea observed during hypoglycaemia by an augmented carotid body and whole body ventilatory CO2 sensitivity.

  May 05, 2016   doi: 10.1113/JP272191   open full text
• The mechanistic bases of the power–time relationship: muscle metabolic responses and relationships to muscle fibre type.
  Anni Vanhatalo, Matthew I. Black, Fred J. DiMenna, Jamie R. Blackwell, Jakob Friis Schmidt, Christopher Thompson, Lee J. Wylie, Magni Mohr, Jens Bangsbo, Peter Krustrup, Andrew M. Jones.
  The Journal of Physiology. May 01, 2016
  Key points The power‐asymptote (critical power; CP) of the hyperbolic power–time relationship for high‐intensity exercise defines a threshold between steady‐state and non‐steady‐state exercise intensities and the curvature constant (W′) indicates a fixed capacity for work >CP that is related to a loss of muscular efficiency. The present study reports novel evidence on the muscle metabolic underpinnings of CP and W′ during whole‐body exercise and their relationships to muscle fibre type. We show that the W′ is not correlated with muscle fibre type distribution and that it represents an elevated energy contribution from both oxidative and glycolytic/glycogenolytic metabolism. We show that there is a positive correlation between CP and highly oxidative type I muscle fibres and that muscle metabolic steady‐state is attainable CP. Our findings indicate a mechanistic link between the bioenergetic characteristics of muscle fibre types and the power–time relationship for high‐intensity exercise. Abstract We hypothesized that: (1) the critical power (CP) will represent a boundary separating steady‐state from non‐steady‐state muscle metabolic responses during whole‐body exercise and (2) that the CP and the curvature constant (W′) of the power–time relationship for high‐intensity exercise will be correlated with type I and type IIx muscle fibre distributions, respectively. Four men and four women performed a 3 min all‐out cycling test for the estimation of CP and constant work rate (CWR) tests slightly >CP until exhaustion (Tlim), slightly CP Tlim isotime to test the first hypothesis. Eleven men performed 3 min all‐out tests and donated muscle biopsies to test the second hypothesis. Below CP, muscle [PCr] [42.6 ± 7.1 vs. 49.4 ± 6.9 mmol (kg d.w.)−1], [La−] [34.8 ± 12.6 vs. 35.5 ± 13.2 mmol (kg d.w.)−1] and pH (7.11 ± 0.08 vs. 7.10 ± 0.11) remained stable between ∼12 and 24 min (P > 0.05 for all), whereas these variables changed with time >CP such that they were greater [[La−] 95.6 ± 14.1 mmol (kg d.w.)−1] and lower [[PCr] 24.2 ± 3.9 mmol (kg d.w.)−1; pH 6.84 ± 0.06] (P < 0.05) at Tlim (740 ± 186 s) than during the

  May 01, 2016   doi: 10.1113/JP271879   open full text
• Role of enteroendocrine L‐cells in arginine vasopressin‐mediated inhibition of colonic anion secretion.
  Ramona Pais, Juraj Rievaj, Claire Meek, Gayan Costa, Samanthie Jayamaha, R. Todd Alexander, Frank Reimann, Fiona Gribble.
  The Journal of Physiology. April 28, 2016
  Key points Arginine vasopressin (AVP) stimulates the release of enteroendocrine L‐cell derived hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY) in vitro from mouse and human colons. This is mediated by the AVP receptor 1B, which is highly enriched in colonic L‐cells and linked to the elevation of L‐cell calcium and cAMP concentrations. By means of Ussing chambers, we show that AVP reduced colonic anion secretion, although this was blocked by a specific neuropeptide Y receptor Y1 receptor antagonist, suggesting that L‐cell‐released PYY acts locally on the epithelium to modulate fluid balance. In human serum samples, PYY concentrations were higher in samples with raised osmolality and copeptin (surrogate marker for AVP). These findings describe, for the first time, the role of L‐cells in AVP regulated intestinal fluid secretion, potentially linking together hormonal control of blood volume and blood glucose levels, and thus adding to our understanding of the complex pathways involved in the gut hormonal response to different stimuli. Abstract Arginine vasopressin (AVP) regulates fluid balance and blood pressure via AVP receptor (AVPR)2 in the kidney and AVP receptor 1A in vascular smooth muscle. Its role in intestinal function has received less attention. We hypothesized that enteroendocrine L‐cells producing glucagon‐like peptide 1 (GLP‐1) and peptide YY (PYY) may be a target of AVP and contribute to the control of fluid balance. Avpr1b expression was assessed by quantitative RT‐PCR on flourescence‐activated cell sorting‐isolated L‐ and control cells and was enriched in colonic L‐cells. AVP stimulated GLP‐1 and PYY release from primary cultured murine and human colonic cells and was associated with elevated calcium and cAMP concentrations in L‐cells as measured in cultures from GLU‐Cre/ROSA26‐GCaMP3 and GLU‐Epac2camps mice. An antagonist of AVPR1B reduced AVP‐triggered hormone secretion from murine and human cells. In Ussing chambers, basolaterally applied AVP reduced colonic anion secretion and this effect was blocked by a specific neuropeptide Y receptor Y1 (NPY1R) antagonist. In human serum, PYY concentrations were higher in samples with raised osmolality or copeptin (a surrogate marker for AVP). In conclusion, we propose that AVP activates L‐cell AVPR1B, causing GLP‐1 and PYY secretion. PYY in turn reduces colonic anion secretion via epithelial NPY1R. Our data suggest L‐cells are active players in the hypothalamic control of intestinal fluid homeostasis, providing a potential link between the regulation of blood volume/pressure/osmolality and blood glucose.

  April 28, 2016   doi: 10.1113/JP272053   open full text
• Physiological adaptations to chronic stress in healthy humans – why might the sexes have evolved different energy utilisation strategies?
  Alexander Jones, Jens C. Pruessner, Merlin R. McMillan, Russell W. Jones, Grzegorz T. Kowalik, Jennifer A. Steeden, Bryan Williams, Andrew M. Taylor, Vivek Muthurangu.
  The Journal of Physiology. April 28, 2016
  Key points The human stress response activates the autonomic nervous system and endocrine systems to increase performance during environmental challenges. This response is usually beneficial, improving the chance of overcoming environmental challenges, but costs resources such as energy. Humans and other animals are known to adapt their responses to acute stress when they are stimulated chronically, presumably to optimise resource utilisation. Characterisation of these adaptations has been limited. Using advanced imaging techniques, we show that cardiovascular and endocrine physiology, reflective of energy utilisation during acute stress, and energy storage (fat) differ between the sexes when they are exposed to chronic stress. We examine possible evolutionary explanations for these differences, related to energy use, and point out how these physiological differences could underpin known disparities between the sexes in their risk of important cardiometabolic disorders such as obesity and cardiovascular disease. Abstract Obesity and associated diseases, such as cardiovascular disease, are the dominant human health problems in the modern era. Humans develop these conditions partly because they consume excess energy and exercise too little. Stress might be one of the factors contributing to these disease‐promoting behaviours. We postulate that sex‐specific primordial energy optimisation strategies exist, which developed to help cope with chronic stress but have become maladaptive in modern societies, worsening health. To demonstrate the existence of these energy optimisation strategies, we recruited 88 healthy adults with varying adiposity and chronic stress exposure. Cardiovascular physiology at rest and during acute stress (Montreal Imaging Stress Task), and body fat distribution were measured using advanced magnetic resonance imaging methods, together with endocrine function, cardiovascular energy use and cognitive performance. Potential confounders such as lifestyle, social class and employment were accounted for. We found that women exposed to chronic stress had lower adiposity, greater acute stress cardiovascular responses and better cognitive performance. Conversely, chronic stress‐exposed men had greater adiposity and lower cardiovascular responses to acute stress. These results provide initial support for our hypothesis that differing sex‐specific energy conservation strategies exist. We propose that these strategies have initially evolved to benefit humans but are now maladaptive and increase the risk of disorders such as obesity, especially in men exposed to chronic stress.

  April 28, 2016   doi: 10.1113/JP272021   open full text
• Divergent in vivo activity of non‐serotonergic and serotonergic VGluT3–neurones in the median raphe region.
  Andor Domonkos, Litsa Nikitidou Ledri, Tamás Laszlovszky, Csaba Cserép, Zsolt Borhegyi, Edit Papp, Gábor Nyiri, Tamás F. Freund, Viktor Varga.
  The Journal of Physiology. April 28, 2016
  Key points The median raphe is a key subcortical modulatory centre involved in several brain functions, such as regulation of the sleep–wake cycle, emotions and memory storage. A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication compared to serotonergic cells, although their in vivo activity is unknown. We provide the first description of the in vivo, brain state‐dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5‐HT). Glutamatergic populations (VGluT3+/5‐HT– and VGluT3+/5‐HT+) were compared with the purely serotonergic (VGluT3–/5‐HT+ and VGluT3–/5‐HT–) neurones. VGluT3+/5‐HT+ neurones fired similar to VGluT3–/5‐HT+ cells, whereas they significantly diverged from the VGluT3+/5‐HT– population. Activity of the latter subgroup resembled the spiking of VGluT3–/5‐HT– cells, except for their diverging response to sensory stimulation. The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state‐dependent, tonic modulation. Abstract Subcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. In the present study, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)‐expressing median raphe neurones. Because there is no appropriate genetic marker for the separation of serotonergic (5‐HT+) and non‐serotonergic (5‐HT–) VGluT3+ neurones, we utilized immunohistochemistry after recording and juxtacellular labelling in anaesthetized rats. VGluT3+/5‐HT– neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3–/5‐HT+ subgroup. VGluT3+/5‐HT– cells were also more active during hippocampal theta. In addition, the VGluT3–/5‐HT– population, comprising putative GABAergic cells, resembled the firing of VGluT3+/5‐HT– neurones but without any significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5‐HT+ group, spiking slower than the VGluT3+/5‐HT– population, exhibited a mixed response (i.e. the initial transient activation was followed by a sustained elevation of firing). Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5‐HT– neurones, also differentiating them from the VGluT3+/5‐HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory‐evoked rapid transients, as well as a fast one capable of conveying complex patterns influenced by sensory inputs.

  April 28, 2016   doi: 10.1113/JP272036   open full text
• Stimulation of dopamine D2‐like receptors in the lumbosacral defaecation centre causes propulsive colorectal contractions in rats.
  Kiyotada Naitou, Hiroyuki Nakamori, Takahiko Shiina, Azusa Ikeda, Yuuta Nozue, Yuuki Sano, Takuya Yokoyama, Yoshio Yamamoto, Akihiro Yamada, Nozomi Akimoto, Hidemasa Furue, Yasutake Shimizu.
  The Journal of Physiology. April 28, 2016
  Key points The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure. In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum. The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors. Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons. Abstract We have recently demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum. We hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. Because dopamine is one of the major neurotransmitters of descending pain inhibitory pathways in the spinal cord, we examined the effects of the intrathecal application of dopamine to the spinal defaecation centre on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anaesthetized rats. Slice patch clamp and immunohistochemistry were used to confirm the existence of dopamine‐sensitive neurons in the sacral parasympathetic nuclei. Intrathecal application of dopamine into the L6–S1 spinal cord, where the lumbosacral defaecation centre is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons using TTX blocked the effect of dopamine. Although thoracic spinal transection had no effect on the enhancement of colorectal motility by intrathecal dopamine, the severing of the pelvic nerves abolished the enhanced motility. Pharmacological experiments revealed that the effect of dopamine is mediated primarily by D2‐like dopamine receptors. Neurons labelled with retrograde dye injected at the colorectum showed an inward current in response to dopamine in slice patch clamp recordings. Furthermore, immunohistochemical analysis revealed that neurons immunoreactive to choline acetyltransferase express D2‐like dopamine receptors. Taken together, our findings demonstrate that dopamine activates sacral parasympathetic preganglionic neurons via D2‐like dopamine receptors and causes propulsive motility of the colorectum in rats. The present study supports the hypothesis that descending pain inhibitory pathways regulate defaecation reflexes.

  April 28, 2016   doi: 10.1113/JP272073   open full text
• Origins of the vagal drive controlling left ventricular contractility.
  Asif Machhada, Nephtali Marina, Alla Korsak, Daniel J. Stuckey, Mark F. Lythgoe, Alexander V. Gourine.
  The Journal of Physiology. April 28, 2016
  Key points The strength, functional significance and origins of parasympathetic innervation of the left ventricle remain controversial. This study tested the hypothesis that parasympathetic control of left ventricular contractility is provided by vagal preganglionic neurones of the dorsal motor nucleus (DVMN). Under β‐adrenoceptor blockade combined with spinal cord (C1) transection (to remove sympathetic influences), systemic administration of atropine increased left ventricular contractility in rats anaesthetized with urethane, confirming the existence of a tonic inhibitory muscarinic influence on cardiac inotropy. Increased left ventricular contractility in anaesthetized rats was observed when DVMN neurones were silenced. Functional neuroanatomical mapping revealed that vagal preganglionic neurones that have an impact on left ventricular contractility are located in the caudal region of the left DVMN. These neurones provide functionally significant parasympathetic control of left ventricular inotropy. Abstract The strength, functional significance and origins of direct parasympathetic innervation of the left ventricle (LV) remain controversial. In the present study we used an anaesthetized rat model to first confirm the presence of tonic inhibitory vagal influence on LV inotropy. Using genetic neuronal targeting and functional neuroanatomical mapping we tested the hypothesis that parasympathetic control of LV contractility is provided by vagal preganglionic neurones located in the dorsal motor nucleus (DVMN). It was found that under systemic β‐adrenoceptor blockade (atenolol) combined with spinal cord (C1) transection (to remove sympathetic influences), intravenous administration of atropine increases LV contractility in rats anaesthetized with urethane, but not in animals anaesthetized with pentobarbital. Increased LV contractility in rats anaesthetized with urethane was also observed when DVMN neurones targeted bilaterally to express an inhibitory Drosophila allatostatin receptor were silenced by application of an insect peptide allatostatin. Microinjections of glutamate and muscimol to activate or inhibit neuronal cell bodies in distinct locations along the rostro‐caudal extent of the left and right DVMN revealed that vagal preganglionic neurones, which have an impact on LV contractility, are located in the caudal region of the left DVMN. Changes in LV contractility were only observed when this subpopulation of DVMN neurones was activated or inhibited. These data confirm the existence of a tonic inhibitory muscarinic influence on LV contractility. Activity of a subpopulation of DVMN neurones provides functionally significant parasympathetic control of LV contractile function.

  April 28, 2016   doi: 10.1113/JP270984   open full text
• Mechano‐signalling pathways in an experimental intensive critical illness myopathy model.
  Rebeca Corpeno Kalamgi, Heba Salah, Stefano Gastaldello, Vicente Martinez‐Redondo, Jorge L. Ruas, Wen Fury, Yu Bai, Jesper Gromada, Roberta Sartori, Denis C. Guttridge, Marco Sandri, Lars Larsson.
  The Journal of Physiology. April 24, 2016
  Key points Using an experimental rat intensive care unit (ICU) model, not limited by early mortality, we have previously shown that passive mechanical loading attenuates the loss of muscle mass and force‐generation capacity associated with the ICU intervention. Mitochondrial dynamics have recently been shown to play a more important role in muscle atrophy than previously recognized. In this study we demonstrate that mitochondrial dynamics, as well as mitophagy, is affected by mechanosensing at the transcriptional level, and muscle changes induced by unloading are counteracted by passive mechanical loading. The recently discovered ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that similarly is prevented by passive mechanical loading. Abstract The complete loss of mechanical stimuli of skeletal muscles, i.e. loss of external strain related to weight bearing and internal strain related to activation of contractile proteins, in mechanically ventilated, deeply sedated and/or pharmacologically paralysed intensive care unit (ICU) patients is an important factor triggering the critical illness myopathy (CIM). Using a unique experimental ICU rat model, mimicking basic ICU conditions, we have recently shown that mechanical silencing is a dominant factor triggering the preferential loss of myosin, muscle atrophy and decreased specific force in fast‐ and slow‐twitch muscles and muscle fibres. The aim of this study is to gain improved understanding of the gene signature and molecular pathways regulating the process of mechanical activation of skeletal muscle that are affected by the ICU condition. We have focused on pathways controlling myofibrillar protein synthesis and degradation, mitochondrial homeostasis and apoptosis. We demonstrate that genes regulating mitochondrial dynamics, as well as mitophagy are induced by mechanical silencing and that these effects are counteracted by passive mechanical loading. In addition, the recently identified ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that is reversed by passive mechanical loading. Thus, mechano‐cell signalling events are identified which may play an important role for the improved clinical outcomes reported in response to the early mobilization and physical therapy in immobilized ICU patients.

  April 24, 2016   doi: 10.1113/JP271973   open full text
• Purinergic signalling underlies transforming growth factor‐β‐mediated bladder afferent nerve hyperexcitability.
  Eric J. Gonzalez, Thomas J. Heppner, Mark T. Nelson, Margaret A. Vizzard.
  The Journal of Physiology. April 24, 2016
  Key points The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor‐β1 (TGF‐β1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin‐1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP‐induced cystitis is decreased with TGF‐β inhibition. These results establish a causal link between an inflammatory mediator, TGF‐β, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling. Abstract The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor‐β (TGF‐β) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF‐β in bladder inflammation, the precise mechanisms of TGF‐β mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF‐β activation. We utilized bladder–pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF‐β1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin‐1 channels. Furthermore, blocking aberrant TGF‐β signalling in cyclophosphamide‐induced cystitis with TβR‐1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF‐β‐mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.

  April 24, 2016   doi: 10.1113/JP272148   open full text
• Opsin spectral sensitivity determines the effectiveness of optogenetic termination of ventricular fibrillation in the human heart: a simulation study.
  Thomas V. Karathanos, Jason D. Bayer, Dafang Wang, Patrick M. Boyle, Natalia A. Trayanova.
  The Journal of Physiology. April 24, 2016
  Key points Optogenetics‐based defibrillation, a theoretical alternative to electrotherapy, involves expression of light‐sensitive ion channels in the heart (via gene or cell therapy) and illumination of the cardiac surfaces (via implanted LED arrays) to elicit light‐induced activations. We used a biophysically detailed human ventricular model to determine whether such a therapy could terminate fibrillation (VF) and identify which combinations of light‐sensitive ion channel properties and illumination configurations would be effective. Defibrillation was successful when a large proportion (> 16.6%) of ventricular tissue was directly stimulated by light that was bright enough to induce an action potential in an uncoupled cell. While illumination with blue light never successfully terminated VF, illumination of red light‐sensitive ion channels with dense arrays of implanted red light sources resulted in successful defibrillation. Our results suggest that cardiac expression of red light‐sensitive ion channels is necessary for the development of effective optogenetics‐based defibrillation therapy using LED arrays. Abstract Optogenetics‐based defibrillation has been proposed as a novel and potentially pain‐free approach to enable cardiomyocyte‐selective defibrillation in humans, but the feasibility of such a therapy remains unknown. This study aimed to (1) assess the feasibility of terminating sustained ventricular fibrillation (VF) via light‐induced excitation of opsins expressed throughout the myocardium and (2) identify the ideal (theoretically possible) opsin properties and light source configurations that would maximise therapeutic efficacy. We conducted electrophysiological simulations in an MRI‐based human ventricular model with VF induced by rapid pacing; light sensitisation via systemic, cardiac‐specific gene transfer of channelrhodopsin‐2 (ChR2) was simulated. In addition to the widely used blue light‐sensitive ChR2‐H134R, we also modelled theoretical ChR2 variants with augmented light sensitivity (ChR2+), red‐shifted spectral sensitivity (ChR2‐RED) or both (ChR2‐RED+). Light sources were modelled as synchronously activating LED arrays (LED radius: 1 mm; optical power: 10 mW mm–2; array density: 1.15–4.61 cm–2). For each unique optogenetic configuration, defibrillation was attempted with two different optical pulse durations (25 and 500 ms). VF termination was only successful for configurations involving ChR2‐RED and ChR2‐RED+ (for LED arrays with density ≥ 2.30 cm–2), suggesting that opsin spectral sensitivity was the most important determinant of optogenetic defibrillation efficacy. This was due to the deeper penetration of red light in cardiac tissue compared with blue light, which resulted in more widespread light‐induced propagating wavefronts. Longer pulse duration and higher LED array density were associated with increased optogenetic defibrillation efficacy. In all cases observed, the defibrillation mechanism was light‐induced depolarisation of the excitable gap, which led to block of reentrant wavefronts.

  April 24, 2016   doi: 10.1113/JP271739   open full text
• Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2‐associated Andersen–Tawil Syndrome.
  Dany Spencer Adams, Sebastien G. M. Uzel, Jin Akagi, Donald Wlodkowic, Viktoria Andreeva, Pamela Crotty Yelick, Adrian Devitt‐Lee, Jean‐Francois Pare, Michael Levin.
  The Journal of Physiology. April 13, 2016
  Key points Xenopus laevis craniofacial development is a good system for the study of Andersen–Tawil Syndrome (ATS)‐associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular‐genetic and biophysical techniques are available for the study of ion‐dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light‐activated channels, all of which have an effect on Vmem, causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl− channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion‐ or channel‐specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS‐associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux‐modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. Abstract Variants in potassium channel KCNJ2 cause Andersen–Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS‐associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells’ resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non‐neural cells in embryos, we show that developmentally patterned K+ flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

  April 13, 2016   doi: 10.1113/JP271930   open full text
• Single cell transcriptome analysis of mouse carotid body glomus cells.
  Ting Zhou, Ming‐Shan Chien, Safa Kaleem, Hiroaki Matsunami.
  The Journal of Physiology. April 13, 2016
  Key points Carotid body (CB) glomus cells mediate acute oxygen sensing and the initiation of the hypoxic ventilatory response, yet the gene expression profile of these cells is not available. We demonstrate that the single cell RNA‐Seq method is a powerful tool for identifying highly expressed genes in CB glomus cells. Our single cell RNA‐Seq results characterized novel CB glomus cell genes, including members of the G protein‐coupled receptor signalling pathway, ion channels and atypical mitochondrial electron transport chain subunits. A heterologous cell‐based screening identified acetate (which is known to affect CB glomus cell activity) as an agonist for the most highly abundant G protein‐coupled receptor (Olfr78) in CB glomus cells. These data established the first transcriptome profile of CB glomus cells, highlighting genes with potential implications in CB chemosensory function. Abstract The carotid body (CB) is a major arterial chemoreceptor containing glomus cells whose activities are regulated by changes in arterial blood content, including oxygen. Despite significant advancements in the characterization of their physiological properties, our understanding of the underlying molecular machinery and signalling pathway in CB glomus cells is still limited. To overcome this, we employed the single cell RNA­Seq method by performing next‐generation sequencing on single glomus cell­derived cDNAs to eliminate contamination of genes derived from other cell types present in the CB. Using this method, we identified a set of genes abundantly expressed in glomus cells, which contained novel glomus cell‐specific genes. Transcriptome and subsequent in situ hybridization and immunohistochemistry analyses identified abundant G protein‐coupled receptor signalling pathway components and various types of ion channels, as well as members of the hypoxia‐inducible factors pathway. A short‐chain fatty acid olfactory receptor Olfr78, recently implicated in CB function, was the most abundant G protein‐coupled receptor. Two atypical mitochondrial electron transport chain subunits (Ndufa4l2 and Cox4i2) were among the most specifically expressed genes in CB glomus cells, highlighting their potential roles in mitochondria‐mediated oxygen sensing. The wealth of information provided by the present study offers a valuable foundation for identifying molecules functioning in the CB.

  April 13, 2016   doi: 10.1113/JP271936   open full text
• Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox‐sensitive genes in human kidney proximal tubular cells.
  Rodolfo Sumayao, Bernadette McEvoy, Philip Newsholme, Tara McMorrow.
  The Journal of Physiology. April 10, 2016
  Key points Cystine is a disulphide amino acid that is normally generated in the lysosomes by the breakdown of cystine‐containing proteins. Previously, we demonstrated that lysosomal cystine accumulation in kidney proximal tubular epithelial cells (PTECs) dramatically reduced glutathione (GSH) levels, which may result in the disruption of cellular redox balance. In the present study, we show that lysosomal cystine accumulation following CTNS gene silencing in kidney PTECs resulted in elevated intracellular reactive oxygen species production, reduced antioxidant capacity, induction of redox‐sensitive proteins, altered mitochondrial integrity and augmented cell death. These alterations may represent different facets of a unique cascade leading to tubular dysfunction initiated by lysosomal cystine accumulation and may present a clear disadvantage for cystinotic PTECs in vivo. Cystine depletion by cysteamine afforded cytoprotection in CTNS knockdown cells by reducing oxidative stress, normalizing intracellular GSH and ATP content, and preserving cell viability. Abstract Cystine is a disulphide amino acid that is normally generated within the lysosomes through lysosomal‐based protein degradation and via extracellular uptake of free cystine. In the autosomal recessive disorder, cystinosis, a defect in the CTNS gene results in excessive lysosomal accumulation of cystine, with early kidney failure a hallmark of the disease. Previously, we demonstrated that silencing of the CTNS gene in kidney proximal tubular epithelial cells (PTECs) resulted in an increase in intracellular cystine concentration coupled with a dramatic reduction in the total GSH content. Because of the crucial role of GSH in maintaining the redox status and viability of kidney PTECs, we assessed the effects of CTNS knockdown‐induced lysosomal cystine accumulation on intracellular reactive oxygen species (ROS) production, activity of classical redox‐sensitive genes, mitochondrial integrity and cell viability. Our results showed that lysosomal cystine accumulation increased ROS production and solicitation to oxidative stress (OS). This was associated with the induction of classical redox‐sensitive proteins, NF‐κB, NRF2, HSP32 and HSP70. Cystine‐loaded PTECs also displayed depolarized mitochondria, reduced ATP content and augmented apoptosis. Treatment of CTNS knockdown PTECs with the cystine‐depleting agent cysteamine resulted in the normalization of OS index, increased GSH and ATP content, and preservation of cell viability. Taken together, the alterations observed in cystinotic cells may represent different facets of a cascade leading to tubular dysfunction and, in combination with cysteamine therapy, may offer a novel link for the attenuation of renal injury and preservation of functions of other organs affected in cystinosis.

  April 10, 2016   doi: 10.1113/JP271858   open full text
• Age decreases mitochondrial motility and increases mitochondrial size in vascular smooth muscle.
  Susan Chalmers, Christopher D. Saunter, John M. Girkin, John G. McCarron.
  The Journal of Physiology. April 09, 2016
  Key points Age is proposed to be associated with altered structure and function of mitochondria; however, in fully‐differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel‐by‐pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during ‘flickers’ of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly‐elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly‐motile. Mitochondria that are motile are located more peripherally in the cell than non‐motile mitochondria. Abstract Mitochondrial function, motility and architecture are each central to cell function. Age‐associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill‐defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic ‘flickers’ of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker‐assisted localization microscopy; FaLM). In freshly‐isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X‐Y areas in both young adult (3 months; 0.05–6.58 μm2) and aged rats (18 months; 0.05–13.4 μm2). In cells from young animals, most mitochondria were small (mode area 0.051 μm2) compared to aged animals (0.710 μm2). Cells from older animals contained a subpopulation of highly‐elongated mitochondria (5.3% were >2 μm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 μm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 μm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian‐like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 μm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly‐elongated. These age‐associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.

  April 09, 2016   doi: 10.1113/JP271942   open full text
• Exercise training improves obesity‐related lymphatic dysfunction.
  Geoffrey E. Hespe, Raghu P. Kataru, Ira L. Savetsky, Gabriela D. García Nores, Jeremy S. Torrisi, Matthew D. Nitti, Jason C. Gardenier, Jie Zhou, Jessie Z. Yu, Lee W. Jones, Babak J. Mehrara.
  The Journal of Physiology. April 09, 2016
  Key points Obesity results in perilymphatic inflammation and lymphatic dysfunction. Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells. Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. Abstract Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti‐inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR‐3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity‐induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications.

  April 09, 2016   doi: 10.1113/JP271757   open full text
• Carbon dioxide‐mediated vasomotion of extra‐cranial cerebral arteries in humans: a role for prostaglandins?
  Ryan L. Hoiland, Michael M. Tymko, Anthony R. Bain, Kevin W. Wildfong, Brad Monteleone, Philip N. Ainslie.
  The Journal of Physiology. April 06, 2016
  Key points Cerebral blood flow increases during hypercapnia and decreases during hypocapnia; it is unknown if vasomotion of the internal carotid artery is implicated in these responses. Indomethacin, a non‐selective cyclooxygenase inhibitor (used to inhibit prostaglandin synthesis), has a unique ability to blunt cerebrovascular carbon dioxide reactivity, while other cyclooxygenase inhibitors have no effect. We show significant dilatation and constriction of the internal carotid artery during hypercapnia and hypocapnia, respectively. Indomethacin, but not ketorolac or naproxen, reduced the dilatatory response of the internal carotid artery to hypercapnia The differential effect of indomethacin compared to ketorolac and naproxen suggests that indomethacin inhibits vasomotion of the internal carotid artery independent of prostaglandin synthesis inhibition. Abstract Extra‐cranial cerebral blood vessels are implicated in the regulation of cerebral blood flow during changes in arterial CO2; however, the mechanisms governing CO2‐mediated vasomotion of these vessels in humans remain unclear. We determined if cyclooxygenase inhibition with indomethacin (INDO) reduces the vasomotor response of the internal carotid artery (ICA) to changes in end‐tidal CO2 (P ETC O2). Using a randomized single‐blinded placebo‐controlled study, participants (n = 10) were tested on two occasions, before and 90 min following oral INDO (1.2 mg kg–1) or placebo. Concurrent measurements of beat‐by‐beat velocity, diameter and blood flow of the ICA were made at rest and during steady‐state stages (4 min) of iso‐oxic hypercapnia (+3, +6, +9 mmHg P ETC O2) and hypocapnia (−3, −6, −9 mmHg P ETC O2). To examine if INDO affects ICA vasomotion independent of cyclooxygenase inhibition, two participant subsets (each n = 5) were tested before and following oral ketorolac (post 45 min, 0.25 mg kg–1) or naproxen (post 90 min, 4.2 mg kg–1). During pre‐drug testing in the INDO trial, the ICA dilatated during hypercapnia at +6 mmHg (4.72 ± 0.45 vs. 4.95 ± 0.51 mm; P < 0.001) and +9 mmHg (4.72 ± 0.45 mm vs. 5.12 ± 0.47 mm; P < 0.001), and constricted during hypocapnia at −6 mmHg (4.95 ± 0.33 vs. 4.88 ± 0.27 mm; P < 0.05) and −9 mmHg (4.95 ± 0.33 vs. 4.82 ± 0.27 mm; P < 0.001). Following INDO, vasomotor responsiveness of the ICA to hypercapnia was reduced by 67 ± 28% (0.045 ± 0.015 vs. 0.015 ± 0.012 mm mmHg P ETC O2−1). There was no effect of the drug in the ketorolac and naproxen trials. We conclude that: (1) INDO markedly reduces the vasomotor response of the ICA to changes in P ETC O2; and (2) INDO may be reducing CO2‐mediated vasomotion via a mechanism(s) independent of cyclooxygenase inhibition.

  April 06, 2016   doi: 10.1113/JP272012   open full text
• The interactive contributions of Na+/K+‐ATPase and nitric oxide synthase to sweating and cutaneous vasodilatation during exercise in the heat.
  Jeffrey C. Louie, Naoto Fujii, Robert D. Meade, Glen P. Kenny.
  The Journal of Physiology. March 29, 2016
  Key points Nitric oxide synthase (NOS) contributes to sweating and cutaneous vasodilatation during exercise in the heat. Similarly, reports show that Na+/K+‐ATPase activation can modulate sweating and microvascular circulation. In light of the fact that NO can activate Na+/K+‐ATPase, we evaluated whether there is an interaction between Na+/K+‐ATPase and NOS in the regulation of heat loss responses during an exercise‐induced heat stress. We demonstrate that Na+/K+‐ATPase and NOS do not synergistically influence local forearm sweating during moderate intensity (fixed rate of metabolic heat production of 500 W) exercise in the heat (35°C). Conversely, we show an interactive role between NOS and Na+/K+‐ATPase in the modulation of cutaneous vasodilatation. These findings provide novel insight regarding the mechanisms underpinning the control of sweating and cutaneous vasodilatation during exercise in the heat. Given that ouabain may be prescribed as a cardiac glycoside in clinical settings, potential heat loss impairments with ouabain administration should be explored. Abstract Nitric oxide (NO) synthase (NOS) contributes to the heat loss responses of sweating and cutaneous vasodilatation. Given that NO can activate Na+/K+‐ATPase, which also contributes to sweating and microvasculature regulation, we evaluated the separate and combined influence of Na+/K+‐ATPase and NOS on sweating and cutaneous vasodilatation. Thirteen young (23±3 years) males performed two 30 min semi‐recumbent cycling bouts in the heat (35°C) at a fixed rate of metabolic heat production (500 W) followed by 20 and 40 min recoveries, respectively. Local sweat rate (LSR) and cutaneous vascular conductance (CVC) were measured at four forearm skin sites continuously perfused via intradermal microdialysis with either: (1) lactated Ringer solution (Control); (2) 6 mᴍ ouabain (Ouabain), a Na+/K+‐ATPase inhibitor; (3) 10 mᴍ l‐NG‐nitroarginine methyl ester (l‐NAME), a NOS inhibitor; or (4) 6 mᴍ ouabain and 10 mᴍ l‐NAME (Ouabain+l‐NAME). At the end of both exercise bouts relative to Control, LSR was attenuated with Ouabain (54–60%), l‐NAME (12–13%) and Ouabain+l‐NAME (68–74%; all P < 0.05). Moreover, the sum of attenuations from Control induced by independent administration of Ouabain and l‐NAME was similar to the combined infusion of Ouabain+l‐NAME (both P ≥ 0.74). Compared to Control, CVC at the end of both exercise bouts was similar with Ouabain (both P ≥ 0.30), but attenuated with l‐NAME (%CVCmax reduction from Control, 24–25%). Furthermore, CVC at the Ouabain+l‐NAME site (38–39%; all P < 0.01) was attenuated compared to Control and did not differ from baseline resting values (both P ≥ 0.81). We show that Na+/K+‐ATPase and NOS do not synergistically mediate sweating, whereas they influence cutaneous blood flow in an interactive manner during exercise in the heat.

  March 29, 2016   doi: 10.1113/JP271990   open full text
• The relationship of pulmonary vascular resistance and compliance to pulmonary artery wedge pressure during submaximal exercise in healthy older adults.
  Stephen P. Wright, John T. Granton, Sam Esfandiari, Jack M. Goodman, Susanna Mak.
  The Journal of Physiology. March 24, 2016
  Key points A consistent inverse hyperbolic relationship has been observed between pulmonary vascular resistance and compliance, although changes in pulmonary artery wedge pressure (PAWP) may modify this relationship. This relationship predicts that pulmonary artery systolic, diastolic and mean pressure maintain a consistent relationship relative to the PAWP. We show that, in healthy exercising human adults, both pulmonary vascular resistance and compliance decrease in relation to exercise‐associated increases in PAWP. Pulmonary artery systolic, diastolic and mean pressures maintain a consistent relationship with one another, increasing linearly with increasing PAWP. Increases in PAWP in the setting of exercise are directly related to a decrease in pulmonary vascular compliance, despite small decreases in pulmonary vascular resistance, thereby increasing the pulsatile afterload to the right ventricle. Abstract The resistive and pulsatile components of right ventricular afterload (pulmonary vascular resistance, Rp; compliance, Cp) are related by an inverse hyperbolic function, expressed as their product known as RpCp‐time. The RpCp‐time exhibits a narrow range, although it may be altered by the pulmonary artery wedge pressure (PAWP). Identifying the determinants of RpCp‐time should improve our understanding of the physiological behaviour of pulmonary arterial systolic (PASP), diastolic (PADP) and mean (mPAP) pressures in response to perturbations. We examined the effect of exercise in 28 healthy non‐athletic adults (55 ± 6 years) who underwent right heart catheterization to assess haemodynamics and calculate Rp and Cp. Measurements were made at rest and during two consecutive 8–10 min stages of cycle ergometry, at targeted heart‐rates of 100 beats min–1 (Light) and 120 beats min–1 (Moderate). Cardiac output increased progressively during exercise. PASP, PADP, mPAP and PAWP increased for Light exercise, without any further rise for Moderate exercise. RpCp‐time decreased for Light exercise (0.39 ± 0.08 to 0.25 ± 0.08, P < 0.001) without any further change for Moderate exercise, and the decrease in RpCp‐time was related to changes in PAWP (r2 = 0.26, P < 0.001). Changes in PASP (r2 = 0.43, P < 0.001), PADP (r2 = 0.47, P < 0.001) and mPAP (r2 = 0.50, P < 0.001) were linearly correlated with changes in PAWP, although they were not significantly related to changes in cardiac output. In healthy adults, exercise is associated with decreases in Cp and a resultant decline in RpCp‐time, indicating increased pulsatile right ventricular afterload. Changes in RpCp‐time, PASP, PADP and mPAP were systematically related to increases in PAWP.

  March 24, 2016   doi: 10.1113/JP271788   open full text
• On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.
  Ying‐Chun Yu, Yoshiro Sohma, Tzyh‐Chang Hwang.
  The Journal of Physiology. March 23, 2016
  Key points Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single‐channel conductance (g) and a ∼13‐fold lower open probability (Po), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H‐CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H‐CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX‐770 or nitrate). These reagents potentiate synergistically R117H‐CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H‐CFTR, but also provide a mechanistic understanding of how VX‐770 ameliorates the gating defects in the R117H mutant. Abstract Cystic fibrosis (CF) is caused by loss‐of‐function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation‐activated, but ATP‐gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H‐CFTR, an arginine‐to‐histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single‐channel conductance (g) in R117H‐CFTR, but found a ∼13‐fold lower open probability (Po). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild‐type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged ‘burst opening’ conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10‐fold. Moreover, our data demonstrate that a synergistic improvement of R117H‐CFTR function can be accomplished with a combined regiment of VX‐770 (Ivacaftor), nitrate ion (NO3−) and N6‐(2‐phenylethyl)‐2′‐deoxy‐ATP (d‐PATP), which almost completely rectifies the gating defect of R117H‐CFTR. Clinical implications of our results are discussed.

  March 23, 2016   doi: 10.1113/JP271723   open full text
• Protein kinase C‐dependent regulation of ClC‐1 channels in active human muscle and its effect on fast and slow gating.
  Anders Riisager, Frank Vincenzo Paoli, Wei‐Ping Yu, Thomas Holm Pedersen, Tsung‐Yu Chen, Ole Bækgaard Nielsen.
  The Journal of Physiology. March 20, 2016
  Key points Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)‐dependent ClC‐1 Cl− channel inhibition in rodent muscle. While this PKC‐dependent ClC‐1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC‐dependent ClC‐1 inhibition are unclear. Here we present the first demonstration of ClC‐1 inhibition in active human muscle fibres, and we determine the changes in ClC‐1 gating that underlie the PKC‐dependent ClC‐1 inhibition in active muscle using human ClC‐1 expressed in Xenopus oocytes. This activity‐induced ClC‐1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity. Abstract Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)‐dependent inhibition of ClC‐1 Cl− ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC‐dependent ClC‐1 inhibition are unclear. This study first determined whether PKC‐dependent ClC‐1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC‐1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30–60% reduction of ClC‐1 function, which could be completely prevented by PKC inhibition (1 μm GF109203X). The role of the PKC‐dependent ClC‐1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC‐dependent ClC‐1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC‐1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC‐1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC‐1. Indeed, there was no effect of PKC activation in C277S mutated ClC‐1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC‐dependent ClC‐1 inhibition via a gating mechanism.

  March 20, 2016   doi: 10.1113/JP271556   open full text
• Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon.
  Izumi Kaji, Yasutada Akiba, Kohtarou Konno, Masahiko Watanabe, Shunsuke Kimura, Toshihiko Iwanaga, Ayaka Kuri, Ken‐ichi Iwamoto, Atsukazu Kuwahara, Jonathan D. Kaunitz.
  The Journal of Physiology. March 20, 2016
  Key points Luminal short‐chain fatty acids (SCFAs) influence gut physiological function via SCFA receptors and transporters. The contribution of an SCFA receptor, free fatty acid receptor (FFA)3, to the enteric nervous system is unknown. FFA3 is expressed in enteric cholinergic neurons. Activation of neural FFA3 suppresses Cl− secretion induced by nicotinic ACh receptor activation via a Gi/o pathway. Neural FFA3 may have an anti‐secretory function by modulating cholinergic neural reflexes in the enteric nervous system. Abstract The proximal colonic mucosa is constantly exposed to high concentrations of microbially‐produced short‐chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non‐neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic‐mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3‐immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high‐affinity choline transporter CHT1. In Ussing chambered mucosa–submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)‐induced Cl− secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3‐chloropropionate. Serosal application of a selective FFA3 agonist (N‐[2‐methylphenyl]‐[4‐furan‐3‐yl]‐2‐methyl‐5‐oxo‐1,4,5,6,7,8‐hexahydro‐quinoline‐3‐carboxamide; MQC) dose‐dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13 μm. Pretreatment with MQC inhibited nicotine‐evoked but not bethanechol‐evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the Gi/o pathway. Luminal propionate induced Cl− secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA‐FFA3 pathway has a novel anti‐secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

  March 20, 2016   doi: 10.1113/JP271441   open full text
• Endothelin‐1 modulates methacholine‐induced cutaneous vasodilatation but not sweating in young human skin.
  Lyra Halili, Maya Sarah Singh, Naoto Fujii, Lacy M. Alexander, Glen P. Kenny.
  The Journal of Physiology. March 11, 2016
  Key points Endothelin‐1 (ET‐1) is a potent endothelial‐derived vasoconstrictor that may modulate cholinergic cutaneous vascular regulation. Endothelin receptors are also expressed on the human eccrine sweat gland, although it remains unclear whether ET‐1 modulates cholinergic sweating. We investigated whether ET‐1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)‐dependent mechanism. Our findings show that ET‐1 attenuates methacholine‐induced cutaneous vasodilatation through a NOS‐independent mechanism. We also demonstrate that ET‐1 attenuates cutaneous vasodilatation in response to sodium nitroprusside, suggesting that ET‐1 diminishes the dilatation capacity of vascular smooth muscle cells. We show that ET‐1 does not modulate methacholine‐induced sweating at any of the administered concentrations. Our findings advance our knowledge pertaining to the peripheral control underpinning the regulation of cutaneous blood flow and sweating and infer that ET‐1 may attenuate the heat loss responses of cutaneous blood flow, but not sweating. Abstract The present study investigated the effect of endothelin‐1 (ET‐1) on cholinergic mechanisms of end‐organs (i.e. skin blood vessels and sweat glands) for heat dissipation. We evaluated the hypothesis that ET‐1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)‐dependent mechanism. Cutaneous vascular conductance (CVC) and sweat rate were assessed in three protocols: in Protocol 1 (n = 8), microdialysis sites were perfused with lactated Ringer solution (Control), 40 pm, 4 nm or 400 nm ET‐1; in Protocol 2 (n = 11) sites were perfused with lactated Ringer solution (Control), 400 nm ET‐1, 10 mm NG‐nitro‐l‐arginine (l‐NNA; a NOS inhibitor) or a combination of 400 nm ET‐1 and 10 mm l‐NNA; in Protocol 3 (n = 8), only two sites (Control and 400 nm ET‐1) were utilized to assess the influence of ET‐1 on the dilatation capacity of vascular smooth muscle cells (sodium nitroprusside; SNP). Methacholine (MCh) was co‐administered in a dose‐dependent manner (0.0125, 0.25, 5, 100, 2000 mm, each for 25 min) at all skin sites. ET‐1 at 400 nm (P < 0.05) compared to lower doses (40 pm and 4 nm) (all P > 0.05) significantly attenuated increases in CVC in response to 0.25 and 5 mm MCh. A high dose of ET‐1 (400 nm) co‐infused with l‐NNA further attenuated CVC during 0.25, 5 and 100 mm MCh administration relative to the ET‐1 site (all P < 0.05). Cutaneous vasodilatation in response to SNP was significantly blunted after administration of 400 nm ET‐1 (P < 0.05). We show that ET‐1 attenuates cutaneous vasodilatation through a NOS‐independent mechanism, possibly through a vascular smooth muscle cell‐dependent mechanism, and methacholine‐induced sweating is not altered by ET‐1.

  March 11, 2016   doi: 10.1113/JP271735   open full text
• Spontaneous Ca2+ transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine.
  Salah A. Baker, Bernard T. Drumm, Dieter Saur, Grant W. Hennig, Sean M. Ward, Kenton M. Sanders.
  The Journal of Physiology. March 11, 2016
  Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC‐DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves. Ca2+ transients in ICC‐DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca2+ transients in ICC‐DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Abstract Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC‐DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC‐DMP are mediated by activation of Ca2+‐activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC‐DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC‐DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca2+ transients, suggesting that ICC‐DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high‐resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC‐DMP in situ.

  March 11, 2016   doi: 10.1113/JP271699   open full text
• Asymmetric dimethylarginine (ADMA) elevation and arginase up‐regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans.
  Mariam El Assar, Javier Angulo, Marta Santos‐Ruiz, Juan Carlos Ruiz de Adana, María Luz Pindado, Alberto Sánchez‐Ferrer, Alberto Hernández, Leocadio Rodríguez‐Mañas.
  The Journal of Physiology. March 04, 2016
  Key points The presence of insulin resistance (IR) is determinant for endothelial dysfunction associated with obesity. Although recent studies have implicated the involvement of mitochondrial superoxide and inflammation in the defective nitric oxide (NO)‐mediated responses and subsequent endothelial dysfunction in IR, other mechanisms could compromise this pathway. In the present study, we assessed the role of asymmetric dimethylarginine (ADMA) and arginase with respect to IR‐induced impairment of endothelium‐dependent vasodilatation in human morbid obesity and in a non‐obese rat model of IR. We show that both increased ADMA and up‐regulated arginase are determinant factors in the alteration of the l‐arginine/NO pathway associated with IR in both models and also that acute treatment of arteries with arginase inhibitor or with l‐arginine significantly alleviate endothelial dysfunction. These results help to expand our knowledge regarding the mechanisms of endothelial dysfunction that are related to obesity and IR and establish potential therapeutic targets for intervention. Abstract Insulin resistance (IR) is determinant for endothelial dysfunction in human obesity. Although we have previously reported the involvement of mitochondrial superoxide and inflammation, other mechanisms could compromise NO‐mediated responses in IR. We evaluated the role of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and arginase with respect to IR‐induced impairment of l‐arginine/NO‐mediated vasodilatation in human morbid obesity and in a non‐obese rat model of IR. Bradykinin‐induced vasodilatation was evaluated in microarteries derived from insulin‐resistant morbidly obese (IR‐MO) and non‐insulin‐resistant MO (NIR‐MO) subjects. Defective endothelial vasodilatation in IR‐MO was improved by l‐arginine supplementation. Increased levels of ADMA were detected in serum and adipose tissue from IR‐MO. Serum ADMA positively correlated with IR score and negatively with pD2 for bradykinin. Gene expression determination by RT‐PCR revealed not only the decreased expression of ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH)1/2 in IR‐MO microarteries, but also increased expression of arginase‐2. Arginase inhibition improved endothelial vasodilatation in IR‐MO. Analysis of endothelial vasodilatation in a non‐obese IR model (fructose‐fed rat) confirmed an elevation of circulating and aortic ADMA concentrations, as well as reduced DDAH aortic content and increased aortic arginase activity in IR. Improvement of endothelial vasodilatation in IR rats by l‐arginine supplementation and arginase inhibition provided functional corroboration. These results demonstrate that increased ADMA and up‐regulated arginase contribute to endothelial dysfunction as determined by the presence of IR in human obesity, most probably by compromising arginine availability. The results provide novel insights regarding the mechanisms of endothelial dysfunction related to obesity and IR and establish potential therapeutic targets for intervention.

  March 04, 2016   doi: 10.1113/JP271836   open full text
• Perpetuation of torsade de pointes in heterogeneous hearts: competing foci or re‐entry?
  Nele Vandersickel, Teun P. Boer, Marc A. Vos, Alexander V. Panfilov.
  The Journal of Physiology. March 04, 2016
  Key points The underlying mechanism of torsade de pointes (TdP) remains of debate: perpetuation may be due to (1) focal activity or (2) re‐entrant activity. The onset of TdP correlates with action potential heterogeneities in different regions of the heart. We studied the mechanism of perpetuation of TdP in silico using a 2D model of human cardiac tissue and an anatomically accurate model of the ventricles of the human heart. We found that the mechanism of perpetuation TdP depends on the degree of heterogeneity. If the degree of heterogeneity is large, focal activity alone can sustain a TdP, otherwise re‐entrant activity emerges. This result can help to understand the relationship between the mechanisms of TdP and tissue properties and may help in developing new drugs against it. Abstract Torsade de pointes (TdP) can be the consequence of cardiac remodelling, drug effects or a combination of both. The mechanism underlying TdP is unclear, and may involve triggered focal activity or re‐entry. Recent work by our group has indicated that both cases may exist, i.e. TdPs induced in the chronic atrioventricular block (CAVB) dog model may have a focal origin or are due to re‐entry. Also it was found that heterogeneities might play an important role. In the current study we have used computational modelling to further investigate the mechanisms involved in TdP initiation and perpetuation, especially in the CAVB dog model, by the addition of heterogeneities with reduced repolarization reserve in comparison with the surrounding tissue. For this, the TNNP computer model was used for computations. We demonstrated in 2D and 3D simulations that ECGs with the typical TdP morphology can be caused by both multiple competing foci and re‐entry circuits as a result of introduction of heterogeneities, depending on whether the heterogeneities have a large or a smaller reduced repolarization reserve in comparison with the surrounding tissue. Large heterogeneities can produce ectopic TdP, while smaller heterogeneities will produce re‐entry‐type TdP.

  March 04, 2016   doi: 10.1113/JP271728   open full text
• Is plasticity within the retrotrapezoid nucleus responsible for the recovery of the PCO2 set‐point after carotid body denervation in rats?
  Tyler M. Basting, Chikara Abe, Kenneth E. Viar, Ruth L. Stornetta, Patrice G. Guyenet.
  The Journal of Physiology. March 04, 2016
  Key points Arterial PCO2 is kept constant via breathing adjustments elicited, at least partly, by central chemoreceptors (CCRs) and the carotid bodies (CBs). The CBs may be active in a normal oxygen environment because their removal reduces breathing. Thereafter, breathing slowly returns to normal. In the present study, we investigated whether an increase in the activity of CCRs accounts for this return. One week after CB excision, the hypoxic ventilatory reflex was greatly reduced as expected, whereas ventilation and blood gases at rest under normoxia were normal. Optogenetic inhibition of Phox2b‐expressing neurons including the retrotrapezoid nucleus, a cluster of CCRs, reduced breathing proportionally to arterial pH. The hypopnoea was greater after CB excision but only in a normal or hypoxic environment. The difference could be simply explained by the loss of fast feedback from the CBs. We conclude that, in rats, CB denervation may not produce CCR plasticity. We also question whether the transient hypoventilation elicited by CB denervation means that these afferents are active under normoxia. Abstract Carotid body denervation (CBD) causes hypoventilation and increases the arterial PCO2 set‐point; these effects eventually subside. The hypoventilation is attributed to reduced CB afferent activity and the PCO2 set‐point recovery to CNS plasticity. In the present study, we investigated whether the retrotrapezoid nucleus (RTN), a group of non‐catecholaminergic Phox2b‐expressing central respiratory chemoreceptors (CCRs), is the site of such plasticity. We evaluated the contribution of the RTN to breathing frequency (FR), tidal volume (VT) and minute volume (VE) by inhibiting this nucleus optogenetically for 10 s (archaerhodopsinT3.0) in unanaesthetized rats breathing various levels of O2 and/or CO2. The measurements were made in seven rats before and 6–7 days after CBD and were repeated in seven sham‐operated rats. Seven days post‐CBD, blood gases and ventilation in 21% O2 were normal, whereas the hypoxic ventilatory reflex was still depressed (95.3%) and hypoxia no longer evoked sighs. Sham surgery had no effect. In normoxia or hypoxia, RTN inhibition produced a more sustained hypopnoea post‐CBD than before; in hyperoxia, the responses were identical. Post‐CBD, RTN inhibition reduced FR and VE in proportion to arterial pH or PCO2 (ΔVE: 3.3 ± 1.5% resting VE/0.01 pHa). In these rats, 20.7 ± 8.9% of RTN neurons expressed archaerhodopsinT3.0. Hypercapnia (3–6% FiCO2) increased FR and VT in CBD rats (n = 4). In conclusion, RTN regulates FR and VE in a pH‐dependent manner after CBD, consistent with its postulated CCR function. RTN inhibition produces a more sustained hypopnoea after CBD than before, although this change may simply result from the loss of the fast feedback action of the CBs.

  March 04, 2016   doi: 10.1113/JP272046   open full text
• Inward rectifier potassium (Kir2.1) channels as end‐stage boosters of endothelium‐dependent vasodilators.
  Swapnil K. Sonkusare, Thomas Dalsgaard, Adrian D. Bonev, Mark T. Nelson.
  The Journal of Physiology. March 04, 2016
  Key points Increase in endothelial cell (EC) calcium activates calcium‐sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium‐dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium‐dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium‐dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. Abstract Endothelium‐dependent vasodilators, such as acetylcholine, increase intracellular Ca2+ through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca2+‐sensitive intermediate and small conductance K+ (IK and SK, respectively) channels. Although strong inward rectifier K+ (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC‐dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC‐dependent vasodilatation of resistance‐sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC‐specific Kir2.1 knockdown (EC‐Kir2.1−/−) mice. Elevation of extracellular K+ to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba2+, ML‐133) or in the arteries from EC‐Kir2.1−/− mice. Potassium‐induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba2+, did not affect currents through TRPV4, IK or SK channels. Endothelial cell‐dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC‐Kir2.1−/−. In angiotensin II‐induced hypertension, the Kir channel function was not altered, although the endothelium‐dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca2+‐dependent activation of IK and SK channels.

  March 04, 2016   doi: 10.1113/JP271652   open full text
• Physiological signalling to myosin phosphatase targeting subunit‐1 phosphorylation in ileal smooth muscle.
  Ning Gao, Audrey N. Chang, Weiqi He, Cai‐Ping Chen, Yan‐Ning Qiao, Minsheng Zhu, Kristine E. Kamm, James T. Stull.
  The Journal of Physiology. March 04, 2016
  Key points The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca2+/calmodulin‐dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency‐maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation. Abstract Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle‐specific deletion of MYPT1; (2) non‐phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1‐deficient tissues from MYPT1SM−/− mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild‐type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1‐deficient tissues may be similar to attenuated MLCP activity in wild‐type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild‐type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.

  March 04, 2016   doi: 10.1113/JP271703   open full text
• Mechanisms of in vivo muscle fatigue in humans: investigating age‐related fatigue resistance with a computational model.
  Damien M. Callahan, Brian R. Umberger, Jane A. Kent.
  The Journal of Physiology. March 02, 2016
  Key points Muscle fatigue can be defined as the transient decrease in maximal force that occurs in response to muscle use. Fatigue develops because of a complex set of changes within the neuromuscular system that are difficult to evaluate simultaneously in humans. The skeletal muscle of older adults fatigues less than that of young adults during static contractions. The potential sources of this difference are multiple and intertwined. To evaluate the individual mechanisms of fatigue, we developed an integrative computational model based on neural, biochemical, morphological and physiological properties of human skeletal muscle. Our results indicate first that the model provides accurate predictions of fatigue and second that the age‐related resistance to fatigue is due largely to a lower reliance on glycolytic metabolism during contraction. This model should prove useful for generating hypotheses for future experimental studies into the mechanisms of muscle fatigue. Abstract During repeated or sustained muscle activation, force‐generating capacity becomes limited in a process referred to as fatigue. Multiple factors, including motor unit activation patterns, muscle fibre contractile properties and bioenergetic function, can impact force‐generating capacity and thus the potential to resist fatigue. Given that neuromuscular fatigue depends on interrelated factors, quantifying their independent effects on force‐generating capacity is not possible in vivo. Computational models can provide insight into complex systems in which multiple inputs determine discrete outputs. However, few computational models to date have investigated neuromuscular fatigue by incorporating the multiple levels of neuromuscular function known to impact human in vivo function. To address this limitation, we present a computational model that predicts neural activation, biomechanical forces, intracellular metabolic perturbations and, ultimately, fatigue during repeated isometric contractions. This model was compared with metabolic and contractile responses to repeated activation using values reported in the literature. Once validated in this way, the model was modified to reflect age‐related changes in neuromuscular function. Comparisons between initial and age‐modified simulations indicated that the age‐modified model predicted less fatigue during repeated isometric contractions, consistent with reports in the literature. Together, our simulations suggest that reduced glycolytic flux is the greatest contributor to the phenomenon of age‐related fatigue resistance. In contrast, oxidative resynthesis of phosphocreatine between intermittent contractions and inherent buffering capacity had minimal impact on predicted fatigue during isometric contractions. The insights gained from these simulations cannot be achieved through traditional in vivo or in vitro experimentation alone.

  March 02, 2016   doi: 10.1113/JP271400   open full text
• Teaching neurons to respond to placebos.
  Fabrizio Benedetti, Elisa Frisaldi, Elisa Carlino, Lucia Giudetti, Alan Pampallona, Maurizio Zibetti, Michele Lanotte, Leonardo Lopiano.
  The Journal of Physiology. February 24, 2016
  Key points We analysed the placebo response at the single‐neuron level in the thalamus of Parkinson patients to see the differences between first‐time administration of placebo and administration after pharmacological pre‐conditioning. When the placebo was given for the first time, it induced neither clinical improvement, as assessed through muscle rigidity reduction at the wrist, nor neuronal changes in thalamic neurons. However, if placebo was given after two, three or four prior administrations of an anti‐Parkinson drug, apomorphine, it produced both clinical and neuronal responses. Both the magnitude and the duration of these placebo responses depended on the number of prior exposures to apomorphine, according to the rule: the greater the number of previous apomorphine administrations, the larger the magnitude and the longer the duration of the clinical and neuronal placebo responses. These findings show that learning plays a crucial role in the placebo response and suggest that placebo non‐responders can be turned into placebo responders, with important clinical implications. Abstract Placebos have been found to affect the patient's brain in several conditions, such as pain and motor disorders. For example, in Parkinson's disease, a placebo treatment induces a release of dopamine in the striatum and changes the activity of neurons in both thalamic and subthalamic nuclei. The present study shows that placebo administration for the first time induces neither clinical nor neuronal improvement in Parkinson patients who undergo implantation of electrodes for deep brain stimulation. However, this lack of placebo responsiveness can be turned into substantial placebo responses following previous exposure to repeated administrations of the anti‐Parkinson agent apomorphine. As the number of apomorphine administrations increased from one to four, both the clinical response and the neuronal activity in the ventral anterior and anterior ventrolateral thalamus increased. In fact, after four apomorphine exposures, placebo administration induced clinical responses that were as large as those to apomorphine, along with long‐lasting neuronal changes. These clinical placebo responses following four apomorphine administrations were again elicited after a re‐exposure to a placebo 24 h after surgery, but not after 48 h. These data indicate that learning plays a crucial role in placebo responsiveness and suggest that placebo non‐responders can be turned into responders, with important implications in the clinical setting.

  February 24, 2016   doi: 10.1113/JP271322   open full text
• Corticospinal excitability is associated with hypocapnia but not changes in cerebral blood flow.
  Geoffrey L. Hartley, Cody L. Watson, Philip N. Ainslie, Craig D. Tokuno, Matthew J. Greenway, David A. Gabriel, Deborah D. O'Leary, Stephen S. Cheung.
  The Journal of Physiology. February 24, 2016
  Key points Reductions in cerebral blood flow (CBF) may be implicated in the development of neuromuscular fatigue; however, the contribution from hypocapnic‐induced reductions (i.e. P ETC O2) in CBF versus reductions in CBF per se has yet to be isolated. We assessed neuromuscular function while using indomethacin to selectively reduce CBF without changes in P ETC O2 and controlled hyperventilation‐induced hypocapnia to reduce both CBF and P ETC O2. Increased corticospinal excitability appears to be exclusive to reductions in P ETC O2 but not reductions in CBF, whereas sub‐optimal voluntary output from the motor cortex is moderately associated with decreased CBF independent of changes in P ETC O2. These findings suggest that changes in CBF and P ETC O2 have distinct roles in modulating neuromuscular function. Abstract Although reductions in cerebral blood flow (CBF) may be involved in central fatigue, the contribution from hypocapnia‐induced reductions in CBF versus reductions in CBF per se has not been isolated. This study examined whether reduced arterial PCO2 (P aC O2), independent of concomitant reductions in CBF, impairs neuromuscular function. Neuromuscular function, as indicated by motor‐evoked potentials (MEPs), maximal M‐wave (Mmax) and cortical voluntary activation (cVA) of the flexor carpi radialis muscle during isometric wrist flexion, was assessed in ten males (29 ± 10 years) during three separate conditions: (1) cyclooxygenase inhibition using indomethacin (Indomethacin, 1.2 mg kg−1) to selectively reduce CBF by 28.8 ± 10.3% (estimated using transcranial Doppler ultrasound) without changes in end‐tidal PCO2 (P ETC O2); (2) controlled iso‐oxic hyperventilation‐induced reductions in P aC O2 (Hypocapnia), P ETC O2 = 30.1 ± 4.5 mmHg with related reductions in CBF (21.7 ± 6.3%); and (3) isocapnic hyperventilation (Isocapnia) to examine the potential direct influence of hyperventilation‐mediated activation of respiratory control centres on CBF and changes in neuromuscular function. Change in MEP amplitude (%Mmax) from baseline was greater in Hypocapnia tha in Isocapnia (11.7 ± 9.8%, 95% confidence interval (CI) [2.6, 20.7], P = 0.01) and Indomethacin (13.3 ± 11.3%, 95% CI [2.8, 23.7], P = 0.01) with a large Cohen's effect size (d ≥ 1.17). Although not statistically significant, cVA was reduced with a moderate effect size in Indomethacin (d = 0.7) and Hypocapnia (d = 0.9) compared to Isocapnia. In summary, increased corticospinal excitability – as reflected by larger MEP amplitude – appears to be exclusive to reduced P aC O2, but not reductions in CBF per se. Sub‐optimal voluntary output from the motor cortex is moderately associated with decreased CBF, independent of reduced P aC O2.

  February 24, 2016   doi: 10.1113/JP271914   open full text
• Microglia modulate brainstem serotonergic expression following neonatal sustained hypoxia exposure: implications for sudden infant death syndrome.
  P. M. MacFarlane, C. A. Mayer, D. G. Litvin.
  The Journal of Physiology. February 21, 2016
  Key points Neonatal sustained hypoxia exposure modifies brainstem microglia and serotonin expression. The altered brainstem neurochemistry is associated with impaired ventilatory responses to acute hypoxia and mortality. The deleterious effects of sustained hypoxia exposure can be prevented by an inhibitor of activated microglia. These observations demonstrate a potential cause of the brainstem serotonin abnormalities thought to be involved in sudden infant death syndrome. Abstract We showed previously that the end of the second postnatal week (days P11–15) represents a period of development during which the respiratory neural control system exhibits a heightened vulnerability to sustained hypoxia (SH, 11% O2, 5 days) exposure. In the current study, we investigated whether the vulnerability to SH during the same developmental time period is associated with changes in brainstem serotonin (5‐HT) expression and whether it can be prevented by the microglia inhibitor minocycline. Using whole‐body plethysmography, SH attenuated the acute (5 min) hypoxic ventilatory response (HVR) and caused a high incidence of mortality compared to normoxia rats. SH also increased microglia cell numbers and decreased 5‐HT immunoreactivity in the nucleus of the solitary tract (nTS) and dorsal motor nucleus of the vagus (DMNV). The attenuated HVR, mortality, and changes in nTS and DMNV immunoreactivity was prevented by minocycline (25 mg kg−1/2 days during SH). These data demonstrate that the 5‐HT abnormalities in distinct respiratory neural control regions can be initiated by prolonged hypoxia exposure and may be modulated by microglia activity. These observations share several commonalities with the risk factors thought to underlie the aetiology of sudden infant death syndrome, including: (1) a vulnerable neonate; (2) a critical period of development; (3) evidence of hypoxia; (4) brainstem gliosis (particularly the nTS and DMNV); and (5) 5‐HT abnormalities.

  February 21, 2016   doi: 10.1113/JP271845   open full text
• Influence of exercise intensity and duration on functional and biochemical perturbations in the human heart.
  Glenn M. Stewart, Akira Yamada, Luke J. Haseler, Justin J. Kavanagh, Jonathan Chan, Gus Koerbin, Cameron Wood, Surendran Sabapathy.
  The Journal of Physiology. February 18, 2016
  Key points Strenuous endurance exercise induces transient functional and biochemical cardiac perturbations that persist for 24–48 h. The magnitude and time‐course of exercise‐induced reductions in ventricular function and increases in cardiac injury markers are influenced by the intensity and duration of exercise. In a human experimental model, exercise‐induced reductions in ventricular strain and increases in cardiac troponin are greater, and persist for longer, when exercise is performed within the heavy‐ compared to moderate‐intensity exercise domain, despite matching for total mechanical work. The results of the present study help us better understand the dose–response relationship between endurance exercise and acute cardiac stress/injury, a finding that has implications for the prescription of day‐to‐day endurance exercise regimes. Abstract Strenuous endurance exercise induces transient cardiac perturbations with ambiguous health outcomes. The present study investigated the magnitude and time‐course of exercise‐induced functional and biochemical cardiac perturbations by manipulating the exercise intensity–duration matrix. Echocardiograph‐derived left (LV) and right (RV) ventricular global longitudinal strain (GLS), and serum high‐sensitivity cardiac troponin (hs‐cTnI) concentration, were examined in 10 males (age: 27 ± 4 years; V̇O2, peak : 4.0 ± 0.8 l min−1) before, throughout (50%, 75% and 100%), and during recovery (1, 3, 6 and 24 h) from two exercise trials. The two exercise trials consisted of 90 and 120 min of heavy‐ and moderate‐intensity cycling, respectively, with total mechanical work matched. LVGLS decreased (P < 0.01) during the 90 min trial only, with reductions peaking at 1 h post (pre: −19.9 ± 0.6%; 1 h post: −18.5 ± 0.7%) and persisting for >24 h into recovery. RVGLS decreased (P < 0.05) during both exercise trials with reductions in the 90 min trial peaking at 1 h post (pre: −27.5 ± 0.7%; 1 h post: −25.1 ± 0.8%) and persisting for >24 h into recovery. Serum hs‐cTnI increased (P < 0.01) during both exercise trials, with concentrations peaking at 3 h post but only exceeding cardio‐healthy reference limits (14 ng l−1) in the 90 min trial (pre: 4.2 ± 2.4 ng l−1; 3 h post: 25.1 ± 7.9 ng l−1). Exercise‐induced reductions in ventricular strain and increases in cardiac injury markers persist for 24 h following exercise that is typical of day‐to‐day endurance exercise training; however, the magnitude and time‐course of this response can be altered by manipulating the intensity–duration matrix.

  February 18, 2016   doi: 10.1113/JP271889   open full text
• Age‐dependent impact of CaV3.2 T‐type calcium channel deletion on myogenic tone and flow‐mediated vasodilatation in small arteries.
  Miriam F. Mikkelsen, Karl Björling, Lars Jørn Jensen.
  The Journal of Physiology. February 18, 2016
  Key points Blood pressure and flow exert mechanical forces on the walls of small arteries, which are detected by the endothelial and smooth muscle cells, and lead to regulation of the diameter (basal tone) of an artery. CaV3.2 T‐type calcium channels are expressed in the wall of small arteries, although their function remains poorly understood because of the low specificity of T‐type blockers. We used mice deficient in CaV3.2 channels to study their role in pressure‐ and flow‐dependent tone regulation and the possible impact of ageing on this role. In young mice, CaV3.2 channels oppose pressure‐induced vasoconstriction and participate in endothelium‐dependent, flow‐mediated dilatation. These effects were not seen in mature adult mice. The results of the present study demonstrate an age‐dependent impact of CaV3.2 T‐type calcium channel deletion in rodents and suggest that the loss of CaV3.2 channel function leads to more constricted arteries, which is a risk factor for cardiovascular disease. Abstract The myogenic response and flow‐mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. CaV3.2 T‐type calcium channels are expressed in both VSMCs and ECs of small arteries. The T‐type channels are important drug targets but, as a result of the lack of specific antagonists, our understanding of the role of CaV3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow‐mediated vasodilatation, structural remodelling and mRNA + protein expression in small mesenteric arteries from CaV3.2 knockout (CaV3.2KO) vs. wild‐type mice at a young vs. mature adult age. In young mice only, deletion of CaV3.2 led to an enhanced myogenic response and a ∼50% reduction of flow‐mediated vasodilatation. Ni2+ had both CaV3.2‐dependent and independent effects. No changes in mRNA expression of several important K+ and Ca2+ channel genes were induced by CaV3.2KO However, the expression of the other T‐type channel isoform (CaV3.1) was reduced at the mRNA and protein level in mature adult compared to young wild‐type arteries. The results of the present study demonstrate the important roles of the CaV3.2 T‐type calcium channels in myogenic tone and flow‐mediated vasodilatation that disappear with ageing. Because increased arterial tone is a risk factor for cardiovascular disease, we conclude that CaV3.2 channels, by modulating pressure‐ and flow‐mediated vasomotor responses to prevent excess arterial tone, protect against cardiovascular disease.

  February 18, 2016   doi: 10.1113/JP271470   open full text
• Individual variability of cerebral autoregulation, posterior cerebral circulation and white matter hyperintensity.
  Jie Liu, Benjamin Y. Tseng, Muhammad Ayaz Khan, Takashi Tarumi, Candace Hill, Niki Mirshams, Timea M. Hodics, Linda S. Hynan, Rong Zhang.
  The Journal of Physiology. February 09, 2016
  Key points Cerebral autoregulation (CA) is a key mechanism to protect brain perfusion in the face of changes in arterial blood pressure, but little is known about individual variability of CA and its relationship to the presence of brain white matter hyperintensity (WMH) in older adults, a type of white matter lesion related to cerebral small vessel disease (SVD). This study demonstrated the presence of large individual variability of CA in healthy older adults during vasoactive drug‐induced changes in arterial pressure assessed at the internal carotid and vertebral arteries. We also observed, unexpectedly, that it was the ‘over‐’ rather than the ‘less‐reactive’ CA measured at the vertebral artery that was associated with WMH severity. These findings challenge the traditional concept of CA and suggest that the presence of cerebral SVD, manifested as WMH, is associated with posterior brain hypoperfusion during acute increase in arterial pressure. Abstract This study measured the individual variability of static cerebral autoregulation (CA) and determined its associations with brain white matter hyperintensity (WMH) in older adults. Twenty‐seven healthy older adults (13 females, 66 ± 6 years) underwent assessment of CA during steady‐state changes in mean arterial pressure (MAP) induced by intravenous infusion of sodium nitroprusside (SNP) and phenylephrine. Cerebral blood flow (CBF) was measured using colour‐coded duplex ultrasonography at the internal carotid (ICA) and vertebral arteries (VA). CA was quantified by a linear regression slope (CA slope) between percentage changes in cerebrovascular resistance (CVR = MAP/CBF) and MAP relative to baseline values. Periventricular and deep WMH volumes were measured with T2‐weighted magnetic resonance imaging. MAP was reduced by −11 ± 7% during SNP, and increased by 21 ± 8% during phenylephrine infusion. CA demonstrated large individual variability with the CA slopes ranging from 0.37 to 2.20 at the ICA and from 0.17 to 3.18 at the VA; no differences in CA were found between the ICA and VA. CA slopes measured at the VA had positive correlations with the total and periventricular WMH volume (r = 0.55 and 0.59, P < 0.01). Collectively, these findings demonstrated the presence of large individual variability of CA in older adults, and that, when measured in the posterior cerebral circulation, it is the higher rather than lower CA reactivity that is associated with WMH severity.

  February 09, 2016   doi: 10.1113/JP271068   open full text
• Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5‐trisphosphate receptors and Ca2+ release.
  Min‐Jeong Son, Joon‐Chul Kim, Sung Woo Kim, Bojjibabu Chidipi, Jeyaraj Muniyandi, Thoudam Debraj Singh, Insuk So, Krishna P. Subedi, Sun‐Hee Woo.
  The Journal of Physiology. February 09, 2016
  Key points During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress‐sensitive membrane current in atrial myocytes using the whole‐cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release in subsarcolemmal domains of atrial myocytes. Ca2+‐mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. Abstract Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress‐sensitive current (Ishear) in rat atrial myocytes. A shear stress of ∼16 dyn cm−2 was applied to single myocytes using a pressurized microflow system, and the current was measured by whole‐cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current–voltage relationship (reversal at −2 mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca2+. It was suppressed by intracellular Ca2+ buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca2+ release through the ryanodine receptors or inositol 1,4,5‐trisphosphate receptors (IP3R) and upon depletion of sarcoplasmic reticulum Ca2+. In type 2 IP3R (IP3R2) knockout atrial myocytes, Ishear was 10–20% of that in wild‐type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3R2 were expressed at peripheral sites with co‐localization, although they are not localized within 40 nm. Peripheral localization of TRPM4 was intact in IP3R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca2+ release from the IP3R2 in the peripheral domains of atrial myocytes.

  February 09, 2016   doi: 10.1113/JP270887   open full text
• Modelling in vivo creatine/phosphocreatine in vitro reveals divergent adaptations in human muscle mitochondrial respiratory control by ADP after acute and chronic exercise.
  Mia Ydfors, Meghan C. Hughes, Robert Laham, Uwe Schlattner, Jessica Norrbom, Christopher G. R. Perry.
  The Journal of Physiology. February 04, 2016
  Key points Mitochondrial respiratory sensitivity to ADP is thought to influence muscle fitness and is partly regulated by cytosolic–mitochondrial diffusion of ADP or phosphate shuttling via creatine/phosphocreatine (Cr/PCr) through mitochondrial creatine kinase (mtCK). Previous measurements of respiration in vitro with Cr (saturate mtCK) or without (ADP/ATP diffusion) show mixed responses of ADP sensitivity following acute exercise vs. less sensitivity after chronic exercise. In human muscle, modelling in vivo ‘exercising’ [Cr:PCr] during in vitro assessments revealed novel responses to exercise that differ from detections with or without Cr (±Cr). Acute exercise increased ADP sensitivity when measured without Cr but had no effect ±Cr or with +Cr:PCr, whereas chronic exercise increased sensitivity ±Cr but lowered sensitivity with +Cr:PCr despite increased markers of mitochondrial oxidative capacity. Controlling in vivo conditions during in vitro respiratory assessments reveals responses to exercise that differ from typical ±Cr comparisons and challenges our understanding of how exercise improves metabolic control in human muscle. Abstract Mitochondrial respiratory control by ADP (Kmapp) is viewed as a critical regulator of muscle energy homeostasis. However, acute exercise increases, decreases or has no effect on Kmapp in human muscle, whereas chronic exercise surprisingly decreases sensitivity despite greater mitochondrial content. We hypothesized that modelling in vivo mitochondrial creatine kinase (mtCK)‐dependent phosphate‐shuttling conditions in vitro would reveal increased sensitivity (lower Kmapp) after acute and chronic exercise. The Kmapp was determined in vitro with 20 mm Cr (+Cr), 0 mm Cr (−Cr) or ‘in vivo exercising’ 20 mm Cr/2.4 mm PCr (Cr:PCr) on vastus lateralis biopsies sampled from 11 men before, immediately after and 3 h after exercise on the first, fifth and ninth sessions over 3 weeks. Dynamic responses to acute exercise occurred throughout training, whereby the first session did not change Kmapp with in vivo Cr:PCr despite increases in −Cr. The fifth session decreased sensitivity with Cr:PCr or +Cr despite no change in −Cr. Chronic exercise increased sensitivity ±Cr in association with increased electron transport chain content (+33–62% complexes I–V), supporting classic proposals that link increased sensitivity to oxidative capacity. However, in vivo Cr:PCr reveals a perplexing decreased sensitivity, contrasting the increases seen ±Cr. Functional responses occurred without changes in fibre type or proteins regulating mitochondrial–cytosolic energy exchange (mtCK, VDAC and ANT). Despite the dynamic responses seen with ±Cr, modelling in vivo phosphate‐shuttling conditions in vitro reveals that ADP sensitivity is unchanged after high‐intensity exercise and is decreased after training. These findings challenge our understanding of how exercise regulates skeletal muscle energy homeostasis.

  February 04, 2016   doi: 10.1113/JP271259   open full text
• The involvement of transient receptor potential canonical type 1 in skeletal muscle regrowth after unloading‐induced atrophy.
  Lu Xia, Kwok‐Kuen Cheung, Simon S. Yeung, Ella W. Yeung.
  The Journal of Physiology. February 04, 2016
  Key points Decreased mechanical loading results in skeletal muscle atrophy. The transient receptor potential canonical type 1 (TRPC1) protein is implicated in this process. Investigation of the regulation of TRPC1 in vivo has rarely been reported. In the present study, we employ the mouse hindlimb unloading and reloading model to examine the involvement of TRPC1 in the regulation of muscle atrophy and regrowth, respectively. We establish the physiological relevance of the concept that manipulation of TRPC1 could interfere with muscle regrowth processes following an atrophy‐inducing event. Specifically, we show that suppressing TRPC1 expression during reloading impairs the recovery of the muscle mass and slow myosin heavy chain profile. Calcineurin appears to be part of the signalling pathway involved in the regulation of TRPC1 expression during muscle regrowth. These results provide new insights concerning the function of TRPC1. Interventions targeting TRPC1 or its downstream or upstream pathways could be useful for promoting muscle regeneration. Abstract Decreased mechanical loading, such as bed rest, results in skeletal muscle atrophy. The functional consequences of decreased mechanical loading include a loss of muscle mass and decreased muscle strength, particularly in anti‐gravity muscles. The purpose of this investigation was to clarify the regulatory role of the transient receptor potential canonical type 1 (TRPC1) protein during muscle atrophy and regrowth. Mice were subjected to 14 days of hindlimb unloading followed by 3, 7, 14 and 28 days of reloading. Weight‐bearing mice were used as controls. TRPC1 expression in the soleus muscle decreased significantly and persisted at 7 days of reloading. Small interfering RNA (siRNA)‐mediated downregulation of TRPC1 in weight‐bearing soleus muscles resulted in a reduced muscle mass and a reduced myofibre cross‐sectional area (CSA). Microinjecting siRNA into soleus muscles in vivo after 7 days of reloading provided further evidence for the role of TRPC1 in regulating muscle regrowth. Myofibre CSA, as well as the percentage of slow myosin heavy chain‐positive myofibres, was significantly lower in TRPC1‐siRNA‐expressing muscles than in control muscles after 14 days of reloading. Additionally, inhibition of calcineurin (CaN) activity downregulated TRPC1 expression in both weight‐bearing and reloaded muscles, suggesting a possible association between CaN and TRPC1 during skeletal muscle regrowth.

  February 04, 2016   doi: 10.1113/JP271705   open full text
• Endolysosomal two‐pore channels regulate autophagy in cardiomyocytes.
  Vanessa García‐Rúa, Sandra Feijóo‐Bandín, Diego Rodríguez‐Penas, Ana Mosquera‐Leal, Emad Abu‐Assi, Andrés Beiras, Luisa María Seoane, Pamela Lear, John Parrington, Manuel Portolés, Esther Roselló‐Lletí, Miguel Rivera, Oreste Gualillo, Valentina Parra, Joseph A. Hill, Beverly Rothermel, José Ramón González‐Juanatey, Francisca Lago.
  The Journal of Physiology. February 04, 2016
  Key points Two‐pore channels (TPCs) were identified as a novel family of endolysosome‐targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na+ channels able to control endolysosomal fusion, a key process in autophagic flux. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy. We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts. Abstract Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two‐pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3‐II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin A1) occurred either when TPC1 or TPC2 were downregulated. The knockdown of TPCs diminished cardiomyocyte viability under starvation and simulated ischaemia. Electron micrographs of hearts from TPC1/2 double knockout mice showed that cardiomyocytes contained large numbers of immature lysosomes with diameters significantly smaller than those of wild‐type mice. In cardiac tissues from humans and rats, TPC1 and TPC2 transcripts and protein levels were higher in females than in males. Furthermore, transcript levels of TPCs correlated negatively with p62 levels in heart tissues. TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes (i.e. there is a negative effect on cell viability under stress conditions in their absence) and they are differentially expressed in male and female human and murine hearts, where they correlate with markers of autophagy.

  February 04, 2016   doi: 10.1113/JP271332   open full text
• Protection against ventricular fibrillation via cholinergic receptor stimulation and the generation of nitric oxide.
  Manish Kalla, Minesh Chotalia, Charles Coughlan, Guoliang Hao, Mark J. Crabtree, Jakub Tomek, Gil Bub, David J. Paterson, Neil Herring.
  The Journal of Physiology. February 04, 2016
  Key points Animal studies suggest an anti‐fibrillatory action of the vagus nerve on the ventricle, although the exact mechanism is controversial. Using a Langendorff perfused rat heart, we show that the acetylcholine analogue carbamylcholine raises ventricular fibrillation threshold (VFT) and flattens the electrical restitution curve. The anti‐fibrillatory action of carbamylcholine was prevented by the nicotinic receptor antagonist mecamylamine, inhibitors of neuronal nitric oxide synthase (nNOS) and soluble guanylyl cyclase (sGC), and can be mimicked by the nitric oxide (NO) donor sodium nitroprusside. Carbamylcholine increased NO metabolite content in the coronary effluent and this was prevented by mecamylamine. The anti‐fibrillatory action of both carbamylcholine and sodium nitroprusside was ultimately dependent on muscarinic receptor stimulation as all effects were blocked by atropine. These data demonstrate a protective effect of carbamylcholine on VFT that depends upon both muscarinic and nicotinic receptor stimulation, where the generation of NO is likely to be via a neuronal nNOS–sGC dependent pathway. Abstract Implantable cardiac vagal nerve stimulators are a promising treatment for ventricular arrhythmia in patients with heart failure. Animal studies suggest the anti‐fibrillatory effect may be nitric oxide (NO) dependent, although the exact site of action is controversial. We investigated whether a stable analogue of acetylcholine could raise ventricular fibrillation threshold (VFT), and whether this was dependent on NO generation and/or muscarinic/nicotinic receptor stimulation. VFT was determined in Langendorff perfused rat hearts by burst pacing until sustained VF was induced. Carbamylcholine (CCh, 200 nmol l–1, n = 9) significantly (P < 0.05) reduced heart rate from 292 ± 8 to 224 ± 6 b.p.m. Independent of this heart rate change, CCh caused a significant increase in VFT (control 1.5 ± 0.3 mA, CCh 2.4 ± 0.4 mA, wash 1.1 ± 0.2 mA) and flattened the restitution curve (n = 6) derived from optically mapped action potentials. The effect of CCh on VFT was abolished by a muscarinic (atropine, 0.1 μmol l−1, n = 6) or a nicotinic receptor antagonist (mecamylamine, 10 μmol l−1, n = 6). CCh significantly increased NOx content in coronary effluent (n = 8), but not in the presence of mecamylamine (n = 8). The neuronal nitric oxide synthase inhibitor AAAN (N‐(4S)‐4‐amino‐5‐[aminoethyl]aminopentyl‐N′‐nitroguanidine; 10 μmol l−1, n = 6) or soluble guanylate cyclase (sGC) inhibitor ODQ (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one; 10 μmol l−1, n = 6) prevented the rise in VFT with CCh. The NO donor sodium nitrprusside (10 μmol l–1, n = 8) mimicked the action of CCh on VFT, an effect that was also blocked by atropine (n = 10). These data demonstrate a protective effect of CCh on VFT that depends upon both muscarinic and nicotinic receptor stimulation, where the generation of NO is likely to be via a neuronal nNOS/sGC‐dependent pathway.

  February 04, 2016   doi: 10.1113/JP271588   open full text
• Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII‐constitutive phosphorylation of RyR2 at site S2814.
  G. Mazzocchi, L. Sommese, J. Palomeque, J. I. Felice, M. N. Di Carlo, D. Fainstein, P. Gonzalez, P. Contreras, D. Skapura, M. D. McCauley, E. C. Lascano, J. A. Negroni, E. G. Kranias, X. H. T. Wehrens, C. A. Valverde, A. Mattiazzi.
  The Journal of Physiology. February 02, 2016
  Key points Mice with Ca2+–calmodulin‐dependent protein kinase (CaMKII) constitutive pseudo‐phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D+/+ mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca2+ leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)‐deficient S2814D+/+ knock‐in mice by crossing two colonies, S2814D+/+ and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D+/+ mice. PLN ablation partially rescues the altered intracellular Ca2+ dynamics of S2814D+/+ hearts and myocytes, but enhances SR Ca2+ sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca2+ waves of S2814D+/+ and transforms them into non‐propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca2+ uptake required to prevent the arrhythmias induced by a CaMKII‐dependent leaky RyR2. Abstract Mice with constitutive pseudo‐phosphorylation at Ser2814‐RyR2 (S2814D+/+) have increased propensity to arrhythmias under β‐adrenergic stress conditions. Although abnormal Ca2+ release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca2+ uptake remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to rescue the increased arrhythmia propensity of S2814D+/+ mice. We generated phospholamban (PLN)‐deficient/S2814D+/+ knock‐in mice by crossing two colonies, S2814D+/+ and PLNKO mice (SD+/+/KO). SD+/+/KO myocytes exhibited both increased SR Ca2+ uptake seen in PLN knock‐out (PLNKO) myocytes and diminished SR Ca2+ load (relative to PLNKO), a characteristic of S2814D+/+ myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D+/+ mice in vivo or programmed electric stimulation and high extracellular Ca2+ in S2814D+/− hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca2+ waves evoked by high extracellular Ca2+ provocation in S2814D+/+ mice into non‐propagated Ca2+ mini‐waves on confocal microscopy. Myocyte Ca2+ waves, typical of S2814D+/+ mice, could be evoked in SD+/+/KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca2+ uptake required to prevent the arrhythmias induced by a Ca2+–calmodulin‐dependent protein kinase (CaMKII)‐dependent leaky RyR2. Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca2+ leak due to CaMKII‐dependent phosphorylation of the RyR2‐S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca2+‐triggered arrhythmias.

  February 02, 2016   doi: 10.1113/JP271622   open full text
• Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels.
  Ikhyun Jun, Mary Hongying Cheng, Eunji Sim, Jinsei Jung, Bong Lim Suh, Yonjung Kim, Hankil Son, Kyungsoo Park, Chul Hoon Kim, Joo‐Heon Yoon, David C. Whitcomb, Ivet Bahar, Min Goo Lee.
  The Journal of Physiology. February 02, 2016
  Key points Cellular stimuli can modulate the ion selectivity of some anion channels, such as CFTR, ANO1 and the glycine receptor (GlyR), by changing pore size. Ion selectivity of CFTR, ANO1 and GlyR is critically affected by the electric permittivity and diameter of the channel pore. Pore size change affects the energy barriers of ion dehydration as well as that of size‐exclusion of anion permeation. Pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of CFTR, ANO1 and GlyR. Dynamic change in P HC O3/ Cl may mediate many physiological and pathological processes. Abstract Chloride (Cl−) and bicarbonate (HCO3−) are two major anions and their permeation through anion channels plays essential roles in our body. However, the mechanism of ion selection by the anion channels is largely unknown. Here, we provide evidence that pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of anion channels by reducing energy barriers of size‐exclusion and ion dehydration of HCO3− permeation. Molecular, physiological and computational analyses of major anion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR), anoctamin‐1(ANO1/TMEM16A) and the glycine receptor (GlyR), revealed that the ion selectivity of anion channels is basically determined by the electric permittivity and diameter of the pore. Importantly, cellular stimuli dynamically modulate the anion selectivity of CFTR and ANO1 by changing the pore size. In addition, pore dilatation by a mutation in the pore‐lining region alters the anion selectivity of GlyR. Changes in pore size affected not only the energy barriers of size exclusion but that of ion dehydration by altering the electric permittivity of water‐filled cavity in the pore. The dynamic increase in P HC O3/ Cl by pore dilatation may have many physiological and pathophysiological implications ranging from epithelial HCO3− secretion to neuronal excitation.

  February 02, 2016   doi: 10.1113/JP271311   open full text
• TRPM7 kinase activity regulates murine mast cell degranulation.
  Susanna Zierler, Adriana Sumoza‐Toledo, Sayuri Suzuki, Fionán Ó Dúill, Lillia V. Ryazanova, Reinhold Penner, Alexey G. Ryazanov, Andrea Fleig.
  The Journal of Physiology. January 27, 2016
  Key points The Mg2+ and Ca2+ conducting transient receptor potential melastatin 7 (TRPM7) channel–enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca2+ and extracellular Mg2+ sensitivity of mast cell degranulation. Abstract Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C‐terminally located α‐kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7+/∆K) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7KR), are not hyperallergic and are resistant to systemic magnesium (Mg2+) deprivation. Since allergic reactions are triggered by mast cell‐mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild‐type (TRPM7+/+), TRPM7+/∆K and TRPM7KR mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg2+ assured unperturbed IgE‐DNP‐dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE‐DNP‐dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca2+) in G protein‐induced exocytosis. In addition, G protein‐coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg2+ caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca2+ and affecting granular mobility and/or histamine contents.

  January 27, 2016   doi: 10.1113/JP271564   open full text
• Ageing and gastrointestinal sensory function: altered colonic mechanosensory and chemosensory function in the aged mouse.
  Christopher Keating, Linda Nocchi, Yang Yu, Jemma Donovan, David Grundy.
  The Journal of Physiology. January 19, 2016
  Key points Remarkably little is known about how age affects the sensory signalling pathways in the gastrointestinal tract despite age‐related gastrointestinal dysfunction being a prime cause of morbidity amongst the elderly population High‐threshold gastrointestinal sensory nerves play a key role in signalling distressing information from the gut to the brain. We found that ageing is associated with attenuated high‐threshold afferent mechanosensitivity in the murine colon, and associated loss of TRPV1 channel function. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease. Abstract Ageing has a profound effect upon gastrointestinal function through mechanisms that are poorly understood. Here we investigated the effect of age upon gastrointestinal sensory signalling pathways in order to address the mechanisms underlying these changes. In vitro mouse colonic and jejunal preparations with attached splanchnic and mesenteric nerves were used to study mechanosensory and chemosensory afferent function in 3‐, 12‐ and 24‐month‐old C57BL/6 animals. Quantitative RT‐PCR was used to investigate mRNA expression in colonic tissue and dorsal root ganglion (DRG) cells isolated from 3‐ and 24‐month animals, and immunohistochemistry was used to quantify the number of 5‐HT‐expressing enterochromaffin (EC) cells. Colonic and jejunal afferent mechanosensory function was attenuated with age and these effects appeared earlier in the colon compared to the jejunum. Colonic age‐related loss of mechanosensory function was more pronounced in high‐threshold afferents compared to low‐threshold afferents. Chemosensory function was attenuated in the 24‐month colon, affecting TRPV1 and serotonergic signalling pathways. High‐threshold mechanosensory afferent fibres and small‐diameter DRG neurons possessed lower functional TRPV1 receptor responses, which occurred without a change in TRPV1 mRNA expression. Serotonergic signalling was attenuated at 24 months, but TPH1 and TPH2 mRNA expression was elevated in colonic tissue. In conclusion, we saw an age‐associated decrease in afferent mechanosensitivity in the mouse colon affecting HT units. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease.

  January 19, 2016   doi: 10.1113/JP271403   open full text
• Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.
  Jessica R. Terrill, Gavin J. Pinniger, Jamie A. Graves, Miranda D. Grounds, Peter G. Arthur.
  The Journal of Physiology. January 18, 2016
  Key points Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N‐acetyl cysteine (NAC) and l‐2‐oxothiazolidine‐4‐carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. Abstract Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l‐2‐oxothiazolidine‐4‐carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti‐inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology.

  January 18, 2016   doi: 10.1113/JP271418   open full text
• Angiopoietin‐like 4 promotes angiogenesis in the tendon and is increased in cyclically loaded tendon fibroblasts.
  Rouhollah Mousavizadeh, Alex Scott, Alex Lu, Gholamreza S Ardekani, Hayedeh Behzad, Kirsten Lundgreen, Mazyar Ghaffari, Robert G McCormack, Vincent Duronio.
  The Journal of Physiology. January 18, 2016
  Key points Angiopoietin‐like 4 (ANGPTL4) modulates tendon neovascularization. Cyclic loading stimulates the activity of transforming growth factor‐β and hypoxia‐inducible factor 1α and thereby increases the expression and release of ANGPTL4 from human tendon cells. Targeting ANGPTL4 and its regulatory pathways is a potential avenue for regulating tendon vascularization to improve tendon healing or adaptation. Abstract The mechanisms that regulate angiogenic activity in injured or mechanically loaded tendons are poorly understood. The present study examined the potential role of angiopoietin‐like 4 (ANGPTL4) in the angiogenic response of tendons subjected to repetitive mechanical loading or injury. Cyclic stretching of human tendon fibroblasts stimulated the expression and release of ANGPTL4 protein via transforming growth factor‐β (TGF‐β) and hypoxia‐inducible factor 1α (HIF‐1α) signalling, and the released ANGPTL4 was pro‐angiogenic. Angiogenic activity was increased following ANGPTL4 injection into mouse patellar tendons, whereas the patellar tendons of ANGPTL4 knockout mice displayed reduced angiogenesis following injury. In human rotator cuff tendons, the expression of ANGPTL4 was correlated with the density of tendon endothelial cells. To our knowledge, this is the first study characterizing a role of ANGPTL4 in the tendon. ANGPTL4 may assist in the regulation of vascularity in the injured or mechanically loaded tendon. TGF‐β and HIF‐1α comprise two signalling pathways that modulate the expression of ANGPTL4 by mechanically stimulated tendon fibroblasts and, in the future, these could be manipulated to influence tendon healing or adaptation.

  January 18, 2016   doi: 10.1113/JP271752   open full text
• Age‐related neuromuscular changes affecting human vastus lateralis.
  M. Piasecki, A. Ireland, D. Stashuk, A. Hamilton‐Wright, D. A. Jones, J. S. McPhee.
  The Journal of Physiology. December 15, 2015
  Key points Skeletal muscle size and strength decline in older age. The vastus lateralis, a large thigh muscle, undergoes extensive neuromuscular remodelling in healthy ageing, as characterized by a loss of motor neurons, enlargement of surviving motor units and instability of neuromuscular junction transmission. The loss of motor axons and changes to motor unit potential transmission precede a clinically‐relevant loss of muscle mass and function. Abstract The anterior thigh muscles are particularly susceptible to muscle loss and weakness during ageing, although how this is associated with changes to neuromuscular structure and function in terms of motor unit (MU) number, size and MU potential (MUP) stability remains unclear. Intramuscular (I.M.) and surface electromyographic signals were recorded from the vastus lateralis (VL) during voluntary contractions held at 25% maximal knee extensor strength in 22 young (mean ± SD, 25.3 ± 4.8 years) and 20 physically active older men (71.4 ± 6.2 years). MUP size, firing rates, phases, turns and near fibre (NF) jiggle were determined and MU number estimates (MUNEs) were made by comparing average surface MUP with maximal electrically‐evoked compound muscle action potentials. Quadriceps cross‐sectional area was measured by magnetic resonance imaging. In total, 379 individual MUs were sampled in younger men and 346 in older men. Compared to the MU in younger participants, those in older participants had 8% lower firing rates and larger MUP size (+25%), as well as increased complexity, as indicated by phases (+13%), turns (+20%) and NF jiggle (+11%) (all P < 0.0005). The MUNE values (derived from the area of muscle in range of the surface‐electrode) in older participants were ∼70% of those in the young (P < 0.05). Taking into consideration the 30% smaller cross‐sectional area of the VL, the total number of MUs in the older muscles was between 50% and 60% lower compared to in young muscles (P < 0.0005). A large portion of the VL MU pool is lost in older men and those recruited during moderate intensity contractions were enlarged and less stable. These MU changes were evident before clinically relevant changes to muscle function were apparent; nevertheless, the changes in MU number and size are probably a prelude to future movement problems.

  December 15, 2015   doi: 10.1113/JP271087   open full text
• Aberrant regulation of synchronous network activity by the ADHD‐associated human dopamine D4 receptor variant D4.7 in prefrontal cortex.
  Ping Zhong, Wenhua Liu, Zhen Yan.
  The Journal of Physiology. November 06, 2015
  A unique feature of human D4 receptor (hD4R) gene is the existence of a large number of polymorphisms in exon 3 that codes for the third intracellular loop, which consists of a variable number of tandem repeats. The hD4R variants with long repeats have been linked to attention‐deficit/hyperactivity disorder (ADHD), however the underlying mechanism is unknown. Emerging evidence suggests that selective attention is controlled by the rhythmic synchronization in prefrontal cortex (PFC) and its connected networks, so we examined the role of hD4R variants in regulating PFC synchronous network activity. D4R knockout mice with viral infection of hD4.4 or hD4.7 in medial PFC were used. Whole‐cell patch‐clamp recordings were performed to examine the effects of activating hD4.x on the spontaneous large scale correlated activity in PFC pyramidal neurons. We have found that, compared to the normal 4‐repeat variant hD4.4, the ADHD‐linked variant hD4.7 induces more suppression of the glutamatergic excitatory network bursts and less suppression of the GABAergic inhibitory network bursts in the PFC circuitry. Methylphenidate, a psychostimulant drug used to treat ADHD, normalized the effects of hD4.7 on synchronous network bursts in PFC pyramidal neurons. These results have revealed the differential effects of hD4R variants on the integrated excitability of PFC circuits. It suggests that the aberrant regulation of PFC network activity by hD4.7 may underlie its involvement in ADHD. The MPH‐induced normalization of synaptic circuitry regulation may contribute to its effectiveness in ADHD treatment. This article is protected by copyright. All rights reserved

  November 06, 2015   doi: 10.1113/JP271317   open full text
• Structural and molecular regulation of lung maturation by intratracheal VEGF administration in the normally grown and placentally restricted fetus.
  Erin V. McGillick, Sandra Orgeig, Janna L Morrison.
  The Journal of Physiology. November 05, 2015
  Abstract Inhibition of hypoxia signalling leads to respiratory distress syndrome (RDS) whilst administration of vascular endothelial growth factor (VEGF), the most widely characterised hypoxia responsive factor, protects from RDS. In the lung of the chronically hypoxemic placentally restricted (PR) fetus, there is altered regulation of hypoxia signalling. This leads to reduced surfactant maturation in late gestation and provides evidence for increased risk of RDS in growth restricted neonates at birth. We evaluated the effect of recombinant human VEGF administration to bypass endogenous regulation of hypoxia signalling in the lung of the normally grown and PR sheep fetus. There was no effect of VEGF administration on fetal blood pressure or fetal breathing movements. We examined the effect on expression of genes regulating VEGF signalling (FLK and KDR), angiogenesis (ANGTP1, AQP1, ADM), alveolarisation (MMP2, MMP9, TIMP1, COL1A1, ELN), proliferation (IGR1, IGF2, IGF1R, MKI67, PCNA), inflammation (CCL2, CCL4, IL1B, TNFA, TGFB1, IL10) and surfactant maturation (SFTP‐A, SFTP‐B, SFTP‐C, SFTP‐D, PCYT1A, LPCAT, LAMP3, ABCA3). Despite effects of PR on expression of genes regulating airway remodelling, inflammatory signalling and surfactant maturation, there were very few effects of VEGF administration on gene expression in the lung of both the normally grown and PR fetus. There were, however, positive effects of VEGF administration on the percent tissue, air space and numerical density of SFTP‐B positive alveolar epithelial cells in fetal lung tissue. These results provide evidence for stimulatory effects of VEGF administration on structural maturation in the lung of both the normally grown and PR fetus. This article is protected by copyright. All rights reserved

  November 05, 2015   doi: 10.1113/JP271113   open full text
• Synapse‐specific expression of calcium‐permeable AMPA receptors in neocortical layer 5.
  Txomin Lalanne, Julia Oyrer, Adamo Mancino, Erica Gregor, Andrew Chung, Louis Huynh, Sasha Burwell, Jérôme Maheux, Mark Farrant, P. Jesper Sjöström.
  The Journal of Physiology. November 05, 2015
  Abstract AMPA‐type glutamate receptors (AMPARs) lacking an edited GluA2 subunit are calcium permeable (CP), and contribute to synaptic plasticity in several hippocampal interneuron types, but their precise role in neocortex is not well described. We explored the presence of CP‐AMPARs at pyramidal cell (PC) inputs to Martinotti cells (MCs) and basket cells (BCs) in layer 5 of developing mouse visual cortex (postnatal days 12–21). GluA2 immunolabelling was stronger in MCs than in BCs. A differential presence of CP‐AMPARs at PC‐BC and PC‐MC synapses was confirmed electrophysiologically, based on measures of spermine‐dependent rectification and CP‐AMPAR block by Naspm using recordings from synaptically connected cell pairs, NPEC‐AMPA uncaging, and miniature current recordings. CP‐AMPAR expression in BCs was in addition correlated with rapidly decaying synaptic currents. Computer modelling predicted that this reduces spike latencies and sharpens suprathreshold responses in BCs, which we verified experimentally using dynamic clamp. The synapse‐specific expression of CP‐AMPARs may thus critically influence both plasticity and information processing in neocortical microcircuits. This article is protected by copyright. All rights reserved

  November 05, 2015   doi: 10.1113/JP271394   open full text
• Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak.
  Rajiv Sankaranarayanan, Yatong Li, David J. Greensmith, David A. Eisner, Luigi Venetucci.
  The Journal of Physiology. November 05, 2015
  In heart failure reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (Ryanodine Receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterized the effects of two forms of Ca leak (Ca‐sensitizing and non‐sensitizing) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitize the RyR to Ca activation) or ryanodine (non‐sensitizing) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the biphasic decay was replaced by slow decay. We conclude that, in the presence of adrenergic stimulation, Ca leak can produce biphasic decay; the slow phase results from the leak opposing Ca uptake by SERCA. The degree of leak determines whether Ca waves, biphasic or monophasic decay occur. This article is protected by copyright. All rights reserved

  November 05, 2015   doi: 10.1113/JP271473   open full text
• The sensory origins of human position sense.
  AJ Tsay, MJ Giummarra, TJ Allen, U Proske.
  The Journal of Physiology. November 05, 2015
  Human limb position sense can be measured in two ways: in a blindfolded matching task, position of one limb is indicated with the other limb. Alternatively, position of a limb, hidden from view, is indicated with a pointer, moved by pressing a lever. These experiments examined the sensory basis of position sense measured in these two ways. Position errors were measured in 14 subjects after elbow flexors or extensors had been conditioned with a half‐maximum voluntary contraction. In agreement with previous studies, in the matching trials, position errors were distributed according to a pattern consistent with the action of muscle spindles as the position sensors. In the pointing trials, all errors lay in the direction of extension of the true position of the hidden arm and their distribution was inconsistent with influences arising in muscle spindles. Vibration of elbow muscles produced an illusion of muscle lengthening during a matching task, while during the pointing task no illusion was present. Finally, the matching : pointing error difference was preserved, even when one arm was loaded with a weight or skin over the elbow was stretched. It is proposed that there are two kinds of position sense, one signalled by muscle spindles, indicating position of one part of the body relative to another. A second sense provides information about the position of the body in extrapersonal space and here we hypothesise that exteroceptors, including vision, touch and hearing, acting via a central map of the body, provide the spatial information. This article is protected by copyright. All rights reserved

  November 05, 2015   doi: 10.1113/JP271498   open full text
• Purinergic signaling mediates bidirectional crosstalk between chemoreceptor type I and glial‐like type II cells of the rat carotid body.
  Sindhubarathi Murali, Colin A. Nurse.
  The Journal of Physiology. November 05, 2015
  The mammalian carotid body (CB) is excited by blood‐borne stimuli including hypoxia and acid hypercapnia, leading to respiratory and cardiovascular reflex responses. This chemosensory organ consists of innervated clusters of receptor type I cells, ensheathed by processes of adjacent glial‐like type II cells. ATP is a major excitatory neurotransmitter released from type I cells and type II cells express purinergic P2Y2 receptors (P2Y2R), activation of which leads to opening of ATP‐permeable, pannexin‐1 (Panx‐1) channels. While these properties support crosstalk between type I and type II cells during chemotransduction, direct evidence is lacking. To address this, we first exposed isolated rat chemoreceptor clusters to acute hypoxia, isohydric hypercapnia, or the depolarizing stimulus high K+,and monitored intracellular [Ca2+] using Fura‐2. As expected, these stimuli induced intracellular [Ca2+] elevations (Δ[Ca2+]i) in type I cells. Interestingly however, there was often a delayed, secondary Δ[Ca2+]i in nearby type II cells that was reversibly inhibited by the P2Y2R antagonist, suramin, or by the nucleoside hydrolase, apyrase. By contrast, type II cell stimulation with the P2Y2R agonist UTP (100 μm) often led to a delayed, secondary Δ[Ca2+]i response in nearby type I cells that was reversibly inhibited by the Panx‐1 blocker, carbenoxolone (5 μm). This Δ[Ca2+]i response was also strongly inhibited by blockers of either adenosine A2AR (SCH58261) or 5’ectonucleotidase (APOCP), suggesting it was due to adenosine arising from breakdown of ATP released through Panx‐1 channels. Collectively, these data strongly suggest that purinergic signalling mechanisms mediate crosstalk between CB chemoreceptor and glial cells during chemotransduction. This article is protected by copyright. All rights reserved

  November 05, 2015   doi: 10.1113/JP271494   open full text
• Shear stress induces a longitudinal Ca2+ wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes.
  Joon‐Chul Kim, Sun‐Hee Woo.
  The Journal of Physiology. November 04, 2015
  Key points Cardiac myocytes are subjected to fluid shear stress during the cardiac cycle and haemodynamic disturbance. A longitudinally propagating, regenerative Ca2+ wave is initiated in atrial myocytes under shear stress. Here we determine the cellular mechanism for this shear‐induced Ca2+ wave using two‐dimensional confocal Ca2+ imaging combined with pressurized fluid flow. Our data suggest that shear stress triggers the Ca2+ wave through ryanodine receptors via P2Y1 purinoceptor–phospholipase C‐type 2 inositol 1,4,5‐trisphosphate receptor signal transduction in atrial myocytes, and that this mechanotransduction is activated by gap junction hemichannel‐mediated ATP release. Shear‐specific mechanotransduction and the subsequent regenerative Ca2+ wave may be one way for atrial myocytes to assess mechanical stimuli directly and alter their Ca2+ signalling accordingly. Abstract Atrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca2+ wave. Here, we investigated the cellular mechanisms underlying the shear stress‐mediated Ca2+ wave, using two‐dimensional confocal Ca2+ imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ∼16 dyn cm−2 for 8 s induced ∼1.2 aperiodic longitudinal Ca2+ waves (∼79 μm s−1) with a delay of 0.2−3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5‐trisphosphate receptors (IP3Rs) abolished shear stress‐induced Ca2+ wave generation. Furthermore, in atrial myocytes from type 2 IP3R (IP3R2) knock‐out mice, shear stress failed to induce longitudinal Ca2+ waves. The phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the shear‐induced longitudinal Ca2+ wave. However, pretreating atrial cells with blockers for stretch‐activated channels, Na+−Ca2+ exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y1 receptor antagonist MRS 2179, both suppressed the Ca2+ wave, whereas the P2X receptor antagonist, iso‐PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP‐metabolizing apyrase inhibited the wave. Removal of external Ca2+ to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca2+ release through RyRs is triggered by P2Y1–PLC–IP3R2 signalling that is activated by gap junction hemichannel‐mediated ATP release in atrial myocytes under shear stress.

  November 04, 2015   doi: 10.1113/JP271016   open full text
• Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal.
  R. E. Kreipke, S. J. Birren.
  The Journal of Physiology. November 04, 2015
  Key points Cardiomyocytes withdraw from the cell cycle late in prenatal and early in postnatal life and subsequent heart growth occurs via cellular hypertrophy. The signals regulating this transition remain unknown. Lesion of the neonatal sympathetic nervous system results in decreased heart size. In vitro investigations show that sympathetic neurons delay cardiomyocyte maturation and cell cycle withdrawal. Early sympathetic innervation may contribute to the determination of adult heart size by regulating the total number of cardiomyocytes via regulation of the proliferative/hypertrophic transition. Early perturbations of sympathetic structure or function could have long‐term effects on adult heart function. Abstract Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6‐hydroxydopamine (6‐OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β‐adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β‐adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

  November 04, 2015   doi: 10.1113/JP270917   open full text
• Effect of resveratrol on metabolic and cardiovascular function in male and female adult offspring exposed to prenatal hypoxia and a high‐fat diet.
  Amin Shah, Laura M. Reyes, Jude S. Morton, David Fung, Jillian Schneider, Sandra T. Davidge.
  The Journal of Physiology. November 04, 2015
  Key points Prenatal hypoxia, a common outcome of many pregnancy complications, predisposes offspring to chronic diseases in later life. We investigated the effect of prenatal hypoxia, on a background of a high‐fat diet, on metabolic and cardiac function in adult male and female rat offspring. We also examined the therapeutic role of resveratrol supplementation in preventing metabolic and cardiac dysfunction. Prenatal hypoxia impaired both metabolic and cardiac function in male and only cardiac function in female rat offspring. We also observed that male rat offspring were more susceptible to metabolic and cardiac dysfunction as compared with their female counterparts; this provides evidence of sexual dichotomy in the fetal programming of diseases due to prenatal hypoxia. Resveratrol supplementation in the diet improved metabolic and cardiac function independent of sex; this provides evidence of a possible therapeutic role of resveratrol in susceptible male and female rat offspring exposed to prenatal hypoxia. Abstract Prenatal hypoxia, a common outcome of pregnancy complications, predisposes offspring to the development of metabolic and cardiovascular disorders in later life. We have previously observed that resveratrol improved cardiovascular and metabolic health in adult male rat offspring exposed to prenatal hypoxia and a postnatal high‐fat (HF) diet; however, the effects of resveratrol in female rat offspring are not known. Our aim was to identify the mechanism(s) by which resveratrol may prevent metabolic and cardiac dysfunction in both male and female rat offspring exposed to prenatal hypoxia and a postnatal HF diet. Offspring that experienced normoxia or hypoxia in utero were fed a HF diet or a HF diet supplemented with resveratrol for 9 weeks following weaning. Body composition, metabolic function, in vivo cardiac function and ex vivo cardiac susceptibility to ischaemia–reperfusion (I/R) injury were assessed at 12 weeks of age. Prenatal hypoxia impaired metabolic function in male, but not female, rat offspring fed a HF diet and this was improved by resveratrol supplementation. Prenatal hypoxia also led to reduced recovery from cardiac I/R injury in male, and to a lesser extent in female, rat offspring fed a HF diet. Indices of cardiac oxidative stress after I/R were enhanced in both male and female rat offspring exposed to prenatal hypoxia. Resveratrol improved cardiac recovery from I/R injury and attenuated superoxide levels in both male and female rat offspring. In conclusion, prenatal hypoxia impaired metabolic and cardiac function in a sex‐specific manner. Resveratrol supplementation may improve metabolic and cardiovascular health in adult male and female rat offspring exposed to prenatal hypoxia.

  November 04, 2015   doi: 10.1113/JP271133   open full text
• Plasma hyperosmolality attenuates skin sympathetic nerve activity during passive heat stress in humans.
  Daniel Gagnon, Steven A. Romero, Hai Ngo, Paula Y.S. Poh, Craig G. Crandall.
  The Journal of Physiology. November 03, 2015
  In humans, plasma hyperosmolality delays the onset of sweating and cutaneous vasodilation during heat stress. However, it remains unknown if hyperosmolality exerts this effect through a central (i.e. central nervous system) and/or peripheral (i.e. effector organ) modulation of thermoregulatory activity. We examined if intravenous infusion of hyperosmotic saline affects skin sympathetic nerve activity (SSNA) during whole‐body passive heating in healthy humans. Furthermore, we examined if local intradermal infusion of hyperosmotic saline affects sweating and cutaneous vasodilation during passive heating. Following intravenous infusion of either 0.9% (ISO) or 3.0% (HYPER) NaCl saline, 12 subjects were passively heated until core temperature increased by ∼0.6°C. During each condition, sweating and cutaneous vascular conductance were measured over two intradermal microdialysis probes, one perfused with ISO saline and the other with HYPER saline. Intravenous infusion of HYPER saline increased plasma osmolality (294 ± 3 to 316 ± 5 mOsm/kgH2O, P ≤ 0.01), which remained greater than ISO throughout heating. Plasma hyperosmolality delayed the mean body temperature onset of sweating (+1.24 ± 0.18 vs. +1.60 ± 0.18ºC, P ≤ 0.01) and cutaneous vasodilation (+1.15 ± 0.18 vs. +1.53 ± 0.22ºC, P ≤ 0.01), and attenuated the increase in SSNA during heating (+147 ± 178 vs. +427 ± 281%, P ≤ 0.01). Intradermal infusion of HYPER saline increased baseline CVC (P ≤ 0.01), which did not increase further during the subsequent heating period (P = 0.11). In contrast, intradermal infusion of HYPER saline did not affect sweating (P = 0.99). These results provide direct evidence that plasma hyperosmolality exerts a central modulatory effect governing efferent thermoregulatory activity in humans. This article is protected by copyright. All rights reserved

  November 03, 2015   doi: 10.1113/JP271497   open full text
• Cord blood mononuclear cells prevent neuronal apoptosis in response to perinatal asphyxia in the newborn lamb.
  James DS Aridas, Courtney A McDonald, Madison CB Paton, Tamara Yawno, Amy E Sutherland, Ilias Nitsos, Yen Pham, Michael Ditchfield, Michael C Fahey, Flora Wong, Atul Malhotra, Margie Castillo‐Melendez, Kishore Bhakoo, Euan M Wallace, Graham Jenkin, Suzanne L Miller.
  The Journal of Physiology. November 03, 2015
  Perinatal asphyxia is a significant cause of death or long‐term neurodevelopmental impairment. Hypothermia, currently the only effective treatment, leads to modest improvements, however new therapeutic strategies are required. Umbilical cord blood (UCB) mononuclear cells have potent anti‐inflammatory properties and may reduce neuropathology. This study examined whether autologous UCB mononuclear cells were neuroprotective when administered to newborn lambs at 12 h after birth asphyxia. At caesarean section, birth asphyxia was induced by clamping the umbilical cord until mean arterial blood pressure decreased to 18–20 mmHg. Asphyxia (n = 20) or control (n = 11) lambs were resuscitated and maintained, with magnetic resonance spectroscropy (MRS) performed at 12 and 72 h, and euthanasia at 72 h. Cord blood was collected once the cord was clamped, mononuclear cells isolated and labelled fluorescently and administered to control (n = 3) or asphyxia (n = 8) lambs. Asphyxia induced a significant increase in cellular apoptosis (caspase‐3 immunopositive) within all brain regions examined, including cortex, hippocampus, thalamus, striatum and subcortical white matter (P < 0.01 versus control). Additionally, asphyxia induced significant and widespread astrogliosis and increased inflammatory cells (activated microglia and macrophages). The administration of UCB mononuclear cells (asphyxia+UCB) significantly decreased neuronal apoptosis, astrogliosis and inflammation (P < 0.05 versus asphyxia alone). Asphyxia+UCB lambs also demonstrated decreased brain metabolites lactate:choline (P = 0.01) and lactate:N‐acetyl aspartate (P < 0.01) from 12 to 72 h, detected using MRS. Autologous UCB mononuclear cell treatment restores normal brain metabolism following perinatal asphyxia, reduces brain inflammation, astrogliosis and neuronal apoptosis, supporting its use as a neuroprotective therapy following asphyxia. This article is protected by copyright. All rights reserved

  November 03, 2015   doi: 10.1113/JP271104   open full text
• Dysferlin deficiency blunts β‐adrenergic‐dependent lusitropic function of mouse heart.
  Bin Wei, Hongguang Wei, J.‐P. Jin.
  The Journal of Physiology. November 02, 2015
  Key points Deficiency of dysferlin causes limb‐girdle muscular dystrophy 2B and Miyoshi myopathy with cardiac involvement that leads to dilated cardiomyopathy and heart failure. The pathogenesis and pathophysiology of dysferlin cardiomyopathy are not fully understood. We studied cardiac phenotypes of young dysferlin gene knockout mice to investigate the primary pathological and pathophysiological changes. In comparison with wild‐type controls, dysferlin‐deficient cardiomyocytes showed slower Ca2+ re‐sequestration, and dysferlin deficiency blunted the β‐adrenergic effect on relaxation and pumping function of ex vivo working hearts. Dysferlin deficiency increased phosphorylation of ventricular myosin light chain 2, suggesting a compensatory response to the impaired cardiac lusitropic function. The data suggest that delayed calcium re‐sequestration and post‐translational modification of myofilament proteins may provide potential targets to develop new treatments for dysferlin cardiomyopathy. Abstract Dysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb‐girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin‐deficient mice showed normal shortening and re‐lengthening velocities in the absence of external load with normal peak systolic Ca2+ but slower Ca2+ re‐sequestration than wild‐type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin‐deficient mouse hearts compared with that in wild‐type hearts. Young dysferlin‐deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β‐adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy.

  November 02, 2015   doi: 10.1113/JP271225   open full text
• Sodium nitrate alleviates functional muscle ischaemia in patients with Becker muscular dystrophy.
  Michael D. Nelson, Ryan Rosenberry, Rita Barresi, Evgeny I. Tsimerinov, Florian Rader, Xiu Tang, O'Neil Mason, Avery Schwartz, Thomas Stabler, Sarah Shidban, Neigena Mobaligh, Shomari Hogan, Robert Elashoff, Jason D. Allen, Ronald G. Victor.
  The Journal of Physiology. November 02, 2015
  Key points Dystrophin deficiency disrupts sarcolemmal targeting of neuronal nitric oxide synathase, resulting in functional muscle ischaemia. Chronic treatment of dystrophic mice with an inorganic nitric oxide (NO) donor alleviates this ischaemia and improves many features of the dystrophic phenotype. The present study translates this preclinical work by showing that a single oral dose of sodium nitrate,which serves as a NO donor when reduced to circulating nitrite by the commensal bacteria in the oral cavity, alleviates functional muscle ischaemia and restores normal blood flow regulation in human patients with dystrophinopathy. The results of the present study further support the mechanistic hypothesis that circulating nitrite serves as an alternative NO donor when reduced by deoxyhaemoglobin and/or deoxymyoglobin in exercising muscle. Abstract Becker muscular dystrophy (BMD) is a progressive X‐linked muscle wasting disease for which there is no treatment. BMD is caused by in‐frame mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the sarcolemma. Among these is neuronal nitric oxide synthase mu (nNOSμ), which requires specific spectrin‐like repeats (SR16/17) in dystrophin's rod domain and the adaptor protein α‐syntrophin for sarcolemmal targeting. When healthy skeletal muscle is exercised, sarcolemmal nNOSμ‐derived nitric oxide (NO) attenuates α‐adrenergic vasoconstriction, thus optimizing perfusion. In the mdx mouse model of dystrophinopathy, this protective mechanism (functional sympatholysis) is defective, resulting in functional muscle ischaemia. Treatment with a NO‐donating non‐steroidal anti‐inflammatory drug (NSAID) alleviates this ischaemia and improves the murine dystrophic phenotype. In the present study, we report that, in 13 men with BMD, sympatholysis is defective mainly in patients whose mutations disrupt sarcolemmal targeting of nNOSμ, with the vasoconstrictor response measured as a decrease in muscle oxygenation (near infrared spectroscopy) to reflex sympathetic activation. Then, in a single‐arm, open‐label trial in 11 BMD patients and a double‐blind, placebo‐controlled cross‐over trial in six patients, we show that acute treatment with oral sodium nitrate, an inorganic NO donor without a NSIAD moiety, restores sympatholysis and improves post‐exercise hyperaemia (Doppler ultrasound). By contrast, sodium nitrate improves neither sympatholysis, nor hyperaemia in healthy controls. Thus, a simple NO donor recapitulates the vasoregulatory actions of sarcolemmal nNOS in BMD patients, and constitutes a putative novel therapy for this disease.

  November 02, 2015   doi: 10.1113/JP271252   open full text
• Neural influences on human intestinal epithelium in vitro.
  Dagmar Krueger, Klaus Michel, Florian Zeller, Ihsan E. Demir, Güralp O. Ceyhan, Julia Slotta‐Huspenina, Michael Schemann.
  The Journal of Physiology. November 02, 2015
  Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R‐basal) and short circuit currents (ISC; secretion) under basal conditions (ISC‐basal) and after electrical field stimulation (ISC‐EFS) of nerves in 2221 resectates from 435 patients. ISC‐EFS was tetrodotoxin‐sensitive and of comparable magnitude in small and large intestine. ISC‐EFS or R‐basal were not influenced by patients´ age, gender or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC‐EFS depended on epithelial cAMP‐driven chloride and bicarbonate secretion, but not on amiloride‐sensitive sodium absorption. While atropine‐sensitive cholinergic components prevailed in ISC‐EFS of duodenum, jejunum and ileum, PG97‐269‐sensitive (VIP receptor1 antagonist) VIPergic together with L‐NAME‐sensitive nitrergic components dominated ISC‐EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors or stimulus frequency dependent transmitter release were not responsible for the region specific transmitter contribution to ISC‐EFS. The low atropine‐sensitivity of ISC‐EFS in the colon was rather due to high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC‐EFS remained unchanged after tachykinin, P2X, P2Y or A1/2 receptor blockade. R‐basal was smaller but ISC‐basal was higher in small intestine. Tetrodotoxin and bumetanide decreased ISC‐basal in all regions suggesting nerve‐dependent secretory tone. ISC‐basal was atropine‐sensitive in the small and PG97‐269‐sensitive in the large intestine. This comprehensive study revealed novel insights into region specific nerve‐mediated secretion in human small and large intestine. This article is protected by copyright. All rights reserved

  November 02, 2015   doi: 10.1113/JP271493   open full text
• In silico prediction of drug therapy in catecholaminergic polymorphic ventricular tachycardia.
  Pei‐Chi Yang, Jonathan D. Moreno, Christina Y. Miyake, Steven B. Vaughn‐Behrens, Mao‐Tsuen Jeng, Eleonora Grandi, Xander H.T. Wehrens, Sergei Noskov, Colleen E. Clancy.
  The Journal of Physiology. October 30, 2015
  Abstract Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by fatal ventricular arrhythmias in structurally normal hearts during β‐adrenergic stimulation. Current treatment strategies include β‐blockade, flecainide, and ICD implementation – none of which is fully effective, and each with associated risk. Recently, flecainide has gained considerable interest in CPVT treatment, but its mechanism of action for therapeutic efficacy is unclear. In this study, we performed in‐silico mutagenesis to construct a CPVT model and then used a computational modeling and simulation approach to make predictions of drug mechanisms and efficacy in the setting of CPVT. Experiments were carried out to validate model results. Our simulations revealed that Na+ channel effects are insufficient to explain flecainide efficacy in CPVT. The pure Na+ channel blocker lidocaine and the antianginal ranolazine were additionally tested and also found to be ineffective. When we tested lower dose combination therapy with flecainide, β‐blockade, and CaMKII inhibition, our model predicted superior therapeutic efficacy than with flecainide monotherapy. Simulations indicate a polytherapeutic approach may mitigate side effects and proarrhythmic potential plaguing CPVT pharmacologic management today. Importantly, our prediction of a novel polytherapy for CPVT was confirmed experimentally. Our simulations suggest that flecainide therapeutic efficacy in CPVT is unlikely to derive from primary interactions with the Na channel and benefit may be gained from an alternative multi‐drug regimen. This article is protected by copyright. All rights reserved

  October 30, 2015   doi: 10.1113/JP271282   open full text
• Early redox imbalance is associated with liver dysfunction at weaning in overfed rats.
  E. P. S. Conceição, E. G. Moura, J. C. Carvalho, E. Oliveira, P. C. Lisboa.
  The Journal of Physiology. October 29, 2015
  Key points Childhood obesity is associated with precocious oxidative stress, which can contribute to future diseases. Early overfed rats have increased oxidative stress in liver and plasma at weaning. The model of litter size reduction causes adipocyte hypertrophy and lower PPARγ at weaning, suggesting a decrease in adipocyte proliferation. Abstract Neonatal overfeeding induced by litter size reduction leads to further obesity and other metabolic disorders, such as liver oxidative stress and microsteatosis at adulthood. We hypothesized that overfeeding causes an early redox imbalance at weaning, which could programme the animals to future liver dysfunction. Thus, we studied lipogenesis, adipogenesis, catecholamine status and oxidative balance in weaned overfed pups. To induce early overfeeding, litters were adjusted to three pups at the 3rd day of lactation (SL group). The control group contained 10 pups per litter until weaning (NL group). Peripheral autonomic nerve function was determined in vivo at 21 days old. Thereafter, pups were killed for further analysis. Differences were considered significant when P < 0.05. The SL pups presented with a higher visceral adipocyte area, higher content of lipogenic enzymes (ACC, FAS) and with a lower content of adipogenic factors (CEBP, PPARγ) in visceral adipose tissue (VAT). Although autonomic nerve activity and adrenal catecholamine production were not significantly altered, catecholamine receptor (β3ADR) content was lower in VAT. The SL pups also presented with higher triglyceride, PPARγ, PPARα and PGC1α contents in liver. In plasma and liver, the SL pups showed an oxidative imbalance, with higher lipid peroxidation and protein oxidation. The SL group presented with a higher serum alanine aminotransferase (ALT). The early increase in lipogenesis in adipose tissue and liver in weaned overfed rats suggests that the higher oxidative stress and lower catecholamine content in VAT are associated with the early development of liver dysfunction and adipocyte hypertrophy.

  October 29, 2015   doi: 10.1113/JP271189   open full text
• 5′‐AMP activated protein kinase α2 controls substrate metabolism during post‐exercise recovery via regulation of pyruvate dehydrogenase kinase 4.
  Andreas Mæchel Fritzen, Anne‐Marie Lundsgaard, Jacob Jeppesen, Mette Landau Brabæk Christiansen, Rasmus Biensø, Jason R. B. Dyck, Henriette Pilegaard, Bente Kiens.
  The Journal of Physiology. October 29, 2015
  Key points There is lower fat oxidation during post‐exercise recovery in mice lacking 5′‐AMP activated protein kinase α2 (AMPKα2). AMPKα2 is involved in post‐transcriptional and not transcriptional regulation of pyruvate dehydrogenase kinase 4 (PDK4) in muscle. Exercise‐induced AMPKα2 activity increases PDK4 protein content, in turn inhibiting pyruvate dehydrogenase activity and glucose oxidation. The mechanism for increased post‐exercise fat oxidation is by inhibition of carbohydrate oxidation allowing increased fat oxidation rather than by direct stimulation of fat oxidation. Abstract It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post‐exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post‐exercise in 5´‐AMP activated protein kinase (AMPK) α2 and α1 knock‐out (KO) and wild‐type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post‐exercise recovery, a lower lipid oxidation (P < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without genotype differences in muscle malonyl‐CoA or free‐carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the exercise‐induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01) inhibitory pyruvate dehydrogenase (PDH)‐E1α Ser293 phosphorylation was observed in AMPKα2 KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism post‐exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation.

  October 29, 2015   doi: 10.1113/JP270821   open full text
• Hydrogen peroxide modulates synaptic transmission in ventral horn neurons of the rat spinal cord.
  Masayuki Ohashi, Toru Hirano, Kei Watanabe, Keiichi Katsumi, Nobuko Ohashi, Hiroshi Baba, Naoto Endo, Tatsuro Kohno.
  The Journal of Physiology. October 29, 2015
  Excessive production of reactive oxygen species (ROS) is a critical component of the cellular and molecular pathophysiology of many central nervous system (CNS) disorders, including trauma, ischemia‐reperfusion injury, and neurodegenerative diseases. Hydrogen peroxide (H2O2), an abundant ROS, modulates synaptic transmission and contributes to neuronal damage in the CNS; however, the pathophysiological role of H2O2 in spinal cord ventral horn (VH) neurons remains poorly understood, despite reports that these neurons are highly vulnerable to oxidative stress and ischemia. This was investigated in the present study using a whole‐cell patch clamp approach in rats. We found that exogenous application of H2O2 increased the release of glutamate from excitatory presynaptic terminals and γ‐aminobutyric acid (GABA) from inhibitory presynaptic terminals. The increase of glutamate release was induced in part by an increase in Ca2+ influx through N‐type voltage‐gated calcium channels (VGCCs) as well as by ryanodine receptor (RyR)‐ and inositol triphosphate receptor‐mediated Ca2+ release from the endoplasmic reticulum (ER). In inhibitory presynaptic neurons, increased IP3R‐mediated Ca2+ release from the ER increased GABAergic transmission, which served to rescue VH neurons from excessive release of glutamate from presynaptic terminals. These findings indicate that inhibiting N‐type VGCCs or RyRs may attenuate excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may therefore represent a novel and targeted strategy for preventing and treating H2O2‐induced motor neuron disorders. This article is protected by copyright. All rights reserved

  October 29, 2015   doi: 10.1113/JP271449   open full text
• Twenty‐eight days of exposure to 3454 m increases mitochondrial volume density in human skeletal muscle.
  Robert A. Jacobs, Anne‐Kristine Meinild Lundby, Simone Fenk, Saskia Gehrig, Christoph Siebenmann, Daniela Flück, Niels Kirk, Matthias P. Hilty, Carsten Lundby.
  The Journal of Physiology. October 28, 2015
  Key points It is generally accepted that mitochondrial volume density in human skeletal muscle diminishes with chronic high altitude exposure. All data supporting this concept were collected during mountaineering expeditions, which are associated with the confounding effects of whole body negative energy balance. Here we examine the effect of 28 days of exposure to 3454 m on skeletal muscle mitochondrial volume density in a setting where whole body weight, whole body composition, leg lean mass, skeletal muscle fibre area and maximal power output were preserved. Our results demonstrate that total skeletal muscle mitochondrial volume density increases in response to high altitude exposure secondary to a preferential increase in intermyofibrillar mitochondrial populations. This study provides direct evidence contradicting the notion that high altitude exposure diminishes skeletal muscle mitochondrial volume density, highlighting an inconsistent understanding of the role of hypoxia on skeletal muscle mitochondria. Abstract The role of hypoxia on skeletal muscle mitochondria is controversial. Studies superimposing exercise training on hypoxic exposure demonstrate an increase in skeletal muscle mitochondrial volume density (MitoVD) over equivalent normoxic training. In contrast, reductions in both skeletal muscle mass and MitoVD have been reported following mountaineering expeditions. These observations may, however, be confounded by negative energy balance, which may obscure the results. Accordingly we sought to examine the effects of high altitude hypoxic exposure on mitochondrial characteristics, with emphasis on MitoVD, while minimizing changes in energy balance. For this purpose, skeletal muscle biopsies were obtained from nine lowlanders at sea level (Pre) and following 7 and 28 days of exposure to 3454 m. Maximal ergometer power output, whole body weight and composition, leg lean mass and skeletal muscle fibre area all remained unchanged following the altitude exposure. Transmission electron microscopy determined that intermyofibrillar (IMF) MitoVD was augmented (P = 0.028) by 11.5 ± 9.2% from Pre (5.05 ± 0.9%) to 28 Days (5.61 ± 0.04%). In contrast, there was no change in subsarcolemmal (SS) MitoVD. As a result, total MitoVD (IMF + SS) was increased (P = 0.031) from 6.20 ± 1.5% at Pre to 6.62 ± 1.4% at 28 Days (7.8 ± 9.3%). At the same time no changes in mass‐specific respiratory capacities, mitochondrial protein or antioxidant content were found. This study demonstrates that skeletal muscle MitoVD may increase with 28 days acclimation to 3454 m.

  October 28, 2015   doi: 10.1113/JP271118   open full text
• Interleukin‐13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity.
  Petra Dames, Theresa Bergann, Anja Fromm, Roland Bücker, Christian Barmeyer, Susanne M. Krug, Michael Fromm, Jörg‐Dieter Schulzke.
  The Journal of Physiology. October 28, 2015
  Key points Interleukin‐13 (IL‐13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2‐driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) – the main limiting factor for sodium absorption in the distal colon – is also influenced by IL‐13 and if so, by what mechanism(s). We demonstrate in an intestinal cell model as well as in mouse distal colon that IL‐13 causes reduced ENaC activity. We show that IL‐13 impairs ENaC‐dependent sodium transport by activating the JAK1/2–STAT6 signalling pathway. These results improve our understanding of the mechanisms through which IL‐13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease. Abstract Interleukin‐13 (IL‐13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL‐13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL‐13 on ENaC was examined in HT‐29/B6‐GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride‐sensitive short‐circuit current (ISC) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT‐PCR and promoter gene assay. We show that IL‐13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa) as a result of decreased transcription levels of β‐ and γ‐ENaC subunits and SGK1, a post‐translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2–STAT6 signalling. This inhibition also affected the IL‐13‐induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL‐13‐mediated ENaC inactivation was confirmed in a STAT6−/− mouse model. In conclusion, these results indicate that IL‐13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.

  October 28, 2015   doi: 10.1113/JP271156   open full text
• Deficiency of GABAergic synaptic inhibition in the Kölliker‐Fuse area underlies respiratory dysrhythmia in a mouse model of Rett syndrome.
  Ana Paula Abdala, Marie A. Toward, Mathias Dutschmann, John M. Bissonnette, Julian F.R. Paton.
  The Journal of Physiology. October 28, 2015
  Central apnoeas and respiratory irregularity are a common feature in Rett syndrome (RTT), a neurodevelopmental disorder most often caused by mutations in the methyl‐CpG‐binding protein 2 gene (MECP2). We used a MECP2 deficient mouse model of RTT as a strategy to obtain insights into the neurobiology of the disease and into mechanisms essential for respiratory rhythmicity during normal breathing. Previously, we showed that, systemic administration of a GABA reuptake blocker in MECP2 deficient mice markedly reduced the occurrence of central apnoeas. Further, we found that, during central apnoeas, post‐inspiratory drive (adductor motor) to the upper airways was enhanced in amplitude and duration in Mecp2 heterozygous female mice. Since pontine Kölliker‐Fuse (KF) region drives post‐inspiration, suppresses inspiration, and can reset the respiratory oscillator phase, we hypothesized that synaptic inhibition in this area is essential for respiratory rhythm regularity. In this study, we found that: (i) Mecp2 heterozygous mice show deficiency of GABA perisomatic bouton‐like puncta and processes in the KF; (ii) blockade of GABA reuptake in the KF of RTT mice reduced breathing irregularity; (iii) conversely, blockade of GABAA receptors in the KF of healthy rats mimicked the RTT respiratory phenotype of recurrent central apnoeas and prolonged post‐inspiratory activity. Our results show that reductions in synaptic inhibition within the KF induce rhythm irregularity whereas boosting GABA transmission reduces respiratory arrhythmia in a murine model of RTT. Our data suggest that manipulation of synaptic inhibition in KF may be a clinically important strategy for alleviating the life threatening respiratory disorders in RTT. This article is protected by copyright. All rights reserved

  October 28, 2015   doi: 10.1113/JP270966   open full text
• Respiratory modulated sympathetic activity: A putative mechanism for developing vascular resistance?
  Linford J.B. Briant, Erin L. O'Callaghan, Alan R. Champneys, Julian F.R. Paton.
  The Journal of Physiology. October 28, 2015
  Sympathetic nerve activity (SNA) exhibits respiratory modulation. This component of SNA is important ‐ being recruited under cardiorespiratory reflex conditions and elevated in the spontaneously hypertensive (SH) rat – and yet, the exact influence of this modulation on vascular tone is not understood, even in normotensive conditions. We constructed a mathematical model of the sympathetic innervation of an arteriole, and used it to test the hypothesis that respiratory modulation of SNA preferentially increases vasoconstriction compared to a frequency‐matched tonic pattern. Simulations supported the hypothesis, where respiratory modulated increases in vasoconstriction were mediated by a noradrenergic mechanism. These predictions were tested in vivo in adult Wistar rats. Stimulation of the sympathetic chain (L3) with respiratory‐modulated bursting patterns, revealed that bursting increases vascular resistance (VR) more than tonic stimulation (57.8 ± 3.3% vs 44.8 ± 4.2%; P < 0.001; n = 8). The onset of the VR response was also quicker for bursting stimulation (rise time‐constant = 1.98 ± 0.09 s vs 2.35 ± 0.20 s; P < 0.01). In adult SH rats (n = 8), the VR response to bursting (44.6 ± 3.9%) was not different to tonic (37.4 ± 3.5%; P = 0.57). Using both mathematical modelling and in vivo techniques, we have shown that VR depends critically on respiratory modulation and revealed that this pattern‐dependency in Wistar rats is due to a noradrenergic mechanism. This respiratory component may therefore contribute to the ontogenesis of hypertension in the pre‐hypertensive SH rat ‐ raising VR and driving vascular remodelling. Why adult SH rats do not exhibit a pattern‐dependent response is not known, but further modelling revealed that this may be due to dysfunctional NA reuptake. This article is protected by copyright. All rights reserved

  October 28, 2015   doi: 10.1113/JP271253   open full text
• Each‐step activation of oxidative phosphorylation is necessary to explain muscle metabolic kinetic responses to exercise and recovery in humans.
  Bernard Korzeniewski, Harry B. Rossiter.
  The Journal of Physiology. October 27, 2015
  To better understand muscle bioenergetic regulation, a previously‐developed model of the skeletal muscle cell bioenergetic system was used to simulate the influence of: 1) each step activation (ESA) of NADH supply (including glycolysis) and oxidative phosphorylation (OXPHOS) complexes; and 2) glycolytic inhibition by protons, on the kinetics of ATP synthesis from OXPHOS, anaerobic glycolysis and creatine kinase (CK). Simulations were fitted to previously published experimental data of ATP production fluxes and metabolite concentrations during moderate and severe intensity exercise transitions in bilateral knee‐extension in humans. Overall, computer simulations agreed well with experimental results. Specifically, a large (>5‐fold) direct activation of all OXPHOS complexes was required to simulate measured phosphocreatine (PCr) and OXPHOS responses to both moderate and severe intensity exercise. In addition, slow decay of ESA was required to fit PCr recovery kinetics, and the time constant of ESA decay was slower following severe (180 s) than moderate (90 s) exercise. Additionally, a strong inhibition of (anaerobic) glycolysis by protons (glycolytic rate inversely proportional to the cube of proton concentration) provided the best fit to the experimental pH kinetics, and may contribute to the progressive increase in oxidative ATP supply during acidifying contractions. During severe‐intensity exercise an ‘additional’ ATP usage (a 27% increase at 8 min, above the initial ATP supply) was necessary to explain the observed VO2 slow component. Thus parallel activation of ATP usage and ATP supply (ESA), and a strong inhibition of ATP supply by anaerobic glycolysis, were necessary to simulate the kinetics of muscle bioenergetics observed in humans. This article is protected by copyright. All rights reserved

  October 27, 2015   doi: 10.1113/JP271299   open full text
• Fundamental role for the KCNE4 ancillary subunit in Kv7.4 regulation of arterial tone.
  Thomas A. Jepps, Georgina Carr, Pia R. Lundegaard, Søren‐Peter Olesen, Iain A. Greenwood.
  The Journal of Physiology. October 27, 2015
  Key points KCNE4 alters the biophysical properties and cellular localisation of Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co‐localises with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarised membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle. Abstract The KCNE ancillary subunits (KCNE1‐5) significantly alter Kv7 channel expression and function; however, their role in the vasculature has yet to be determined. The aim of this study was to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co‐expression of KCNE4 increased membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. QPCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed KCNE4 co‐localised with Kv7.4 in mesenteric artery myocytes. Morpholino‐induced knockdown of KCNE4 depolarised mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine (EC50 decreased from 5.7 ± 0.63 μm to 1.6 ± 0.23 μm), which coincided with the effects of Kv7 modulators, being attenuated. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarised membrane potential and an augmented response to vasoconstrictors. This study is the first to ascertain an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle. This article is protected by copyright. All rights reserved

  October 27, 2015   doi: 10.1113/JP271286   open full text
• Corticospinal axons make direct synaptic connections with spinal motoneurons innervating forearm muscles early during postnatal development in the rat.
  Hitoshi Maeda, Satoshi Fukuda, Hiroshi Kameda, Naoyuki Murabe, Noriko Isoo, Hiroaki Mizukami, Keiya Ozawa, Masaki Sakurai.
  The Journal of Physiology. October 27, 2015
  Recent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7–9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labeled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno‐associated virus vector encoding channelrhodopsin‐2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB‐labeled MNs and CS axons labeled through introduction of fluorescent protein‐conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS‐MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates. This article is protected by copyright. All rights reserved

  October 27, 2015   doi: 10.1113/JP270885   open full text
• Aging‐related changes in GABAergic inhibition in mouse auditory cortex, measured using in vitro flavoprotein autofluorescence imaging.
  K.A. Stebbings, H.W. Choi, A. Ravindra, D.M. Caspary, J.G. Turner, D.A. Llano.
  The Journal of Physiology. October 27, 2015
  To examine aging‐related changes in the earliest stages of auditory cortical processing, population auditory cortical responses to thalamic afferent stimulation were studied in brain slices obtained from young and aged CBA/CAj mice (up to 28 months of age). Cortical responses were measured using flavoprotein autofluorescence imaging, and aging‐related changes in inhibition were assessed by measuring the sensitivity of these responses to blockade of GABAA receptors using bath‐applied SR95531. The maximum auditory cortical response to afferent stimulation was not different between young and aged animals under control conditions, but responses to afferent stimulation in aged animals showed a significantly lower sensitivity to GABA blockade with SR95531. Cortical thickness, but not hearing loss, improved the prediction of all imaging variables when combined with age, particularly sensitivity to GABA blockade for the maximum response. To determine if the observed differences between slices from young and aged animals were due to differences in slice health, the redox state in the auditory cortex was assessed by measuring the FAD+/NADH ratio using fluorescence imaging. We found that this ratio is highly sensitive to known redox stressors such as H2O2 and NaCN; however, no difference was found between young and aged animals. By using a new approach to quantitatively assess pharmacological sensitivity of population‐level cortical responses to afferent stimulation, these data demonstrate that auditory cortical inhibition diminishes with aging. Further, these data establish a significant relationship between cortical thickness and GABAergic sensitivity, which had not previously been observed in an animal model of aging. This article is protected by copyright. All rights reserved

  October 27, 2015   doi: 10.1113/JP271221   open full text
• Placental phenotype and resource allocation to fetal growth are modified by the timing and degree of hypoxia during mouse pregnancy.
  J. S. Higgins, O. R. Vaughan, E. Liger, A. L. Fowden, A. N. Sferruzzi‐Perri.
  The Journal of Physiology. October 26, 2015
  Key points Hypoxia is a major cause of fetal growth restriction, particularly at high altitude, although little is known about its effects on placental phenotype and resource allocation to fetal growth. In the present study, maternal hypoxia induced morphological and functional changes in the mouse placenta, which depended on the timing and severity of hypoxia, as well as the degree of maternal hypophagia. Hypoxia at 13% inspired oxygen induced beneficial changes in placental morphology, nutrient transport and metabolic signalling pathways associated with little or no change in fetal growth, irrespective of gestational age. Hypoxia at 10% inspired oxygen adversely affected placental phenotype and resulted in severe fetal growth restriction, which was due partly to maternal hypophagia. There is a threshold between 13% and 10% inspired oxygen, corresponding to altitudes of ∼3700 m and 5800 m, respectively, at which the mouse placenta no longer adapts to support fetal resource allocation. This has implications for high altitude human pregnancies. Abstract The placenta adapts its transport capacity to nutritional cues developmentally, although relatively little is known about placental transport phenotype in response to hypoxia, a major cause of fetal growth restriction. The present study determined the effects of both moderate hypoxia (13% inspired O2) between days (D)11 and D16 or D14 and D19 of pregnancy and severe hypoxia (10% inspired O2) from D14 to D19 on placental morphology, transport capacity and fetal growth on D16 and D19 (term∼D20.5), relative to normoxic mice in 21% O2. Placental morphology adapted beneficially to 13% O2; fetal capillary volume increased at both ages, exchange area increased at D16 and exchange barrier thickness reduced at D19. Exposure to 13% O2 had no effect on placental nutrient transport on D16 but increased placental uptake and clearance of 3H‐methyl‐d‐glucose at D19. By contrast, 10% O2 impaired fetal vascularity, increased barrier thickness and reduced placental 14C‐methylaminoisobutyric acid clearance at D19. Consequently, fetal growth was only marginally affected in 13% O2 (unchanged at D16 and −5% at D19) but was severely restricted in 10% O2 (−21% at D19). The hypoxia‐induced changes in placental phenotype were accompanied by altered placental insulin‐like growth factor (IGF)‐2 expression and insulin/IGF signalling, as well as by maternal hypophagia depending on the timing and severity of the hypoxia. Overall, the present study shows that the mouse placenta can integrate signals of oxygen and nutrient availability, possibly through the insulin‐IGF pathway, to adapt its phenotype and optimize maternal resource allocation to fetal growth during late pregnancy. It also suggests that there is a threshold between 13% and 10% inspired O2 at which these adaptations no longer occur.

  October 26, 2015   doi: 10.1113/JP271057   open full text
• The role of cyclooxygenase‐1 in high salt diet‐induced microvascular dysfunction in humans.
  Ana Cavka, Anita Cosic, Ivana Jukic, Bojan Jelakovic, Julian H. Lombard, Shane A. Phillips, Vatroslav Seric, Ivan Mihaljevic, Ines Drenjancevic.
  The Journal of Physiology. October 24, 2015
  Objectives To assess the effect of 1‐week high salt (HS) diet on the role of cyclooxygenases (COX‐1,‐2) and vasoconstrictor prostaglandins ‐ thromboxane A2 (TXA2) and prostaglandin F2α (PGF2α) on skin microcirculatory blood flow and to detect its effect on markers of endothelial activation such a soluble cell adhesion molecules (sCAMs). Methods Young women (N = 54) were assigned to either HS diet group (N = 30) (∼14 g of NaCl/day) or low salt (LS) diet group (N = 24) (<2.3 g NaCl/day) for 7 days. Post‐occlusive reactive hyperemia (PORH) in the skin microcirculation was assessed by laser Doppler flowmetry (LDF). Plasma Renin Activity (PRA), plasma aldosterone, plasma and 24 h‐urine sodium and potassium, plasma concentrations of TXB2 (stable TXA2 metabolite) and PGF2α, sCAMs and blood pressure (BP) were measured before and after diet protocols. One HS diet group subset received 100 mg of indomethacin (non‐selective COX‐1,2 inhibitor), and another HS group subset received 200 mg of celecoxib (selective COX‐2 inhibitor) before repeating LDF measurements. Results BP was unchanged after HS diet, but significantly reduced after LS diet. 24 h‐urinary sodium was increased, PRA and plasma aldosterone levels decreased after HS diet. HS diet significantly impaired PORH and increased TXA2, but did not change PGF2α levels. Indomethacin restored microcirculatory blood flow, reduced TXA2. In contrast, celecoxib decreased TXA2 levels, but had no significant effects on blood flow. Conclusions Restoration of of PORH by indomethacin during high salt diet suggest an important role of COX‐1 derived vasoconstrictor metabolites in regulation of microvascular blood flow during high salt intake. This article is protected by copyright. All rights reserved

  October 24, 2015   doi: 10.1113/JP271631   open full text
• Left ventricular AV‐plane displacement is preserved with lifelong endurance training and is the main determinant of maximal cardiac output.
  Katarina Steding‐Ehrenborg, Robert C. Boushel, José A. Calbet, Per Åkeson, Stefan P. Mortensen.
  The Journal of Physiology. October 23, 2015
  Background: Age‐related decline in cardiac function can be prevented or postponed by lifelong endurance training. However, effects of normal ageing as well as of lifelong endurance exercise on longitudinal and radial contribution to stroke volume are unknown. The aim of this study was to determine resting longitudinal and radial pumping in elderly athletes, sedentary elderly and young sedentary subjects. Furthermore, we aimed to investigate determinants of maximal cardiac output in elderly. Methods: 8 elderly athletes (63 ± 4 years), 7 elderly sedentary (66 ± 4 years) and 10 young sedentary subjects (29 ± 4 years) underwent cardiac MR. All subjects underwent maximal exercise testing and for elderly subjects maximal cardiac output during cycling was determined using dye dilution technique. Results: Longitudinal and radial contribution to stroke volume did not differ between groups (longitudinal left ventricle (LV) 52–65%, P = 0.12, right ventricle (RV) 77–87%, P = 0.16, radial 7.9‐8.6%, P = 1.0). Left ventricular atrioventricular plane displacement (AVPD) was higher in elderly athletes and young sedentary compared to elderly sedentary (14 ± 3 mm, 15 ± 2 mm, and 11 ± 1 mm, respectively P < 0.05). There was no difference between groups for RVAVPD (P = 0.2). LVAVPD was an independent predictor of maximal cardiac output (R2 = 0.61, P < 0.01, β = 0.78). Conclusion: Longitudinal and radial contribution to stroke volume did not differ between groups. However, how longitudinal pumping was achieved differed, where elderly athletes and young sedentary showed similar AVPD whilst it was significantly lower in elderly sedentary. Instead elderly sedentary achieved longitudinal pumping through increased short‐axis area of the ventricle. Large AVPD was a determinant of maximal cardiac output and exercise capacity. This article is protected by copyright. All rights reserved

  October 23, 2015   doi: 10.1113/JP271621   open full text
• Ketamine suppresses hypoxia‐induced inflammatory responses in the late‐gestation ovine fetal kidney cortex.
  Eileen I. Chang, Miguel A. Zárate, Maria B. Rabaglino, Elaine M. Richards, Maureen Keller‐Wood, Charles E. Wood.
  The Journal of Physiology. October 23, 2015
  The fetus responds to decreases in arterial partial pressure of oxygen by redirecting the blood flow mainly to the brain and the heart, at a cost to other peripheral organs like the kidneys. Renal hypoxia and ischemia stimulate inflammatory and apoptotic responses. Ketamine, an NMDA receptor antagonist, is able to reduce renal immune and inflammatory gene expressions stimulated by hypoxia. Ketamine may have therapeutic potential for protection against ischemic renal damage in fetuses subjected to acute hypoxia. Abstract Acute fetal hypoxia is a form of fetal stress that stimulates renal vasoconstriction and ischemia as a consequence of the physiological redistribution of combined ventricular output. Because of the potential ischemia/reperfusion injury to the kidney, we hypothesized that it would respond to hypoxia with an increase in the expression of inflammatory genes, and that ketamine (an N‐Methyl‐D‐aspartate receptor antagonist) would reduce or block this response. Hypoxia was induced for 30 min in chronically catheterized fetal sheep (125 ± 3 d), with or without ketamine (3 mg kg‐1) administered intravenously to the fetus 10 min prior to hypoxia. Gene expression in fetal kidney cortex collected 24 hr after the onset of hypoxia was analyzed using ovine Agilent 15.5 k array and validated with qPCR and immunohistochemistry in four groups of ewes: normoxic control, normoxia+ketamine, hypoxic control and hypoxia+ketamine (n = 3‐4/group). Significant differences in gene expression between groups were determined with t‐statistics using the limma package for R (P ≤ 0.05). Enriched biological processes for the 427 upregulated genes were immune and inflammatory responses and for the 946 downregulated genes were metabolic processes. Ketamine countered the effects of hypoxia on upregulated immune/inflammatory responses as well as the downregulated metabolic responses. We conclude that our transcriptomics modeling predicts that hypoxia activates inflammatory pathways and reduces metabolism in the fetal kidney cortex, and ketamine blocks or ameliorates this response. The results suggest that ketamine may have therapeutic potential for protection from ischemic renal damage. This article is protected by copyright. All rights reserved

  October 23, 2015   doi: 10.1113/JP271066   open full text
• Inhibitory respiratory responses to progesterone and allopregnanolone in newborn rats chronically treated with caffeine.
  NagaPraveena Uppari, Vincent Joseph, Aida Bairam.
  The Journal of Physiology. October 23, 2015
  In premature newborn recurrent apnoea are systematically treated with caffeine to prevent long‐term neurocognitive disorders, but a substantial percentage of apnoea persists particularly in neonates born before 28 weeks of gestation Progesterone has been proposed as a respiratory stimulant potentially suitable for the treatment of newborn apnoea persistent to caffeine. Accordingly we asked whether acute progesterone administration reduces apnoea frequency in newborn rats treated with caffeine. Surprisingly our results show that in newborn rats treated with caffeine, administration of progesterone inhibits breathing and increases apnea frequency. Additional experiments showed an enhanced GABAergic inhibitory drive on breathing after caffeine treatment, and that progesterone is converted to allopregnanolone (an allosteric modulator of GABAA receptors) to inhibit breathing. We conclude that combining progesterone and chronic caffeine is not an option in preterm neonates, unless the effects of allopregnanolone could be counteracted. Abstract Caffeine is the main treatment for apnoea in preterm neonates, but its interactions with other respiratory stimulants like progesterone are unknown. We tested the hypothesis that the addition of progesterone to caffeine treatments further stimulates ventilation. Newborn rats were treated with water (control) or caffeine (15 mg kg‐1) by daily gavage between the postnatal (P) days 3–12. At P4 and P12, we measured apnoea frequency, ventilatory responses, and metabolic parameters under both normoxia and hypoxia (12% O2, 20 min) following an acute administration of either saline or progesterone (4 mg kg‐1; i.p.). Progesterone injection increased the serum levels of both progesterone and its neuroactive metabolite allopregnanolone. Progesterone had no effect on ventilation in control rats under normoxia. Progesterone depressed ventilation in P12 caffeine‐treated rats under normoxia and hypoxia and increased apnoea frequency in both P4 and P12 rats. Because allopregnanolone is an allosteric modulator of GABAA receptors and caffeine may enhance GABAergic inhibition in newborns, we studied the effects of the GABAA receptor antagonist bicuculline at 0, 1, 2, and 3 mg kg‐1 doses and allopregnanolone (10 mg kg‐1 dose) in P12 rats. In caffeine‐treated rats, bicuculline enhanced ventilation, while allopregnanolone decreased ventilation and increased total apnoea time. Progesterone had no effect on ventilation and apnoea frequency in caffeine‐treated rats injected with finasteride, which blocks the conversion of progesterone to allopregnanolone. We conclude that combining progesterone and chronic caffeine therapy is not an option for the treatment of persistent apnoea in preterm neonates, unless the effects of allopregnanolone could be counteracted. This article is protected by copyright. All rights reserved

  October 23, 2015   doi: 10.1113/JP270914   open full text
• Intrinsic muscle clock is necessary for musculoskeletal health.
  Elizabeth A. Schroder, Brianna D. Harfmann, Xiping Zhang, Ratchakrit Srikuea, Jonathan H. England, Brian A. Hodge, Yuan Wen, Lance A. Riley, Qi Yu, Alexander Christie, Jeffrey D Smith, Tanya Seward, Erin M. Wolf Horrell, Jyothi Mula, Charlotte A. Peterson, Timothy A. Butterfield, Karyn A. Esser.
  The Journal of Physiology. October 21, 2015
  Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in aging and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription‐translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced aging and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fiber levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle‐specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle is associated with a muscle phenotype including reductions in specific tension, increased oxidative fiber type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1−/− mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifies a novel crosstalk between skeletal muscle, cartilage and bone. This article is protected by copyright. All rights reserved

  October 21, 2015   doi: 10.1113/JP271436   open full text
• Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B.
  Silvia Cruz‐Rangel, José J. Jesús‐Pérez, Juan A. Contreras‐Vite, Patricia Pérez‐Cornejo, H. Criss Hartzell, Jorge Arreola.
  The Journal of Physiology. October 21, 2015
  TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium‐activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (Vm), intracellular calcium concentrations ([Ca2+]i) and external permeant anions. As a consequence, the chloride current (ICl) kinetics is complex. For example, TMEM16A ICl activates slowly with a non‐mono‐exponential time course while TMEM16B ICl activates rapidly following a mono‐exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded ICl from HEK‐293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct Vm‐dependent gating modes were uncovered; a fast‐mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising ICl was recorded in whole‐cell and inside‐out patches. The amplitude of ICl at the end of the long pulse nearly doubled and was blocked by 100 μm tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl−]o from 140 to 30 mm and by altering the sequence of the first intracellular loop. Mutating 480RSQ482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 449AVK451 to RSQ in TMEM16A has little effect. Deleting 448EAVK451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic Vm‐ and Cl−‐sensitive dual gating that elicit complex ICl kinetics. This article is protected by copyright. All rights reserved

  October 21, 2015   doi: 10.1113/JP271256   open full text
• Essential role of carbonic anhydrase XII in secretory glands fluid and HCO3−Secretion revealed by disease causing human mutation.
  Jeong Hee Hong, Emad Muhammad, Changyu Zheng, Eli Hershkovitz, Soliman Alkrinawi, Neta Loewenthal, Ruti Parvari, Shmuel Muallem.
  The Journal of Physiology. October 21, 2015
  Aberrant epithelial fluid and HCO3− secretion is associated with many diseases. The activity of HCO3− transporters depends of HCO3− availability that is determined by carbonic anhydrases (CAs). Which CAs is essential for epithelial function is unknown. CA12 stands out since the CA12(E143K) mutation causes salt wasting in sweat and dehydration in humans. Here, we report that expression of CA12 and of CA12(E143K) in mice salivary glands increased and prominently inhibited, ductal fluid secretion and salivation in vivo. CA12 markedly increases the activity and is the major HCO3− supplier of ductal Cl−/HCO3− exchanger AE2, but not of NBCe1‐B. The E143K mutation alters CA12 glycosylation at N28 and N80, resulting in retention of the basolateral CA12 in the ER. Knockdown of AE2 and of CA12 inhibited pancreatic and salivary glands ductal AE2 activity and fluid secretion. Accordingly, patients homozygous for the CA12(E143K) mutation have dry mouth, dry tongue phenotype. These findings reveals an unsuspected prominent role of CA12 in epithelial function, explains the disease and calls for caution in the use of CA12 inhibitors in cancer treatment. This article is protected by copyright. All rights reserved

  October 21, 2015   doi: 10.1113/JP271378   open full text
• Stimulation‐induced Ca2+ influx at nodes of Ranvier in mouse peripheral motor axons.
  Zhongsheng Zhang, Gavriel David.
  The Journal of Physiology. October 20, 2015
  Key points In peripheral myelinated axons of mammalian spinal motor neurons, Ca2+ influx was thought to occur only in pathological conditions such as ischaemia. Using Ca2+ imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [Ca2+] around nodes of Ranvier. These stimulation‐induced [Ca2+] elevations require Ca2+ influx, and are partially reduced by blocking T‐type Ca2+ channels (e.g. mibefradil) and by blocking the Na+/Ca2+ exchanger (NCX), suggesting an important contribution of Ca2+ influx via reverse‐mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation‐induced [Ca2+] elevations around nodes by allowing activation of sub‐myelin L‐type (nimodipine‐sensitive) Ca2+ channels. The Ca2+ that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca2+ influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Abstract Activity‐dependent Ca2+ signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra‐axonally injected fluorescent [Ca2+] indicator revealed that during repetitive action potential stimulation, [Ca2+] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca2+] elevations (∼0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca2+ from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T‐type Ca2+ channels and reverse mode Na+/Ca2+ exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation‐induced [Ca2+] elevations, which now included a prominent contribution from L‐type Ca2+ channels. These results suggest that the peri‐nodal axolemma of motor axons includes multiple pathways for stimulation‐induced Ca2+ influx, some active in normally‐myelinated axons (T‐type channels, NCX), others active only when exposed by myelin disruption (L‐type channels). The modest axoplasmic peri‐nodal [Ca2+] elevations measured in intact motor axons might mediate local responses to axonal activation. The larger [Ca2+] elevations measured after myelin disruption might, over time, contribute to the axonal degeneration observed in peripheral demyelinating neuropathies.

  October 20, 2015   doi: 10.1113/JP271207   open full text
• Cystine accumulation attenuates insulin release from the pancreatic beta‐cell due to elevated oxidative stress and decreased ATP levels.
  Bernadette McEvoy, Rodolfo Sumayao, Craig Slattery, Tara McMorrow, Philip Newsholme.
  The Journal of Physiology. October 20, 2015
  The pancreatic beta‐cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end‐stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi‐organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN‐BD11 rat clonal pancreatic beta‐cell line using Ctns‐targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient stimulated insulin secretions were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox‐sensitive transcription factor NF‐κB were increased in cells with silenced CTNS. From this study, the cystinotic‐like pancreatic beta‐cell model demonstrated that the altered oxidative status of the cell, resulting in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient‐stimulated insulin secretion. This article is protected by copyright. All rights reserved

  October 20, 2015   doi: 10.1113/JP271237   open full text
• Voltage‐gated Ca2+ influx through L‐type channels contributes to sarcoplasmic reticulum Ca2+ loading in skeletal muscle.
  Gaëlle Robin, Bruno Allard.
  The Journal of Physiology. October 18, 2015
  Key points Muscle contraction is triggered by Ca2+ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also known to be associated with a voltage‐activated trans‐sarcolemmal Ca2+ influx. Because removal of external Ca2+ does not impede fibres from contracting, a negligible role has been initially attributed to this Ca2+ entry. Furthermore, it is not clearly established whether Ca2+ exclusively flows through L‐type channels. By monitoring the quenching of fura‐2 fluorescence resulting from Mn2+ influx in voltage‐controlled mouse and zebrafish muscle fibres, we show that the L‐type current is the only contributor to Ca2+ influx during long‐lasting depolarizations. Calibration of the Mn2+ quenching signal allowed us to estimate an Mn2+ current of 0.31 A F–1 flowing during a train of action potentials. Measurements of SR Ca2+ changes with fluo‐5N in response to depolarization indicated that voltage‐activated Ca2+/Mn2+ influx contributes to SR Ca2+/Mn2+ loading. Abstract Muscle contraction is triggered by Ca2+ ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also associated with a voltage‐activated trans‐sarcolemmal Ca2+ influx early identified as a current flowing through L‐type Ca2+ channels. Because removal of external Ca2+ does not impede fibres from contracting, a negligible role was given to this voltage‐activated Ca2+ entry, although the decline of Ca2+ release is more pronounced in the absence of Ca2+ during long‐lasting activation. Furthermore, it is not clearly established whether Ca2+ exclusively flows through L‐type channels or in addition through a parallel voltage‐activated pathway distinct from L‐type channels. Here, by monitoring the quenching of fura‐2 fluorescence resulting from Mn2+ influx in voltage‐controlled mouse and zebrafish isolated muscle fibres, we show that the L‐type current is the only contributor to Ca2+ influx during long‐lasting depolarizations in skeletal muscle. Calibration of the Mn2+ quenching signal allowed us to estimate a mean Mn2+ current of 0.31 ± 0.06 A F–1 flowing through L‐type channels during a train of action potentials. Measurements of SR Ca2+ changes with fluo‐5N in response to depolarization revealed that an elevated voltage‐activated Ca2+ current potentiated SR Ca2+ loading and addition of external Mn2+ produced quenching of fluo‐5N in the SR, indicating that voltage‐activated Ca2+/Mn2+ influx contributes to SR Ca2+/Mn2+ loading.

  October 18, 2015   doi: 10.1113/JP270252   open full text
• Sleep spindles and human cortical nociception: a surface and intracerebral electrophysiological study.
  Léa Claude, Florian Chouchou, Germán Prados, Maïté Castro, Barbara Blay, Caroline Perchet, Luis García‐Larrea, Stéphanie Mazza, Hélène Bastuji.
  The Journal of Physiology. October 18, 2015
  Key points Sleep spindle are usually considered to play a major role in inhibiting sensory inputs. Using nociceptive stimuli in humans, we tested the effect of spindles on behavioural, autonomic and cortical responses in two experiments using surface and intracerebral electroencephalographic recordings. We found that sleep spindles do not prevent arousal reactions to nociceptive stimuli and that autonomic reactivity to nociceptive inputs is not modulated by spindle activity. Moreover, neither the surface sensory, nor the insular evoked responses were modulated by the spindle, as detected at the surface or within the thalamus. The present study comprises the first investigation of the effect of spindles on nociceptive information processing and the results obtained challenge the classical inhibitory effect of spindles. Abstract Responsiveness to environmental stimuli declines during sleep, and sleep spindles are often considered to play a major role in inhibiting sensory inputs. In the present study, we tested the effect of spindles on behavioural, autonomic and cortical responses to pain, in two experiments assessing surface and intracerebral responses to thermo‐nociceptive laser stimuli during the all‐night N2 sleep stage. The percentage of arousals remained unchanged as a result of the presence of spindles. Neither cortical nociceptive responses, nor autonomic cardiovascular reactivity were depressed when elicited within a spindle. These results could be replicated in human intracerebral recordings, where sleep spindle activity in the posterior thalamus failed to depress the thalamocortical nociceptive transmission, as measured by sensory responses within the posterior insula. Hence, the assumed inhibitory effect of spindles on sensory inputs may not apply to the nociceptive system, possibly as a result of the specificity of spinothalamic pathways and the crucial role of nociceptive information for homeostasis. Intriguingly, a late scalp response commonly considered to reflect high‐order stimulus processing (the ‘P3’ potential) was significantly enhanced during spindling, suggesting a possible spindle‐driven facilitation, rather than attenuation, of cortical nociception.

  October 18, 2015   doi: 10.1113/JP270941   open full text
• Retinol dehydrogenase 8 and ATP‐binding cassette transporter 4 modulate dark adaptation of M‐cones in mammalian retina.
  Alexander V. Kolesnikov, Akiko Maeda, Peter H. Tang, Yoshikazu Imanishi, Krzysztof Palczewski, Vladimir J. Kefalov.
  The Journal of Physiology. October 18, 2015
  Key points This study explores the molecular mechanisms that regulate the recycling of chromophore required for pigment regeneration in mammalian cones. We report that two chromophore binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor‐specific ATP‐binding cassette transporter (ABCA4) accelerate the dark adaptation of cones, first, directly, by facilitating the processing of chromophore in cones, and second, indirectly, by accelerating the turnover of chromophore in rods, which is then recycled and delivered to both rods and cones. Preventing competition with the rods by knocking out rhodopsin accelerated cone dark adaptation, demonstrating the interplay between rod and cone pigment regeneration driven by the retinal pigment epithelium (RPE). This novel interdependence of rod and cone pigment regeneration should be considered when developing therapies targeting the recycling of chromophore for rods, and evaluating residual cone function should be a critical test for such regimens targeting the RPE. Abstract Rapid recycling of visual chromophore and regeneration of the visual pigment are critical for the continuous function of mammalian cone photoreceptors in daylight vision. However, the molecular mechanisms modulating the supply of visual chromophore to cones have remained unclear. Here we explored the roles of two chromophore‐binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor‐specific ATP‐binding cassette transporter 4 (ABCA4), in dark adaptation of mammalian cones. We report that young adult RDH8/ABCA4‐deficient mice have normal M‐cone morphology but reduced visual acuity and photoresponse amplitudes. Notably, the deletion of RDH8 and ABCA4 suppressed the dark adaptation of M‐cones driven by both the intraretinal visual cycle and the retinal pigmented epithelium (RPE) visual cycle. This delay can be caused by two separate mechanisms: direct involvement of RDH8 and ABCA4 in cone chromophore processing, and an indirect effect from the delayed recycling of chromophore by the RPE due to its slow release from RDH8/ABCA4‐deficient rods. Intriguingly, our data suggest that RDH8 could also contribute to the oxidation of cis‐retinoids in cones, a key reaction of the retina visual cycle. Finally, we dissected the roles of rod photoreceptors and RPE for dark adaptation of M‐cones. We found that rods suppress, whereas RPE promotes, cone dark adaptation. Thus, therapeutic approaches targeting the RPE visual cycle could have adverse effects on the function of cones, making the evaluation of residual cone function a critical test for regimens targeting the RPE.

  October 18, 2015   doi: 10.1113/JP271285   open full text
• Contractile properties of developing human fetal cardiac muscle.
  Alice W. Racca, Jordan M. Klaiman, J. Manuel Pioner, Yuanhua Cheng, Anita E. Beck, Farid Moussavi‐Harami, Michael J. Bamshad, Michael Regnier.
  The Journal of Physiology. October 13, 2015
  Key points The contractile properties of human fetal cardiac muscle have not been previously studied. Small‐scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59 days to 134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell‐derived cardiomyocytes. Abstract Little is known about the contractile properties of human fetal cardiac muscle during development. Understanding these contractile properties, and how they change throughout development, can provide valuable insight into human heart development, and provide a framework to study the early stages of cardiac diseases that develop in utero. We characterized the contractile properties of isolated human fetal cardiac myofibrils across 8–19 weeks of gestation. Mechanical measurements revealed that in early stages of gestation there is low specific force and slow rates of force development and relaxation, with increases in force and the rates of activation and relaxation as gestation progresses. The duration and slope of the initial, slow phase of relaxation, related to myosin detachment and thin filament deactivation rates, decreased with gestation age. F‐actin sliding on human fetal cardiac myosin coated surfaces slowed significantly from 108 to 130 days gestation. Electron micrographs showed human fetal muscle myofibrils elongate and widen with age, but features such as the M‐line and Z‐band are apparent even as early as day 52. Protein isoform analysis revealed that β‐myosin is predominantly expressed even in the earliest time point studied, but there is a progressive increase in expression of cardiac troponin I (TnI), with a concomitant decrease in slow skeletal TnI. Together, our results suggest that cardiac myofibril force production and kinetics of activation and relaxation change significantly with gestation age and are influenced by the structural maturation of the sarcomere and changes in contractile filament protein isoforms. This article is protected by copyright. All rights reserved

  October 13, 2015   doi: 10.1113/JP271290   open full text
• Late gestational hypoxia and a postnatal high salt diet programs endothelial dysfunction and arterial stiffness in adult mouse offspring.
  Sarah L. Walton, Reetu R. Singh, Tiffany Tan, Tamara M. Paravicini, Karen M. Moritz.
  The Journal of Physiology. October 12, 2015
  Key points Maternal hypoxia is a common perturbation that leads to growth restriction and may program vascular dysfunction in adult offspring. An adverse prenatal environment may render offspring vulnerable to increased cardiovascular risk when challenged with a ‘second hit’, such as a high salt diet. We investigated whether maternal hypoxia impaired vascular function, structure and mechanics in mouse offspring, and also whether this was exacerbated by excess dietary salt intake in postnatal life. Maternal hypoxia predisposed adult male and female offspring to endothelial dysfunction. The combination of prenatal hypoxia and high dietary salt intake caused significant stiffening of mesenteric arteries, and also altered structural characteristics of the aorta consistent with vascular stiffening. The results of the present study suggest that prenatal hypoxia combined with a high salt diet in postnatal life can contribute to vascular dysfunction. Abstract Gestational hypoxia and high dietary salt intake have both been associated with impaired vascular function in adulthood. Using a mouse model of prenatal hypoxia, we examined whether a chronic high salt diet had an additive effect in promoting vascular dysfunction in offspring. Pregnant CD1 dams were placed in a hypoxic chamber (12% O2) or housed under normal conditions (21% O2) from embryonic day 14.5 until birth. Gestational hypoxia resulted in a reduced body weight for both male and female offspring at birth. This restriction in body weight persisted until weaning, after which the animals underwent catch‐up growth. At 10 weeks of age, a subset of offspring was placed on a high salt diet (5% NaCl). Pressurized myography of mesenteric resistance arteries at 12 months of age showed that both male and female offspring exposed to maternal hypoxia had significantly impaired endothelial function, as demonstrated by impaired vasodilatation to ACh but not sodium nitroprusside. Endothelial dysfunction caused by prenatal hypoxia was not exacerbated by postnatal consumption of a high salt diet. Prenatal hypoxia increased microvascular stiffness in male offspring. The combination of prenatal hypoxia and a postnatal high salt diet caused a leftward shift in the stress–strain relationship in both sexes. Histopathological analysis of aortic sections revealed a loss of elastin integrity and increased collagen, consistent with increased vascular stiffness. These results demonstrate that prenatal hypoxia programs endothelial dysfunction in both sexes. A chronic high salt diet in postnatal life had an additive deleterious effect on vascular mechanics and structural characteristics in both sexes.

  October 12, 2015   doi: 10.1113/JP271067   open full text
• High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons.
  Dominique Engel, Vincent Seutin.
  The Journal of Physiology. October 12, 2015
  Key points The hyperpolarization‐activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell‐attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon‐bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon‐lacking dendrites. Dual current‐clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD 7288 increased the temporal summation in all dendrites with a comparable effect in axon‐ and non‐axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine. Abstract Dendrites of most neurons express voltage‐gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization‐activated cation current Ih present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h‐channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of Ih can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating Ih current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell‐attached patch‐clamp recordings, we find a higher Ih current density in the axon‐bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. Ih is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of Ih in axon‐ and non‐axon‐bearing dendrites. The presence of Ih close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement.

  October 12, 2015   doi: 10.1113/JP271052   open full text
• Ca2+ current facilitation determines short‐term facilitation at inhibitory synapses between cerebellar Purkinje cells.
  Françoise Díaz‐Rojas, Takeshi Sakaba, Shin‐ya Kawaguchi.
  The Journal of Physiology. October 12, 2015
  Key points Short‐term facilitation takes place at GABAergic synapses between cerebellar Purkinje cells (PCs). By directly patch clamp recording from a PC axon terminal, we studied the mechanism of short‐term facilitation. We show that the Ca2+ currents elicited by high‐frequency action potentials were augmented in a [Ca2+]i‐dependent manner. The facilitation of synaptic transmission showed 4–5th power dependence on the Ca2+ current facilitation, and was abolished when the Ca2+ current amplitude was adjusted to be identical. Short‐term facilitation of Ca2+ currents predominantly mediates short‐term facilitation at synapses between PCs. Abstract Short‐term synaptic facilitation is critical for information processing of neuronal circuits. Several Ca2+‐dependent positive regulations of transmitter release have been suggested as candidate mechanisms underlying facilitation. However, the small sizes of presynaptic terminals have hindered the biophysical study of short‐term facilitation. In the present study, by directly recording from the axon terminal of a rat cerebellar Purkinje cell (PC) in culture, we demonstrate a crucial role of [Ca2+]i‐dependent facilitation of Ca2+ currents in short‐term facilitation at inhibitory PC–PC synapses. Voltage clamp recording was performed from a PC axon terminal visualized by enhanced green fluorescent protein, and the Ca2+ currents elicited by the voltage command consisting of action potential waveforms were recorded. The amplitude of presynaptic Ca2+ current was augmented upon high‐frequency paired‐pulse stimulation in a [Ca2+]i‐dependent manner, leading to paired‐pulse facilitation of Ca2+ currents. Paired recordings from a presynaptic PC axon terminal and a postsynaptic PC soma demonstrated that the paired‐pulse facilitation of inhibitory synaptic transmission between PCs showed 4–5th power dependence on that of Ca2+ currents, and was completely abolished when the Ca2+ current amplitude was adjusted to be identical. Thus, short‐term facilitation of Ca2+ currents predominantly mediates short‐term synaptic facilitation at synapses between PCs.

  October 12, 2015   doi: 10.1113/JP270704   open full text
• Recovery of visual functions in amblyopic animals following brief exposure to total darkness.
  Donald E. Mitchell, Kaitlyn MacNeil, Nathan A. Crowder, Katelyn Holman, Kevin R. Duffy.
  The Journal of Physiology. October 09, 2015
  A 10 day period of total darkness has been shown to either block or erase the severe effects on vision of a prior short period of monocular deprivation (MD) in kittens depending on whether darkness is contiguous or is delayed with respect to the period of MD. We have extended these earlier findings from kittens for which the period of MD began at one month and lasted for a week to more clinically relevant situations where MD began near birth and lasted for 6 weeks or more. Despite the far longer MD and the absence of prior binocular vision, all animals recovered normal visual acuity in the previously deprived eye. As before, when the period of darkness followed immediately after MD the vision of both eyes was initially very poor but thereafter the acuity of each eye increased gradually and equally to attain normal levels in about 7 weeks. By contrast, when darkness was introduced 8 weeks after MD, the visual acuity of the deprived eye recovered quickly to normal levels in just a week without any change in the vision of the fellow (non‐deprived) eye. Short (15 or 30 min) periods of illumination each day during an otherwise 10 day period of darkness obliterated all the benefits for vision, and a 5 day period of darkness was also completely ineffective. Measurements of depth perception indicated that despite possessing normal visual acuity in both eyes, only about a quarter showed evidence of having attained normal stereoscopic vision. This article is protected by copyright. All rights reserved

  October 09, 2015   doi: 10.1113/JP270981   open full text
• Differential short‐term regional effects of early high dose erythropoietin on white matter in preterm lambs after mechanical ventilation.
  Samantha K. Barton, Annie R. A. McDougall, Jacqueline M. Melville, Timothy J. M. Moss, Valerie A. Zahra, Tammy Lim, Kelly J. Crossley, Graeme R. Polglase, Mary Tolcos.
  The Journal of Physiology. October 08, 2015
  Key points Erythropoietin is a neuroprotectant undergoing clinical trial for brain injury in term and preterm infants. This is the first experimental study to assess the acute effects of erythropoietin on the cerebral white matter in preterm ventilated lambs. Administration of erythropoietin within minutes of injurious ventilation onset amplified pro‐inflammatory cytokine gene expression in both the periventricular and subcortical white matter compared to the ventilation alone group; erythropoietin had no effect on the area of microglial aggregations in white matter regions with a reduction in cellular density of aggregations in the subcortical white matter only. Administration of erythropoietin in conjunction with injurious ventilation increased gene expression of tight junction proteins and reduced protein extravasation from blood vessels in the cerebral white matter. Given the increase in inflammation after erythropoietin, we recommend further investigation into its use as a treatment for ventilated preterm babies prior to clinical translation. Abstract Inadvertently injurious ventilation of preterm neonates in the delivery room can cause cerebral white matter (WM) inflammation and injury. We investigated the impact of an early high dose of recombinant human erythropoietin (EPO) on ventilation‐induced WM changes in preterm lambs. Injurious ventilation, targeting a VT of 15 ml kg−1 with no positive end‐expiratory pressure, was initiated for 15 min in preterm lambs (0.85 gestation). Conventional ventilation was continued for a further 105 min. Lambs received either 5000 IU kg−1 of EPO (EPREX®; Vent+EPO; n = 6) or vehicle (Vent; n = 8) via an umbilical vein at 4 ± 2 min. Markers of WM injury and inflammation were assessed using quantitative real‐time PCR (qPCR) and immunohistochemistry and compared to a group of unventilated controls (UVC; n = 4). In Vent+EPO lambs compared to Vent lambs: (i) interleukin (IL)‐1β and IL‐6 mRNA levels in the periventricular WM and IL‐8 mRNA levels in the subcortical WM were higher (P < 0.05 for all); (ii) the density of microglia within the aggregations was not different in the periventricular WM and was lower in the subcortical WM (P = 0.001); (iii) the density of astrocytes was lower in the subcortical WM (P = 0.002); (iv) occludin and claudin‐1 mRNA levels were higher in the periventricular WM (P < 0.02 for all) and (vi) the number of blood vessels with protein extravasation was lower (P < 0.05). Recombinant human EPO had variable regional effects within the WM when administered during injurious ventilation. The adverse short‐term outcomes discourage the use of early high dose EPO administration in preterm ventilated babies.

  October 08, 2015   doi: 10.1113/JP271376   open full text
• Perivascular tissue inhibits rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and endothelium‐dependent H2S signalling in rat coronary arteries.
  Filip Aalbaek, Lisbeth Bonde, Sukhan Kim, Ebbe Boedtkjer.
  The Journal of Physiology. October 07, 2015
  Key points Local regulation of vascular resistance adjusts coronary blood flow to metabolic demand, although the mechanisms involved are not comprehensively understood We show that heart tissue surrounding rat coronary arteries releases diffusible factors that regulate vasoconstriction and relaxation Perivascular tissue reduces rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and constriction of coronary arteries to serotonin, the thromboxane analogue U46619 and the α1‐adrenergic agonist phenylephrine Endothelium‐dependent relaxation of coronary arteries in response to cholinergic stimulation is inhibited by perivascular tissue as a result of reduced endothelial Ca2+ responses and attenuated H2S‐dependent signalling These results establish cellular mechanisms by which perivascular heart tissue can modify local vascular tone and coronary blood flow Abstract Interactions between perivascular tissue (PVT) and the vascular wall modify artery tone and contribute to local blood flow regulation. Using isometric myography, fluorescence microscopy, membrane potential recordings and phosphospecific immunoblotting, we investigated the cellular mechanisms by which PVT affects constriction and relaxation of rat coronary septal arteries. PVT inhibited vasoconstriction to thromboxane, serotonin and α1‐adrenergic stimulation but not to depolarization with elevated extracellular [K+]. When PVT was wrapped around isolated arteries or placed at the bottom of the myograph chamber, a smaller yet significant inhibition of vasoconstriction was observed. Resting membrane potential, depolarization to serotonin or thromboxane stimulation, and resting and serotonin‐stimulated vascular smooth muscle [Ca2+]‐levels were unaffected by PVT. Serotonin‐induced vasoconstriction was almost abolished by rho‐kinase inhibitor Y‐27632 and modestly reduced by protein kinase C inhibitor bisindolylmaleimide X. PVT reduced phosphorylation of myosin phosphatase targeting subunit (MYPT) at Thr850 by ∼40% in serotonin‐stimulated arteries but had no effect on MYPT‐phosphorylation in arteries depolarized with elevated extracellular [K+]. The net anti‐contractile effect of PVT was accentuated after endothelial denudation. PVT also impaired vasorelaxation and endothelial Ca2+ responses to cholinergic stimulation. Methacholine‐induced vasorelaxation was mediated by NO and H2S, and particularly the H2S‐dependent (dl‐propargylglycine‐ and XE991‐sensitive) component was attenuated by PVT. Vasorelaxation to NO‐ and H2S‐donors was maintained in arteries with PVT. In conclusion, cardiomyocyte‐rich PVT surrounding coronary arteries releases diffusible factors that reduce rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and endothelial Ca2+ responses. These mechanisms inhibit agonist‐induced vasoconstriction and endothelium‐dependent vasorelaxation and suggest new signalling pathways for metabolic regulation of blood flow.

  October 07, 2015   doi: 10.1113/JP271006   open full text
• Learning from the other limb's experience: Sharing the “Trained” M1's representation of the motor sequence knowledge.
  Ella Gabitov, David Manor, Avi Karni.
  The Journal of Physiology. October 07, 2015
  Abstract Following unimanual training on a novel sequence of movements, sequence‐specific performance may improve overnight not only in the trained hand, but also in the hand afforded no actual physical experience. It is not clear, however, how transfer to the untrained hand is achieved. Here we examined whether and how interaction between the two primary motor cortices contributes to the performance of a sequence of movements, extensively trained a day before, by the untrained hand. To this end, we studied participants during the untrained‐hand performance of a finger‐to‐thumb opposition sequence (FOS), intensively trained a day earlier (T‐FOS), and a similarly constructed, but novel, untrained FOS (U‐FOS). Changes in neural signals driven by task performance were assessed using fMRI. To minimize potential differences due to the rate of sequence execution per se, participants performed both sequences at an identical paced rate. The analyses showed that the superior fluency in executing the T‐FOS, compared to the U‐FOS, was associated with higher activity within the primary motor cortex (M1), bilaterally, for the T‐FOS. The differential responses in the “trained” M1 were positively correlated with experience‐related differences in the functional connectivity between the “trained” M1 and (1) its left homolog, and (2) the left dorsal premotor cortex. However, no significant correlation was evident between the changes in connectivity in these two routes. These results suggest that the transfer of sequence‐specific information between the two primary motor cortices is predominantly mediated by excitatory mechanisms driven by the “trained” M1 through at least two independent neural pathways. This article is protected by copyright. All rights reserved

  October 07, 2015   doi: 10.1113/JP270184   open full text
• Ca2+ signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca2+ channel blockade.
  Oleksiy Gryshchenko, Julia V. Gerasimenko, Oleg V. Gerasimenko, Ole H. Petersen.
  The Journal of Physiology. October 07, 2015
  Abstract Normal pancreatic stellate cells (PSC) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca2+ signals. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The BK‐induced Ca2+ signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca2+ rise in PSCs was due to IP3 receptor ‐ mediated release from internal stores, whereas the sustained phase depended on external Ca2+ entry through Ca2+ release‐activated Ca2+ (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca2+ signal generation in PSCs and this may be helpful in treating acute pancreatitis. This article is protected by copyright. All rights reserved

  October 07, 2015   doi: 10.1113/JP271468   open full text
• Ischaemic concentrations of lactate increase TREK1 channel activity by interacting with a single histidine residue in the carboxy terminal domain.
  Swagata Ghatak, Aditi Banerjee, Sujit Kumar Sikdar.
  The Journal of Physiology. October 07, 2015
  Abstract Rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia, however the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15‐30 mM) at pH 7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell‐free inside‐out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates like pyruvate and acetate at pH 7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at 328th position (H328) in the carboxy terminal domain of TREK1 channel. The interaction of lactate with H328 is dependent on the charge on histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity. This article is protected by copyright. All rights reserved

  October 07, 2015   doi: 10.1113/JP270706   open full text
• Regulation of ventilatory sensitivity and carotid body proliferation in hypoxia by the PHD2/HIF‐2 pathway.
  Emma J. Hodson, Lynn G. Nicholls, Philip J. Turner, Ronan Llyr, James W. Fielding, Gillian Douglas, Indrika Ratnayaka, Peter A. Robbins, Christopher W. Pugh, Keith J. Buckler, Peter J. Ratcliffe, Tammie Bishop.
  The Journal of Physiology. October 06, 2015
  Key points Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation. Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs). Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia. To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α. Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α. Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation. These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology. Abstract Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia‐inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF‐1α and HIF‐2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen‐treated Phd2f/f;Rosa26+/CreERT2 mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min−1 g−1 in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF‐2α, but not HIF‐1α. Furthermore, inducible inactivation of HIF‐2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min−1 g−1 in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine‐positive cells mm−2 in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF‐2α enzyme–substrate couple in modulating ventilatory sensitivity to hypoxia.

  October 06, 2015   doi: 10.1113/JP271050   open full text
• Impact of sympathetic nervous system activity on post‐exercise flow‐mediated dilation in humans.
  Ceri L. Atkinson, Nia C.S. Lewis, Howard H. Carter, Dick H.J. Thijssen, Philip N. Ainslie, Daniel J. Green.
  The Journal of Physiology. October 06, 2015
  Abstract Transient reduction in vascular function following systemic large muscle group exercise has previously been reported in humans. The mechanisms responsible are currently unknown. We hypothesised that sympathetic nervous system activation, induced by cycle ergometer exercise, would contribute to post‐exercise reductions in flow‐mediated dilation (FMD). Ten healthy male subjects (28 ± 5years) undertook two 30 minute sessions of cycle exercise at 75% HRmax. Prior to exercise, individuals ingested either a placebo or an α1‐adrenoreceptor blocker (Prazosin; 0.05mg.kg−1). Central hemodynamics, brachial artery shear rate (SR) and blood flow profiles were assessed throughout each exercise bout and in response to brachial artery FMD, measured prior to‐, immediately after, and 60‐minutes post‐exercise. Cycle exercise increased both mean and antegrade SR (P < 0.001) with retrograde SR also elevated under both conditions (P < 0.001). Pre‐exercise FMD was similar on both occasions, and significantly reduced (27%) immediately following exercise in the placebo condition (t‐test, P = 0.03). In contrast, FMD increased (37%) immediately following exercise in the Prazosin condition (t‐test, P = 0.004, interaction effect P = 0.01). Post‐exercise FMD remained different between conditions after correction for baseline diameters preceding cuff deflation and also post‐deflation shear rate. No differences in FMD or other variables were evident 60‐minutes following recovery. Our results indicate that sympathetic vasoconstriction competes with endothelium‐dependent dilator activity to determine post‐exercise arterial function. These findings have implications for understanding the chronic impacts of interventions, such as exercise training, which affect both sympathetic activity and arterial shear stress. This article is protected by copyright. All rights reserved

  October 06, 2015   doi: 10.1113/JP270946   open full text
• Functional characterization of spikelet activity in the primary visual cortex.
  Benjamin Scholl, Sari Andoni, Nicholas J. Priebe.
  The Journal of Physiology. October 02, 2015
  Key points In vivo whole‐cell patch‐clamp recordings in cat visual cortex revealed small deflections in the membrane potential of neurons, termed spikelets. Spikelet statistics and functional properties suggest these deflections originate from a single, nearby cell. Spikelets shared a number sensory selectivities with the principal neuron including orientation selectivity, receptive field location and eye preference. Principal neurons and spikelets did not, however, generally share preferences for depth (binocular disparity). Cross‐correlation of spikelet activity and membrane potential revealed direct effects on the membrane potential of some principal neurons, suggesting that these cells were synaptically coupled or received common input from the cortical network. Other spikelet–neuron pairs revealed indirect effects, likely to be the result of correlated network events. Abstract Intracellular recordings in the neocortex reveal not only the membrane potential of neurons, but small unipolar or bipolar deflections that are termed spikelets. Spikelets have been proposed to originate from various sources, including active dendritic mechanisms, gap junctions and extracellular signals. Here we examined the functional characteristics of spikelets measured in neurons from cat primary visual cortex in vivo. Spiking statistics and our functional characterization of spikelet activity indicate that spikelets originate from a separate, nearby cell. Spikelet kinetics and lack of a direct effect on spikelet activity from hyperpolarizing current injection suggest they do not arise from electrical coupling to the principal neuron being recorded. Spikelets exhibited matched orientation tuning preference and ocular dominance to the principal neuron. In contrast, binocular disparity preferences of spikelets and the principal neuron were unrelated. Finally, we examined the impact of spikelets on the principal neuron's membrane potential; we did observe some records for which spikelets were correlated with the membrane potential of the principal neuron, suggesting that these neurons were synaptically coupled or received common input from the cortical network.

  October 02, 2015   doi: 10.1113/JP270876   open full text
• GABAB receptor‐mediated feed‐forward circuit dysfunction in the mouse model of fragile X syndrome.
  Sarah Wahlstrom‐Helgren, Vitaly A. Klyachko.
  The Journal of Physiology. October 02, 2015
  Key points Cortico‐hippocampal feed‐forward circuits formed by the temporoammonic (TA) pathway exhibit a marked increase in excitation/inhibition ratio and abnormal spike modulation functions in Fmr1 knock‐out (KO) mice. Inhibitory, but not excitatory, synapse dysfunction underlies cortico‐hippocampal feed‐forward circuit abnormalities in Fmr1 KO mice. GABA release is reduced in TA‐associated inhibitory synapses of Fmr1 KO mice in a GABAB receptor‐dependent manner. Inhibitory synapse and feed‐forward circuit defects are mediated predominately by presynaptic GABAB receptor signalling in the TA pathway of Fmr1 KO mice. GABAB receptor‐mediated inhibitory synapse defects are circuit‐specific and are not observed in the Schaffer collateral pathway‐associated inhibitory synapses in stratum radiatum. Abstract Circuit hyperexcitability has been implicated in neuropathology of fragile X syndrome, the most common inheritable cause of intellectual disability. Yet, how canonical unitary circuits are affected in this disorder remains poorly understood. Here, we examined this question in the context of the canonical feed‐forward inhibitory circuit formed by the temporoammonic (TA) branch of the perforant path, the major cortical input to the hippocampus. TA feed‐forward circuits exhibited a marked increase in excitation/inhibition ratio and major functional defects in spike modulation tasks in Fmr1 knock‐out (KO) mice, a fragile X mouse model. Changes in feed‐forward circuits were caused specifically by inhibitory, but not excitatory, synapse defects. TA‐associated inhibitory synapses exhibited increase in paired‐pulse ratio and in the coefficient of variation of IPSPs, consistent with decreased GABA release probability. TA‐associated inhibitory synaptic transmission in Fmr1 KO mice was also more sensitive to inhibition of GABAB receptors, suggesting an increase in presynaptic GABAB receptor (GABABR) signalling. Indeed, the differences in inhibitory synaptic transmission between Fmr1 KO and wild‐type (WT) mice were eliminated by a GABABR antagonist. Inhibition of GABABRs or selective activation of presynaptic GABABRs also abolished the differences in the TA feed‐forward circuit properties between Fmr1 KO and WT mice. These GABABR‐mediated defects were circuit‐specific and were not observed in the Schaffer collateral pathway‐associated inhibitory synapses. Our results suggest that the inhibitory synapse dysfunction in the cortico‐hippocampal pathway of Fmr1 KO mice causes hyperexcitability and feed‐forward circuit defects, which are mediated in part by a presynaptic GABABR‐dependent reduction in GABA release.

  October 02, 2015   doi: 10.1113/JP271190   open full text
• Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion.
  Dimitrios Kadas, Stefanie Ryglewski, Carsten Duch.
  The Journal of Physiology. October 02, 2015
  Key points We combine in situ electrophysiology with genetic manipulation in Drosophila larvae aiming to investigate the role of fast calcium‐activated potassium currents for motoneurone firing patterns during locomotion. We first demonstrate that slowpoke channels underlie fast calcium‐activated potassium currents in these motoneurones. By conducting recordings in semi‐intact animals that produce crawling‐like movements, we show that slowpoke channels are required specifically in motoneurones for maximum firing rates during locomotion. Such enhancement of maximum firing rates occurs because slowpoke channels prevent depolarization block by limiting the amplitude of motoneurone depolarization in response to synaptic drive. In addition, slowpoke channels mediate a fast afterhyperpolarization that ensures the efficient recovery of sodium channels from inactivation during high frequency firing. The results of the present study provide new insights into the mechanisms by which outward conductances facilitate neuronal excitability and also provide direct confirmation of the functional relevance of precisely regulated slowpoke channel properties in motor control. Abstract A large number of voltage‐gated ion channels, their interactions with accessory subunits, and their post‐transcriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behaviour. The present study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, probably by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability but is in accordance with reports on transient BK and Kv3 channel function in multiple types of vertebrate neurones. Therefore, our finding that ICF enhances firing rates specifically during bursting patterns relevant to behaviour is probably of relevance to all brains.

  October 02, 2015   doi: 10.1113/JP271323   open full text
• Beetroot juice supplementation reduces whole body oxygen consumption but does not improve indices of mitochondrial efficiency in human skeletal muscle.
  J. Whitfield, A. Ludzki, G.J.F. Heigenhauser, J.M.G. Senden, L.B. Verdijk, L.J.C. Loon, L.L. Spriet, G.P. Holloway.
  The Journal of Physiology. October 02, 2015
  Abstract Ingestion of sodium nitrate (NO3−) simultaneously reduces whole‐body oxygen consumption (VO2) during sub‐maximal exercise while improving mitochondrial efficiency, suggesting a causal link. Consumption of beetroot juice (BRJ) elicits similar decreases in VO2 but potential effects on the mitochondria remain unknown. Therefore we examined the effects of 7‐day supplementation with BRJ (280 ml d−1, ∼26 mmol NO3−) in young active males (n = 10) who had muscle biopsies taken before and after supplementation for assessments of mitochondrial bioenergetics. Subjects performed 20 min of cycling (10 min at 50% and 70% VO2peak) 48 h before pre (baseline) and post (day 5 of supplementation) biopsies. Whole‐body VO2 decreased (P < 0.05) by ∼3% at 70% VO2peak following supplementation. Mitochondrial respiration in permeabilized muscle fibres showed no change in leak respiration, the content of proteins associated with uncoupling (UCP3, ANT1, ANT2), maximal substrate‐supported respiration, or ADP sensitivity (apparent Km). In addition, isolated subsarcolemmal and intermyofibrillar mitochondria showed unaltered assessments of mitochondrial efficiency, including ADP consumed/oxygen consumed (P/O Ratio), respiratory control ratios (RCR) and membrane potential determined fluorometrically using Safranine‐O. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased following BRJ. Therefore, in contrast to sodium nitrate, BRJ supplementation does not alter key parameters of mitochondrial efficiency. This occurred despite a decrease in exercise VO2, suggesting that the ergogenic effects of BRJ ingestion are not due to a change in mitochondrial coupling or efficiency. It remains to be determined if increased mitochondrial H2O2 contributes to this response. This article is protected by copyright. All rights reserved

  October 02, 2015   doi: 10.1113/JP270844   open full text
• Influence of developmental nicotine exposure on the ventilatory and metabolic response to hyperthermia.
  Jonathan Ferng, Ralph F. Fregosi.
  The Journal of Physiology. October 02, 2015
  Abstract To determine whether developmental nicotine exposure (DNE) alters the ventilatory and metabolic response to hyperthermia in neonatal rats (postnatal age 2–4 days), pregnant dams were exposed to nicotine (6 mg kg−1 of nicotine tartrate daily) or saline with an osmotic mini‐pump implanted subdermally on the 5th day of gestation. Rat pups (a total of 72 controls and 72 DNE pups) were studied under thermoneutral conditions (chamber temperature 33°C) and during moderate thermal stress (37.5°C). In all pups, core temperature was similar to chamber temperature, with no treatment effects. The rates of pulmonary ventilation (VI), O2 consumption (VO2) and CO2 production (VCO2) did not change with hyperthermia in either control or DNE pups. However, VI was lower in DNE pups at both chamber temperatures, while the duration of spontaneous apnoeas was longer in DNE pups than in controls at 33°C. The VI/ VO2 ratio increased at 37.5°C in control pups, but did not change in DNE pups. To simulate severe thermal stress, additional pups were studied at 33°C and 43°C. VI increased with heating in control pups, but not DNE pups. As heat stress continued, gasping was evoked in both groups, with no effect of DNE on the gasping pattern. Over a 20‐minute recovery period at 33°C, VI returned to baseline in control pups, but remained depressed in DNE pups. In addition to altering baseline VI and apnoea duration, DNE is associated with subtle but significant alterations in the ventilatory response to hyperthermia in neonatal rats. This article is protected by copyright. All rights reserved

  October 02, 2015   doi: 10.1113/JP271374   open full text
• Genetic upregulation of BK channel activity normalizes multiple synaptic and circuit defects in a mouse model of fragile X syndrome.
  Pan‐Yue Deng, Vitaly A. Klyachko.
  The Journal of Physiology. October 02, 2015
  Abstract Loss of Fragile X Mental Retardation Protein (FMRP) causes Fragile X Syndrome (FXS), yet the mechanisms underlying the pathophysiology of FXS are incompletely understood. Recent studies identified important new functions of FMRP in regulating neural excitability and synaptic transmission via both translation‐dependent mechanisms and direct interactions of FMRP with a number of ion channels in the axons and presynaptic terminals. Among these presynaptic FMRP functions, FMRP interaction with BK channels, specifically their auxiliary β4 subunit, regulates action potential waveform and glutamate release in hippocampal and cortical pyramidal neurons. Given the multitude of ion channels and mechanisms that mediate presynaptic FMRP actions, it remains unclear, however, to what extent FMRP‐BK channel interactions contribute to synaptic and circuit defects in FXS. To examine this question, we generated Fmr1/β4 double knock‐out (dKO) mice to genetically upregulate BK channel activity in the absence of FMRP and determine its ability to normalize multilevel defects caused by FMRP loss. Single‐channel analyses revealed that FMRP loss reduced BK channel open probability, and this defect was compensated in dKO mice. Furthermore, dKO mice exhibited normalized action potential duration, glutamate release and short‐term dynamics during naturalistic stimulus trains in hippocampal pyramidal neurons. BK channel upregulation was also sufficient to correct excessive seizure susceptibility in an in vitro model of seizure activity in hippocampal slices. Our studies thus suggest that upregulation of BK channel activity normalizes multi‐level deficits caused by FMRP loss. This article is protected by copyright. All rights reserved

  October 02, 2015   doi: 10.1113/JP271031   open full text
• Hypoxic induction of T‐type Ca2+ channels in rat cardiac myocytes: role of HIF‐1α and RhoA/ROCK signalling.
  P. González‐Rodríguez, D. Falcón, M. J. Castro, J. Ureña, J. López‐Barneo, A. Castellano.
  The Journal of Physiology. October 01, 2015
  Key points T‐type Ca2+ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are downregulated as development progresses. However, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. Hypoxia induces the upregulation of the T‐type Ca2+ channel Cav3.2 mRNA in cardiac myocytes, whereas Cav3.1 mRNA is not significantly altered. The effect of hypoxia on Cav3.2 mRNA requires hypoxia inducible factor‐1α (HIF‐1α) stabilization and involves the small monomeric G‐protein RhoA and its effector ROCKI. Our results suggest that the hypoxic regulation of the Cav3.2 channels may be involved in the increased probability of developing arrhythmias observed in ischemic situations, and in the pathogenesis of diseases associated with hypoxic Ca2+ overload. Abstract T‐type Ca2+ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage‐activated T‐type Ca2+ channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Cav3.2 mRNA, whereas Cav3.1 mRNA is not significantly altered. The effect of hypoxia on Cav3.2 mRNA was time‐ and dose‐dependent, and required hypoxia inducible factor‐1α (HIF‐1α) stabilization. Patch‐clamp recordings confirmed that T‐type Ca2+ channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl2 (a T‐type channel blocker) demonstrated that the Cav3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Cav3.2 channel electrophysiological properties. The small monomeric G‐protein RhoA and its effector Rho‐associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Cav3.2 channel and the stabilization of HIF‐1α that occurred in response to hypoxia. These results suggest a possible role for Cav3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca2+ overload.

  October 01, 2015   doi: 10.1113/JP271053   open full text
• Cardiovascular function in term fetal sheep conceived, gestated and studied in the hypobaric hypoxia of the Andean altiplano.
  Emilio A. Herrera, Rodrigo T. Rojas, Bernardo J. Krause, Germán Ebensperger, Roberto V. Reyes, Dino A. Giussani, Julian T. Parer, Aníbal J. Llanos.
  The Journal of Physiology. October 01, 2015
  Key points High altitude developmental hypoxia causes intrauterine growth restriction and cardiovascular programming. However, some mammals exposed chronically to high‐altitude hypoxia have less growth restriction suggesting certain protection. Cardiovascular defence mechanisms during acute fetal hypoxia divert blood flow from the periphery towards the brain, heart and adrenals. In contrast, little is known about the cardiovascular defence mechanisms during chronic fetal hypoxia. Here, we established the cardiovascular responses in fetal sheep that were conceived, gestated, born and studied at 3600 m. The data suggest that chronically hypoxic pregnant ewes and their fetuses have evolved different mechanisms from sea level pregnancies to withstand chronic hypoxia. The cardiovascular responses to acute hypoxia are blunted in the chronically hypoxic fetus. These findings points towards compensatory mechanisms in the highland fetus at the level of the cells and molecules rather than mounting major cardiovascular responses, saving oxygen not easily available in the Alto Andino. Abstract High‐altitude hypoxia causes intrauterine growth restriction and cardiovascular programming. However, adult humans and animals that have evolved at altitude show certain protection against the effects of chronic hypoxia. Whether the highland fetus shows similar protection against high altitude gestation is unclear. We tested the hypothesis that high‐altitude fetal sheep have evolved cardiovascular compensatory mechanisms to withstand chronic hypoxia that are different from lowland sheep. We studied seven high‐altitude (HA; 3600 m) and eight low‐altitude (LA; 520 m) pregnant sheep at ∼90% gestation. Pregnant ewes and fetuses were instrumented for cardiovascular investigation. A three‐period experimental protocol was performed in vivo: 30 min of basal, 1 h of acute superimposed hypoxia (∼10% O2) and 30 min of recovery. Further, we determined ex vivo fetal cerebral and femoral arterial function. HA pregnancy led to chronic fetal hypoxia, growth restriction and altered cardiovascular function. During acute superimposed hypoxia, LA fetuses redistributed blood flow favouring the brain, heart and adrenals, whereas HA fetuses showed a blunted cardiovascular response. Importantly, HA fetuses have a marked reduction in umbilical blood flow versus LA. Isolated cerebral arteries from HA fetuses showed a higher contractile capacity but a diminished response to catecholamines. In contrast, femoral arteries from HA fetuses showed decreased contractile capacity and increased adrenergic contractility. The blunting of the cardiovascular responses to hypoxia in fetuses raised in the Alto Andino may indicate a change in control strategy triggered by chronic hypoxia, switching towards compensatory mechanisms that are more cost‐effective in terms of oxygen uptake.

  October 01, 2015   doi: 10.1113/JP271110   open full text
• Homeostatic regulation of h‐conductance controls intrinsic excitability and stabilizes the threshold for synaptic modification in CA1 neurons.
  Célia Gasselin, Yanis Inglebert, Dominique Debanne.
  The Journal of Physiology. October 01, 2015
  Key points We determined the contribution of the hyperpolarization‐activated cationic (h) current (Ih) to the homeostatic regulation of CA1 pyramidal cells in vitro using chronic treatments (48 h) that either increase (picrotoxin) or decrease (kynurenate) neuronal activity. The h‐conductance was found to be up‐ or down‐regulated following chronic activity enhancement or activity deprivation, respectively. This bidirectional plasticity of Ih was found to subsequently alter both apparent input resistance and intrinsic neuronal excitability. Bidirectional homeostatic plasticity of Ih also determined EPSP waveform and EPSP summation tested at 5–30 Hz. Long‐term synaptic modification induced by repetitive stimulation of the Schaffer collaterals was found to be constant across treatments in the presence of Ih but not when Ih was blocked pharmacologically. Thus, bidirectional homeostatic regulation of Ih stabilizes induction of long‐term synaptic modification in CA1 pyramidal neurons that depends on EPSP summation. Abstract The hyperpolarization‐activated cationic (h) current is a voltage‐shock absorber, highly expressed in the dendrites of CA1 pyramidal neurons. Up‐regulation of Ih has been reported following episodes of intense network activity but the effect of activity deprivation on Ih and the functional consequence of homeostatic regulation of Ih remain unclear. We determined here the contribution of Ih to the homeostatic regulation of CA1 pyramidal cell excitability. Intrinsic neuronal excitability was decreased in neurons treated for 2–3 days with the GABAA channel blocker picrotoxin (PiTx) but increased in neurons treated (2–3 days) with the glutamate receptor antagonist kynurenate (Kyn). Membrane capacitance remained unchanged after treatment but the apparent input resistance was reduced for PiTx‐treated neurons and enhanced for Kyn‐treated neurons. Maximal Ih conductance was up‐regulated after chronic hyperactivity but down‐regulated following chronic hypoactivity. Up‐regulation of Ih in PiTx‐treated cultures was found to accelerate EPSP kinetics and reduce temporal summation of EPSPs whereas opposite effects were observed in Kyn‐treated cultures, indicating that homeostatic regulation of Ih may control the induction of synaptic modification depending on EPSP summation. In fact, stimulation of the Schaffer collaterals at 3–10 Hz induced differential levels of plasticity in PiTx‐treated and Kyn‐treated neurons when Ih was blocked pharmacologically but not in control conditions. These data indicate that homeostatic regulation of Ih normalizes the threshold for long‐term synaptic modification that depends on EPSP summation. In conclusion, bidirectional homeostatic regulation of Ih not only controls spiking activity but also stabilizes the threshold for long‐term potentiation induced in CA1 pyramidal neurons by repetitive stimulation.

  October 01, 2015   doi: 10.1113/JP271369   open full text
• Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of guanine nucleotide binding in CaV1.2 regulation.
  Donald D. Chang, Henry M. Colecraft.
  The Journal of Physiology. October 01, 2015
  Abstract Rad and Rem are Ras‐like G‐proteins linked to diverse cardiovascular functions and patho‐physiology. Understanding how Rad and Rem are regulated is important for deepened insights into their patho‐physiological roles. Like other Ras‐like G‐proteins, Rad and Rem contain a conserved guanine‐nucleotide binding domain (G‐domain). Canonically, G‐domains are key control modules, functioning as nucleotide‐regulated switches of G‐protein activity. Whether Rad and Rem G‐domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (CaV1.2 currents, Ca2+ transients, CaVβ binding) as biosensors to probe the role of the G‐domain in regulation of Rad and Rem function. We utilized RadS105N and RemT94N which are the cognate mutants to RasS17N, a dominant‐negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over‐expression of either RadS105N or RemT94N strongly inhibited reconstituted CaV1.2 currents to the same extent as their wild‐type (wt) counterparts, contrasting with reports that RadS105N is functionally inert in HEK293 cells. Adenovirus‐mediated expression of either wt Rad or RadS105N in cardiomyocytes dramatically blocked ICa,L and inhibited Ca2+‐induced Ca2+ release, contradicting reports that RadS105N acts as a dominant negative in heart. By contrast, RemT94N was significantly less effective than wt Rem at inhibiting ICa,L and Ca2+ transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both RadS105N and RemT94N had moderately reduced binding affinity for CaVβs relative to their wt counterparts. The results indicate Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of their G‐domain in CaV1.2 regulation. This article is protected by copyright. All rights reserved

  October 01, 2015   doi: 10.1113/JP270889   open full text
• Potassium inhibits nitric oxide and adenosine arteriolar vasodilatation via KIR and Na+/K+ATPase: Implications for redundancy in active hyperaemia.
  Iain R. Lamb, Coral L. Murrant.
  The Journal of Physiology. October 01, 2015
  Abstract Redundancy, in active hyperaemia, where one vasodilator can compensate for another if the first is missing, would require that one vasodilator inhibits the effects of another, therefore if the first vasodilator is inhibited, its inhibitory influence on the second vasodilator is removed and the second vasodilator exerts a greater vasodilatory effect. We sought to test if vasodilators relevant to skeletal muscle contraction, (potassium (K+), adenosine (ADO) and nitric oxide (NO)) inhibit one another and, further, investigate the mechanisms for this interaction. We used the hamster cremaster muscle and intravital microscopy to directly visualize 2A arterioles when exposed to a range of concentrations of one vasodilator (10−8M‐10−5 M SNAP, 10−8M‐10−5 M ADO, 10 and 20 mm KCl) in the absence and then in the presence of a second vasodilator (10−7 M ADO, 10−7 M SNAP, 10 mm KCl). We found that KCl significantly attenuated SNAP‐induced vasodilatations by ∼65.8% and vasodilatations induced by 10−8M‐10−6 M ADO by ∼72.8%. Further, we observed that inhibition of KCl vasodilatation, by antagonizing either Na+/K+ATPase using ouabain or KIR channels using barium chloride, could restore the SNAP‐induced vasodilatation by up to ∼53.9% and 30.6% respectively, and restore the ADO‐induced vasodilatations by up to ∼107% and 76.7% respectively. Our data show that vasodilators relevant to muscle contraction can interact in a way that alters eachothers vasodilatory effectiveness. These data suggest that active hyperaemia may be the result of complex interactions between multiple vasodilators through a redundant control paradigm. This article is protected by copyright. All rights reserved

  October 01, 2015   doi: 10.1113/JP270613   open full text
• High‐frequency focal repetitive cerebellar stimulation induces prolonged increases in human pharyngeal motor cortex excitability.
  Dipesh H. Vasant, Emilia Michou, Satish Mistry, John C Rothwell, Shaheen Hamdy.
  The Journal of Physiology. September 30, 2015
  Key points Neurostimulation is a rapidly emerging approach to swallowing rehabilitation, but cerebellar stimulation has not been explored as a treatment. Such proposed therapies for post‐stroke dysphagia have required confirmation of physiological effects and optimisation of parameters in healthy humans prior to translational progression into patient groups. There is strong evidence for a role of the cerebellum in swallowing physiology, but this relationship has been under‐explored. Recently, single pulses of cerebellar magnetic stimulation have been shown to directly evoke responses from pharyngeal musculature and produce short‐term enhancement of cortico‐pharyngeal motor evoked potentials, suggesting the feasibility of a cerebellar approach to neurostimulation in the swallowing system. We therefore examined multiple parameters of repetitive cerebellar magnetic stimulation and have described the optimal settings to provoke longer‐lasting changes in swallowing neurophysiology. Based on evidence from the post‐stroke dysphagia neurostimulation literature, these changes may have a therapeutic potential for swallowing rehabilitation. Abstract Brain neurostimulation has been shown to modulate cortical swallowing neurophysiology in post‐stroke dysphagia with therapeutic effects which are critically dependent on the stimulation parameters. Cerebellar neurostimulation is, however, a novel, unexplored approach to modulation of swallowing pathways as a prelude to therapy for dysphagia. Here, we randomised healthy human subjects (n = 17) to receive one of five cerebellar repetitive TMS (rTMS) interventions (Sham, 1 Hz, 5 Hz, 10 Hz and 20 Hz) on separate visits to our laboratory. Additionally, a subset of subjects randomly received each of three different durations (50, 250, 500 pulses) of optimal frequency versus sham cerebellar rTMS. Prior to interventions subjects underwent MRI‐guided single‐pulse transcranial magnetic stimulation (TMS) to co‐localise pharyngeal and thenar representation in the cortex and cerebellum (midline and hemispheric) before acquisition of baseline motor evoked potential (MEP) recordings from each site as a measure of excitability. Post‐interventional MEPs were recorded for an hour and compared to sham using repeated measures ANOVA. Only 10 Hz cerebellar rTMS increased cortico‐pharyngeal MEP amplitudes (mean bilateral increase 52%, P = 0.007) with effects lasting 30 min post‐intervention with an optimal train length of 250 pulses (P = 0.019). These optimised parameters of cerebellar rTMS can produce sustained increases in corticobulbar excitability and may have clinical translation in future studies of neurogenic dysphagia.

  September 30, 2015   doi: 10.1113/JP270817   open full text
• Dual action of leptin on rest‐firing and stimulated catecholamine release via phosphoinositide 3‐kinase‐driven BK channel up‐regulation in mouse chromaffin cells.
  Daniela Gavello, David Vandael, Sara Gosso, Emilio Carbone, Valentina Carabelli.
  The Journal of Physiology. September 27, 2015
  Key points Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite‐suppressing effect and, as such, exerts a relevant action on the adipo‐adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3‐kinase signalling cascade. This inhibitory effect is absent in db–/db– mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well‐adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready‐releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo‐adrenal axis in the leptin‐mediated increase of sympathetic tone and catecholamine release. Abstract Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite‐suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1 nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1 μm), suggesting the involvement of BK potassium channels. Single‐channel recordings in ‘perforated microvesicles’ confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up‐regulation was associated with the phosphoinositide 3‐kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100 nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca2+‐driven exocytosis. Although leptin preserves well‐adapted AP trains of lower frequency, APs are broader and depolarization‐evoked exocytosis is increased as a result of the larger size of the ready‐releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db–/db– mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo‐adrenal axis in the leptin‐mediated increase of sympathetic tone and catecholamine release.

  September 27, 2015   doi: 10.1113/JP271078   open full text
• Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.
  Theanne N. Griffith, Geoffrey T. Swanson.
  The Journal of Physiology. September 20, 2015
  Key points Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin‐ and tolloid‐like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3–S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR‐specific modulators to alter receptor function. Abstract Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit‐ and Neto isoform‐specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3–S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3–S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand‐binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3–S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3–S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR‐specific modulators that alter the function of auxiliary proteins in native receptors.

  September 20, 2015   doi: 10.1113/JP271103   open full text
• Contrasting actions of a convulsant barbiturate and its anti‐convulsant enantiomer on the α1β3γ2L GABAA receptor account for their in vivo effects.
  Rooma Desai, Pavel Y. Savechenkov, Dorota Zolkowska, Ri Le Ge, Michael A. Rogawski, Karol S. Bruzik, Stuart A. Forman, Douglas E. Raines, Keith W. Miller.
  The Journal of Physiology. September 17, 2015
  Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABAARs by photolabeling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, mTFD‐MPPB (1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid). In mice, S–mTFD‐MPPB acted as a convulsant, whereas R–mTFD‐MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α1β3γ2L GABAARs expressed in HEK cells, we found that S–mTFD‐MPPB inhibited GABA–induced currents, whereas R–mTFD‐MPPB enhanced them. S–mTFD‐MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration‐response curve to the right. R–mTFD‐MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate‐limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration‐response curve to the left. Under conditions when most GABAARs were open, an inhibitory action of R–mTFD‐MPPB was revealed that had a similar IC50 to S–mTFD‐MPPB's. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S–mTFD‐MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate–binding sites on GABAARs underlie the enantiomers’ different pharmacological activities in mice. This article is protected by copyright. All rights reserved

  September 17, 2015   doi: 10.1113/JP270971   open full text
• Morphological and molecular changes in the murine placenta exposed to normobaric hypoxia throughout pregnancy.
  Hannah Matheson, Jan H. W. Veerbeek, D. Stephen Charnock‐Jones, Graham J. Burton, Hong Wa Yung.
  The Journal of Physiology. September 15, 2015
  Key points Exposure of pregnant mice to chronic hypoxia at 13% O2 induces fetal growth restriction but increases placental weight. Sex dimorphism induces differential responses in placental weight to hypoxia. The male placenta is heavier than the female and is associated with less severe fetal growth restriction. Increases in maternal arterial/venous blood spaces and higher protein kinase B (Akt)‐mechanistic target of rapamycin growth signalling could contribute to the heavier hypoxic placenta. Placental endoplasmic reticulum stress is elevated equally in both sexes in response to hypoxia. In comparison, oxidative stress is only apparent in female placentas. Chronic hypoxia induces down‐regulation of placental mitochondrial electron transport chain complexes protein subunits but does not cause intracellular energy depletion. Abstract Chronic hypoxia is a common complication of pregnancy, arising through malperfusion of the placenta or pregnancy at high altitude. The present study investigated the effects of hypoxia on the growth of the placenta, which is the organ that interfaces between the mother and her fetus. Mice were housed in an hypoxic environment for the whole of gestation. An atmosphere of 13% oxygen induced fetal growth restriction (1182 ± 9 mg, n = 90 vs. 1044 ± 11 mg, n = 62, P < 0.05) but enhanced placental weight (907 ± 11 mg, n = 90 vs. 998 ± 15 mg, n = 62,P < 0.05). Stereological analyses revealed an increase in the volume of maternal blood spaces in the placenta, consistent with increased flow. At the molecular level, we observed activation of the protein kinase B (Akt)‐mechanistic target of rapamycin growth and proliferation pathway. Chronic hypoxia also triggered mild endoplasmic reticulum stress, a conserved homeostatic response that mediates translational arrest through phosphorylation of eukaryotic initiation factor 2 subunit α. Surprisingly, although subunits of the mitochondrial electron transport chain complexes were reduced at the protein level, there was no evidence of intracellular energy depletion. Finally, we demonstrated sex‐specific placental responses to chronic hypoxia. Placentas from male fetuses were heavier (1082 ± 2 mg, n = 30 vs. 928 ± 2 mg, n = 34, P < 0.05) and less susceptible to hypoxia‐induced oxidative stress than those from females. Their capacity to adapt may explain why male fetuses were significantly less growth restricted at embryonic day 18.5 than their female counterparts. These findings are consistent with the concept that male fetuses are more aggressive with respect to their nutrient demands, which may place them at greater risk of adverse outcomes under limiting conditions.

  September 15, 2015   doi: 10.1113/JP271073   open full text
• Human skeletal muscle fibre contractile properties and proteomic profile: Adaptations to 3‐week unilateral lower limb suspension and active recovery.
  Lorenza Brocca, Emanuela Longa, Jessica Cannavino, Olivier Seynnes, Giuseppe Vito, Jamie McPhee, Marco Narici, Maria Antonietta Pellegrino, Roberto Bottinelli.
  The Journal of Physiology. September 15, 2015
  Abstract Following disuse muscle fibres function goes through adaptations such as loss of specific force (Po/CSA) and increase in unloaded shortening velocity (Vo), which could be due to both quantitative changes, i.e. atrophy, and qualitative changes in protein pattern. The underlying mechanisms have not been settled yet. In addition, little is known about the recovery of muscle mass and strength following disuse. Here we report an extensive data set describing in detail functional and protein content adaptations of skeletal muscle in response to both disuse and retraining. Eight young healthy subjects were subjected to 3 weeks unilateral lower limb suspension (ULLS), a widely used human model of disuse skeletal muscle atrophy. Needle biopsies samples were taken from the vastus lateralis muscle pre‐ULLS, post‐ULLS and after 3 weeks recovery during which heavy resistance training was performed (post‐RT). After disuse, cross sectional area (CSA), specific force (Po/CSA) and myosin concentration (MC) decreased both in type 1 and 2A skinned muscle fibres. After recovery, CSA and MC returned to levels comparable to those observed before disuse, whereas Po/CSA and unloading shortening velocity reached a higher level. Myosin heavy chain (MHC) isoform composition of muscle samples did not differ among experimental groups. To study the mechanisms underlying such adaptations, a 2Dproteomic analysis was performed. ULLS induced reduction of myofibrillar, metabolic (glycolytic and oxidative) and antioxidant defense system proteins content. Resistance training was very effective in counteracting ULLS induced alterations indicating that long term ULLS did not prevent the positive effect of exercise on human muscle. This article is protected by copyright. All rights reserved

  September 15, 2015   doi: 10.1113/JP271188   open full text
• Increase in pulmonary blood flow at birth: role of oxygen and lung aeration.
  Justin A.R. Lang, James T. Pearson, Corinna Binder‐Heschl, Megan J. Wallace, Melissa L. Siew, Marcus J. Kitchen, Arjan B. te Pas, Andreas Fouras, Robert A. Lewis, Graeme R. Polglase, Mikiyasu Shirai, Stuart B. Hooper.
  The Journal of Physiology. September 10, 2015
  Key points There is no well‐established, direct correlation between local aeration and perfusion in the lungs immediately following birth. In a new study of simultaneous X‐ray imaging and angiography in near‐term rabbits, we investigated the relative contributions of lung aeration and increased oxygenation in the increase in pulmonary perfusion at birth. We demonstrated that partial lung aeration induces a global increase in pulmonary blood flow that is independent of changes in inspired oxygen. These results show that mechanisms unrelated to oxygenation or the spatial relationships that match ventilation to perfusion initiate the large increase in pulmonary blood flow at birth. Abstract Lung aeration stimulates the increase in pulmonary blood flow (PBF) at birth, but the spatial relationships between PBF and lung aeration and the role of increased oxygenation remain unclear. Using simultaneous phase‐contrast X‐ray imaging and angiography, we have investigated the separate roles of lung aeration and increased oxygenation in PBF changes at birth using near‐term (30 days of gestation) rabbit kits (n = 18). Rabbits were imaged before ventilation, then the right lung was ventilated with 100% nitrogen (N2), air or 100% O2 (oxygen), before all kits were switched to ventilation in air, followed by ventilation of both lungs using air. Unilateral ventilation of the right lung with 100% N2 significantly increased heart rate (from 69.4 ± 4.9 to 93.0 ± 15.0 bpm), the diameters of both left and right pulmonary axial arteries, number of visible vessels in both left and right lungs, relative PBF index in both pulmonary arteries, and reduced bolus transit time for both left and right axial arteries (from 1.34 ± 0.39 and 1.81 ± 0.43 s to 0.52 ± 0.17 and 0.89 ± 0.21 s in the left and right axial arteries, respectively). Similar changes were observed with 100% oxygen, but increases in visible vessel number and vessel diameter of the axial arteries were greater in the ventilated right lung during unilateral ventilation. These findings confirm that PBF increase at birth is not spatially related to lung aeration and that the increase in PBF to unventilated regions is unrelated to oxygenation, although oxygen can potentiate this increase.

  September 10, 2015   doi: 10.1113/JP270926   open full text
• Development of an experimental model of maternal allergic asthma during pregnancy.
  Vicki L. Clifton, Timothy J. M. Moss, Amy L. Wooldridge, Kathryn L. Gatford, Bahar Liravi, Dasom Kim, Beverly S. Muhlhausler, Janna L. Morrison, Andrew Davies, Robert Matteo, Megan J. Wallace, Robert J. Bischof.
  The Journal of Physiology. September 02, 2015
  Key points We studied the effects of preconceptional allergen sensitisation and repeated airway allergen challenges during pregnancy on maternal immune and airway functions during pregnancy, and maternal, fetal and placental phenotype in late pregnancy in sheep. This protocol induced maternal responses consistent with an allergic asthmatic phenotype. During pregnancy, lung resistance and the eosinophil influx induced by allergen challenges increased progressively in allergic sheep, and in late pregnancy airway smooth muscle content was greater in allergic than control ewes. Effects on fetal growth and development were consistent with those of maternal asthma in humans. Maternal allergic asthma decreased relative fetal weight by 12%, reduced fetal lung expression of surfactant protein B, and altered placental morphology. This provides an animal model in which to identify mechanisms underlying fetal effects of maternal asthma in pregnancy, including fetal physiological responses to exacerbations, and to evaluate responses to clinically used treatments and novel interventions. Abstract Maternal asthma during pregnancy adversely affects pregnancy outcomes but identification of the cause/s, and the ability to evaluate interventions, is limited by the lack of an appropriate animal model. We therefore aimed to characterise maternal lung and cardiovascular responses and fetal–placental growth and lung surfactant levels in a sheep model of allergic asthma. Immune and airway functions were studied in singleton‐bearing ewes, either sensitised before pregnancy to house dust mite (HDM, allergic, n = 7) or non‐allergic (control, n = 5), and subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Maternal lung, fetal and placental phenotypes were characterised at 140 ± 1 days gestational age (term, ∼147 days). The eosinophil influx into lungs was greater after HDM challenge in allergic ewes than after saline challenge in control ewes before mating and in late gestation. Airway resistance increased throughout pregnancy in allergic but not control ewes, consistent with increased airway smooth muscle in allergic ewes. Maternal allergic asthma decreased relative fetal weight (−12%) and altered placental phenotype to a more mature form. Expression of surfactant protein B mRNA was 48% lower in fetuses from allergic ewes than controls, with a similar trend for surfactant protein D. Thus, allergic asthma in pregnant sheep modifies placental phenotype, and inhibits fetal growth and lung development consistent with observations from human pregnancies. Preconceptional allergen sensitisation and repeated airway challenges in pregnant sheep therefore provides an animal model to identify mechanisms of altered fetal development and adverse pregnancy outcomes caused by maternal asthma in pregnancy.

  September 02, 2015   doi: 10.1113/JP270752   open full text
• Hypoxia, AMPK activation and uterine artery vasoreactivity.
  K. L. Skeffington, J. S. Higgins, A. D. Mahmoud, A. M. Evans, A. N. Sferruzzi‐Perri, A. L. Fowden, H. W. Yung, G. J. Burton, D. A. Giussani, L. G. Moore.
  The Journal of Physiology. July 31, 2015
  Key points Uterine artery vasodilatation is a key mechanism for increasing utero‐placental blood flow and fetal nutrient supply. Since the pioneering work of Joseph Barcroft, the natural laboratory of high altitude has been used to study the mechanisms regulating uterine artery blood supply and fetal growth. Genes near the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) have been implicated in genetic protection from high altitude‐associated fetal growth restriction. We show that AMPK is present in utero‐placental tissues and has vasodilator effects in murine uterine arteries, and that exposure to chronic hypoxia sufficient to decrease fetal growth increases the vasodilator actions of AMPK in opposing phenylephrine‐induced vasoconstriction. These results point to AMPK as being a key link between maternal vascular responses to pregnancy and fetal growth. Manipulation of AMPK may be a novel mechanism for developing new therapies in pregnancies complicated by chronic hypoxia. Abstract Genes near adenosine monophosphate‐activated protein kinase‐α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude‐adapted Andean populations. Adenosine monophosphate‐activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near‐term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7–9) from day 14 to day 19, and placentas from non‐labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near‐term murine UtA and placental tissue. RT‐PCR products for AMPK‐α1 and ‐α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK‐α1 and ‐α2 expression in the high‐ compared with low‐altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre‐constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l‐NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO‐dependent and NO‐independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero‐placental blood flow in hypoxic pregnancy.

  July 31, 2015   doi: 10.1113/JP270995   open full text
• Magnesium sulphate and cardiovascular and cerebrovascular adaptations to asphyxia in preterm fetal sheep.
  Robert Galinsky, Joanne O. Davidson, Paul P. Drury, Guido Wassink, Christopher A. Lear, Lotte G. den Heuij, Alistair J. Gunn, Laura Bennet.
  The Journal of Physiology. July 08, 2015
  Key points Magnesium sulphate is the recommended treatment for pre‐eclampsia and is now widely recommended for perinatal neuroprotection. MgSO4 has vasodilatory and negative inotropic effects; however, it is unknown whether it impairs the cardiovascular and cerebrovascular adaptations to acute asphyxia in preterm fetuses. Intravenous infusion of a clinically comparable dose of MgSO4 to the preterm fetus was associated with no change in blood pressure, reduced fetal heart rate and increased femoral arterial conductance and blood flow; femoral arterial waveform height and width were increased, consistent with increased stroke volume during MgSO4 infusion. During asphyxia MgSO4 was associated with increased carotid and femoral arterial conductance and blood flows; after asphyxia, fetal heart rate was lower and carotid and femoral blood flows and vascular conductance were greater in MgSO4‐treated fetuses. These data demonstrate that MgSO4 may increase perfusion of peripheral vascular beds during adverse perinatal events such as asphyxia. Abstract Magnesium sulphate is a standard therapy for eclampsia in pregnancy and is widely recommended for perinatal neuroprotection during threatened preterm labour. MgSO4 is a vasodilator and negative inotrope. Therefore the aim of this study was to investigate the effect of MgSO4 on the cardiovascular and cerebrovascular responses of the preterm fetus to asphyxia. Fetal sheep were instrumented at 98 ± 1 days of gestation (term = 147 days). At 104 days, unanaesthetised fetuses were randomly assigned to receive an intravenous infusion of MgSO4 (n = 6) or saline (n = 9). At 105 days all fetuses underwent umbilical cord occlusion for 25 min. Before occlusion, MgSO4 treatment reduced heart rate and increased femoral blood flow (FBF) and vascular conductance compared to controls. During occlusion, carotid and femoral arterial conductance and blood flows were higher in MgSO4‐treated fetuses than controls. After occlusion, fetal heart rate was lower and carotid and femoral arterial conductance and blood flows were higher in MgSO4‐treated fetuses than controls. Femoral arterial waveform height and width were increased during MgSO4 infusion, consistent with increased stroke volume. MgSO4 did not alter the fetal neurophysiological or nuchal electromyographic responses to asphyxia. These data demonstrate that a clinically comparable dose of MgSO4 increased FBF and stroke volume without impairing mean arterial pressure (MAP) or carotid blood flow (CaBF) during and immediately after profound asphyxia. Thus, MgSO4 may increase perfusion of peripheral vascular beds during adverse perinatal events.

  July 08, 2015   doi: 10.1113/JP270614   open full text
• Depressed perivascular sensory innervation of mouse mesenteric arteries with advanced age.
  Erika M. Boerman, Steven S. Segal.
  The Journal of Physiology. June 30, 2015
  Key points The dilatory role for sensory innervation of mesenteric arteries (MAs) is impaired in Old (∼24 months) versus Young (∼4 months) mice. We investigated the nature of this impairment in isolated pressurized MAs. With perivascular sensory nerve stimulation, dilatation and inhibition of sympathetic vasoconstriction observed in Young MAs were lost in Old MAs along with impaired dilatation to calcitonin gene‐related peptide (CGRP). Inhibiting NO and prostaglandin synthesis increased CGRP EC50 in Young and Old MAs. Endothelial denudation attenuated dilatation to CGRP in Old MAs yet enhanced dilatation to CGRP in Young MAs while abolishing all dilatations to ACh. In Old MAs, sensory nerve density was reduced and RAMP1 (CGRP receptor component) associated with nuclear regions of endothelial cells in a manner not seen in Young MAs or in smooth muscle cells of either age. With advanced age, loss of dilatory signalling mediated through perivascular sensory nerves may compromise perfusion of visceral organs. Abstract Vascular dysfunction and sympathetic nerve activity increase with advancing age. In the gut, blood flow is governed by perivascular sensory and sympathetic nerves but little is known of how their functional role is affected by advanced age. We tested the hypothesis that functional sensory innervation of mesenteric arteries (MAs) is impaired for Old (24 months) versus Young (4 months) C57BL/6 male mice. In cannulated pressurized MAs preconstricted 50% with noradrenaline and treated with guanethidine (to inhibit sympathetic neurotransmission), perivascular nerve stimulation (PNS) evoked dilatation in Young but not Old MAs while dilatations to ACh were not different between age groups. In Young MAs, capsaicin (to inhibit sensory neurotransmission) blocked dilatation and increased constriction during PNS. With no difference in efficacy, the EC50 of CGRP as a vasodilator was ∼6‐fold greater in Old versus Young MAs. Inhibiting nitric oxide (l‐NAME) and prostaglandin (indomethacin) synthesis increased CGRP EC50 in both age groups. Endothelial denudation reduced the efficacy of dilatation to CGRP by ∼30% in Old MAs yet increased this efficacy ∼15% in Young MAs while all dilatations to ACh were abolished. Immunolabelling revealed reduced density of sensory (CGRP) but not sympathetic (tyrosine hydroxylase) innervation for Old versus Young MAs. Whereas the distribution of CGRP receptor proteins was similar in SMCs, RAMP1 associated with nuclear regions of endothelial cells of Old but not Young MAs. With advanced age, the loss of sensory nerve function and diminished effectiveness of CGRP as a vasodilator is multifaceted and may adversely affect splanchnic perfusion.

  June 30, 2015   doi: 10.1113/JP270710   open full text
• Determinants of ventilation and pulmonary artery pressure during early acclimatization to hypoxia in humans.
  Marzieh Fatemian, Mari Herigstad, Quentin P. P. Croft, Federico Formenti, Rosa Cardenas, Carly Wheeler, Thomas G. Smith, Maria Friedmannova, Keith L. Dorrington, Peter A. Robbins.
  The Journal of Physiology. June 05, 2015
  Key points Lung ventilation and pulmonary artery pressure rise progressively in response to 8 h of hypoxia, changes described as ‘acclimatization to hypoxia’. Acclimatization responses differ markedly between humans for unknown reasons. We explored whether the magnitudes of the ventilatory and vascular responses were related, and whether the degree of acclimatization could be predicted by acute measurements of ventilatory and vascular sensitivities. In 80 healthy human volunteers measurements of acclimatization were made before, during, and after a sustained exposure to 8 h of isocapnic hypoxia. No correlation was found between measures of ventilatory and pulmonary vascular acclimatization. The ventilatory chemoreflex sensitivities to acute hypoxia and hypercapnia all increased in proportion to their pre‐acclimatization values following 8 h of hypoxia. The peripheral (rapid) chemoreflex sensitivity to CO2, measured before sustained hypoxia against a background of hyperoxia, was a modest predictor of ventilatory acclimatization to hypoxia. This finding has relevance to predicting human acclimatization to the hypoxia of altitude. Abstract Pulmonary ventilation and pulmonary arterial pressure both rise progressively during the first few hours of human acclimatization to hypoxia. These responses are highly variable between individuals, but the origin of this variability is unknown. Here, we sought to determine whether the variabilities between different measures of response to sustained hypoxia were related, which would suggest a common source of variability. Eighty volunteers individually underwent an 8‐h isocapnic exposure to hypoxia (end‐tidal PO2=55 Torr) in a purpose‐built chamber. Measurements of ventilation and pulmonary artery systolic pressure (PASP) assessed by Doppler echocardiography were made during the exposure. Before and after the exposure, measurements were made of the ventilatory sensitivities to acute isocapnic hypoxia (GpO2) and hyperoxic hypercapnia, the latter divided into peripheral (G pC O2) and central (G cC O2) components. Substantial acclimatization was observed in both ventilation and PASP, the latter being 40% greater in women than men. No correlation was found between the magnitudes of pulmonary ventilatory and pulmonary vascular responses. For GpO2, G pC O2 and G cC O2, but not the sensitivity of PASP to acute hypoxia, the magnitude of the increase during acclimatization was proportional to the pre‐acclimatization value. Additionally, the change in GpO2 during acclimatization to hypoxia correlated well with most other measures of ventilatory acclimatization. Of the initial measurements prior to sustained hypoxia, only G pC O2 predicted the subsequent rise in ventilation and change in GpO2 during acclimatization. We conclude that the magnitudes of the ventilatory and pulmonary vascular responses to sustained hypoxia are predominantly determined by different factors and that the initial G pC O2 is a modest predictor of ventilatory acclimatization.

  June 05, 2015   doi: 10.1113/JP270061   open full text
• Sympathetic neural activation does not mediate heart rate variability during repeated brief umbilical cord occlusions in near‐term fetal sheep.
  Christopher A. Lear, Robert Galinsky, Guido Wassink, Clinton J. Mitchell, Joanne O. Davidson, Jennifer A. Westgate, Laura Bennet, Alistair J. Gunn.
  The Journal of Physiology. May 22, 2015
  Key points Fetal heart rate variability and changes in the ST segment of the electrocardiogram are used clinically during labour to identify fetuses at risk of severe metabolic acidosis or death. Sympathetic nervous system activity contributes to heart rate variability in healthy normoxic fetuses, and is critical for the rapid haemodynamic adaptations to repeated episodes of asphyxia induced by brief complete umbilical cord occlusions at rates consistent with active labour. We now show that chemical sympathectomy did not alter fetal heart rate variability between episodes of brief repeated asphyxia or elevation of the ST segment during asphyxia. The lack of influence of the sympathetic system on fetal heart rate variability between episodes of brief asphyxia suggests that measures of fetal heart rate variability are unlikely to help monitor changes in sympathetic nervous system activity during active labour. Abstract Changes in fetal heart rate variability (FHRV) and ST segment elevation (measured as the T/QRS ratio) are used to evaluate fetal adaptation to labour. The sympathetic nervous system (SNS) is an important contributor to FHRV under healthy normoxic conditions, and is critical for rapid support of blood pressure during brief labour‐like asphyxia. However, although it has been assumed that SNS activity contributes to FHRV during labour; this has never been tested, and it is unclear whether the SNS contributes to the rapid increase in T/QRS ratio during brief asphyxia. Thirteen chronically instrumented fetal sheep at 0.85 of gestation received either chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA; n = 6) or sham treatment (control; n = 7), followed 4–5 days later by 2 min episodes of complete umbilical cord occlusion repeated every 5 min for up to 4 h, or until mean arterial blood pressure fell to <20 mmHg for two successive occlusions. FHRV was decreased before occlusions in the 6‐OHDA group (P < 0.05) and 2–4.5 h during recovery after occlusions (P < 0.05) compared to the control group. During each occlusion there was a rapid increase in T/QRS ratio. Between successive occlusions the T/QRS ratio rapidly returned to baseline, and FHRV increased above baseline in both groups (P < 0.05), with no significant effect of sympathectomy on FHRV or T/QRS ratio. In conclusion, these data show that SNS activity does not mediate the increase in FHRV between repeated episodes of brief umbilical cord occlusion or the transient increase in T/QRS ratio during occlusions.

  May 22, 2015   doi: 10.1113/JP270125   open full text
• Disrupted hippocampal sharp‐wave ripple‐associated spike dynamics in a transgenic mouse model of dementia.
  Jonathan Witton, Lydia E. Staniaszek, Ullrich Bartsch, Andrew D. Randall, Matthew W. Jones, Jonathan T. Brown.
  The Journal of Physiology. January 02, 2015
  Key points High frequency (100–250 Hz) neuronal oscillations in the hippocampus, known as sharp‐wave ripples (SWRs), synchronise the firing behaviour of groups of neurons and play a key role in memory consolidation. Learning and memory are severely compromised in dementias such as Alzheimer's disease; however, the effects of dementia‐related pathology on SWRs are unknown. The frequency and temporal structure of SWRs was disrupted in a transgenic mouse model of tauopathy (one of the major hallmarks of several dementias). Excitatory pyramidal neurons were more likely to fire action potentials in a phase‐locked manner during SWRs in the mouse model of tauopathy; conversely, inhibitory interneurons were less likely to fire phase‐locked spikes during SWRs. These findings indicate there is reduced inhibitory control of hippocampal network events and point to a novel mechanism which may underlie the cognitive impairments in this model of dementia. Abstract Neurons within the CA1 region of the hippocampus are co‐activated during high frequency (100–250 Hz) sharp‐wave ripple (SWR) activity in a manner that probably drives synaptic plasticity and promotes memory consolidation. In this study we have used a transgenic mouse model of dementia (rTg4510 mice), which overexpresses a mutant form of tau protein, to examine the effects of tauopathy on hippocampal SWRs and associated neuronal firing. Tetrodes were used to record simultaneous extracellular action potentials and local field potentials from the dorsal CA1 pyramidal cell layer of 7‐ to 8‐month‐old wild‐type and rTg4510 mice at rest in their home cage. At this age point these mice exhibit neurofibrillary tangles, neurodegeneration and cognitive deficits. Epochs of sleep or quiet restfulness were characterised by minimal locomotor activity and a low theta/delta ratio in the local field potential power spectrum. SWRs detected off‐line were significantly lower in amplitude and had an altered temporal structure in rTg4510 mice. Nevertheless, the average frequency profile and duration of the SWRs were relatively unaltered. Putative interneurons displayed significantly less temporal and phase locking to SWRs in rTg4510 mice, whilst putative pyramidal neurons showed increased temporal and phase locking to SWRs. These findings indicate there is reduced inhibitory control of hippocampal network events and point to a novel mechanism which may contribute to impairments in memory consolidation in this model of dementia.

  January 02, 2015   doi: 10.1113/jphysiol.2014.282889   open full text
• Biophysical constraints on the evolution of tissue structure and function.
  P. J. Hunter, B. Bono.
  The Journal of Physiology. May 30, 2014
  Key points We outline an extension of phylogenetic studies of protein interaction networks to take into account biophysical constraints to interaction imposed by tissues regulating these networks and we define these constraints explicitly as a means to track the evolution of tissue structure and function objectively. For biophysical constraints that are associated at the lowest spatial scales with molecular diffusion within and between cells that are less than 100 μm apart, we define a cylinder of 40 μm radius centred on a small vessel as the primary functional tissue unit (pFTU). Molecular transport and communication between distributed or contiguous pFTUs via the endothelial or epithelial vessels is characterised at the level of secondary functional tissue units (sFTUs). sFTUs represent units of physiological function that are replicated multiple times in a whole organ. Non‐dimensional analysis, which expresses the relative importance of biophysical processes, provides the metrics to quantify and compare the function of FTUs for phylogenetic analysis. We lay the ground for a systematic approach to the measurement of tissue structure and function, based on biophysical principles, at the level of primary and secondary FTUs. Abstract Phylogenetic analyses based on models of molecular sequence evolution have driven to industrial scale the generation, cataloguing and modelling of nucleic acid and polypeptide structure. The recent application of these techniques to study the evolution of protein interaction networks extends this analytical rigour to the study of nucleic acid and protein function. Can we further extend phylogenetic analysis of protein networks to the study of tissue structure and function? If the study of tissue phylogeny is to join up with mainstream efforts in the molecular evolution domain, the continuum field description of tissue biophysics must be linked to discrete descriptions of molecular biochemistry. In support of this goal we discuss tissue units, and biophysical constraints to molecular function associated with these units, to present a rationale with which to model tissue evolution. Our rationale combines a multiscale hierarchy of functional tissue units (FTUs) with the corresponding application of physical laws to describe molecular interaction networks and flow processes over continuum fields within these units. Non‐dimensional numbers, derived from the equations governing biophysical processes in FTUs, are proposed as metrics for comparative studies across individuals, species or evolutionary time. We also outline the challenges inherent to the systematic cataloguing and phylogenetic analysis of tissue features relevant to the maintenance and regulation of molecular interaction networks. These features are key to understanding the core biophysical constraints on tissue evolution.

  May 30, 2014   doi: 10.1113/jphysiol.2014.273235   open full text
• The degree of acute descending control of spinal nociception in an area of primary hyperalgesia is dependent on the peripheral domain of afferent input.
  R.A.R. Drake, R.P. Hulse, B.M. Lumb, L.F. Donaldson.
  The Journal of Physiology. May 28, 2014
  Descending controls of spinal nociceptive processing play a critical role in the development of inflammatory hyperalgesia. Acute peripheral nociceptor sensitisation drives spinal sensitisation, and activates spino‐supraspinal‐spinal loops leading to descending inhibitory and facilitatory controls of spinal neuronal activity, that further modify the extent and degree of the pain state. The afferent inputs from hairy and glabrous skin are distinct with respect to both the profile of primary afferents classes and the degree of their peripheral sensitisation. It is not known whether these differences in afferent input differentially engage descending control systems to different extents or in different ways. Injection of Complete Freund's adjuvant resulted in inflammation and swelling of hairy hindpaw skin in rats, a transient thermal hyperalgesia lasting <2 hours, and long‐lasting primary mechanical hyperalgesia (at least 7d). Much longer lasting thermal hyperalgesia was apparent in glabrous skin (1h up to >72h). In hairy skin, transient hyperalgesia was associated with sensitisation of withdrawal reflexes to thermal activation of either A‐ and C‐nociceptors. The transience of the hyperalgesia was attributable to a rapidly engaged descending inhibitory noradrenergic mechanism, which affected withdrawal responses to both A‐ and C‐nociceptor activation and this could be reversed by intrathecal administration of yohimbine (alpha‐2‐adrenoceptor antagonist). In glabrous skin, yohimbine had no effect on an equivalent thermal inflammatory hyperalgesia. We conclude that acute inflammation and peripheral nociceptor sensitisation in hind paw hairy skin, but not glabrous skin, rapidly activates a descending inhibitory noradrenergic system. This may result from differences in the engagement of descending controls systems following sensitisation of different primary afferent classes that innervate glabrous and hairy skin. This article is protected by copyright. All rights reserved

  May 28, 2014   doi: 10.1113/jphysiol.2013.266494   open full text
• Utrophin regulates modal gating of mechanosensitive ion channels in dystrophic skeletal muscle.
  Nhi Tan, Jeffry B. Lansman.
  The Journal of Physiology. May 28, 2014
  Dystrophin is a large, submembrane cytoskeletal protein, an absence of which causes Duchenne muscular dystrophy. Utrophin is a dystrophin homologue found in both muscle and brain whose physiological function is unknown. Recordings of single‐channel activity were made from membrane patches on skeletal muscle from mdx, mdx/utrn+/− heterozygotes, and mdx/utrn−/− double knockout mice to investigate the role of these cytoskeletal proteins in mechanosensitive channel gating. We find complex, gene dose‐dependent effects of utophin depletion in dystrophin‐deficient mdx muscle: 1) increased MS channel open probability, 2) a shift of MS chanel gating to larger pressures, 3) appearance of modal gating of MS channels and small conductance channels, and 4) expression of large conductance MS channels. We suggest a physical model in which utrophin acts as a scaffolding protein that stabilizes lipid microdomains and clusters MS channel subunits. Depletion of utrophin disrupts domain composition in a manner that favors open channel area expansion, as well as allowing diffusion and aggregation of addditional MS channel subunits. This article is protected by copyright. All rights reserved

  May 28, 2014   doi: 10.1113/jphysiol.2014.274332   open full text
• Direct excitation of parvalbumin‐positive interneurons by M1 muscarinic acetylcholine receptors: roles in cellular excitability, inhibitory transmission and cognition.
  Feng Yi, Jackson Ball, Kurt E. Stoll, Vaishali C. Satpute, Samantha M. Mitchell, Jordan L. Pauli, Benjamin B. Holloway, April D. Johnston, Neil M. Nathanson, Karl Deisseroth, David J. Gerber, Susumu Tonegawa, J. Josh Lawrence.
  The Journal of Physiology. May 28, 2014
  Parvalbumin‐containing (PV) neurons, a major class of GABAergic interneurons, are essential circuit elements of learning networks. As levels of acetylcholine rise during active learning tasks, PV neurons become increasingly engaged in network dynamics. Conversely, impairment of either cholinergic or PV interneuron function induces learning deficits. Here, we examined PV interneurons in hippocampus (HC) and prefrontal cortex (PFC) and their modulation by muscarinic acetylcholine receptors (mAChRs). HC PV cells, visualized by crossing PV‐CRE mice with Rosa26YFP mice, were anatomically identified as basket cells and PV bistratified cells in stratum pyramidale; in stratum oriens, HC PV cells were electrophysiologically distinct from somatostatin‐containing cells. With glutamatergic transmission pharmacologically blocked, mAChR activation enhanced PV cell excitability in both CA1 HC and PFC; however, CA1 HC PV cells exhibited a stronger postsynaptic depolarization than PFC PV cells. To genetically delete M1 mAChRs from PV interneurons, we created PV‐M1KO mice by crossing PV‐CRE and floxed M1 mice. The elimination of M1 mAChRs from PV cells diminished M1 mAChR immunoreactivity and muscarinic excitation of HC PV cells. Selective cholinergic activation of HC PV interneurons using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology enhanced the frequency and amplitude of inhibitory synaptic currents in CA1 pyramidal cells. Finally, relative to WT controls, PV‐M1KO mice exhibited impaired novel object recognition and, to a lesser extent, impaired spatial working memory, but reference memory remained intact. Therefore, the direct activation of M1 mAChRs on PV cells contributes to some forms of learning and memory. This article is protected by copyright. All rights reserved

  May 28, 2014   doi: 10.1113/jphysiol.2014.275453   open full text
• Rhodopsin in the rod surface membrane regenerates more rapidly than bulk rhodopsin in the disc membranes in vivo.
  Christopher Kessler, Megan Tillman, Marie E. Burns, Edward N. Pugh.
  The Journal of Physiology. May 28, 2014
  Key points The early receptor potential (ERP) of the mouse electroretinogram (ERG) was measured in wild‐type (WT) mice, in mice (Opn1sw−/−) that lack S‐cone opsin and overexpress M‐cone opsin, and in mice heterozygous for the retinal pigment epithelium isomerase Rpe65. The amplitude of the ERP saturated exponentially with flash intensity. In WT mice the saturated amplitude was ∼1000 μV, about 20% larger than the saturated amplitude of the ERG a‐wave. The ERP was 13% larger in mice overexpressing M‐opsin than in WT mice, indicating that 26% of the ERP of WT mice in these experiments arises from M‐opsin. After complete bleaching, the ERP of WT mice recovered in two phases, a fast phase responsible for ∼20% of the recovery having a time constant of ∼1 min, and a complementary slower phase with a time constant of 23 min. The fast phase of ERP recovery did not depend on the expression level of Rpe65, but the slow phase did. The fast phase of ERP recovery is concluded to arise from M‐opsin regeneration, and the slow phase from the regeneration of rod plasma membrane rhodopsin. The slower phase of ERP recovery is faster than the regeneration of bulk rhodopsin in the internal disc membranes, consistent with the hypothesis that delivery of 11‐cis retinal across the cytoplasmic gap between plasma and disc membranes retards regeneration of disc membrane rhodopsin. Abstract Sustained vertebrate vision requires that opsin chromophores isomerized by light to the all‐trans form be replaced with 11‐cis retinal to regenerate the visual pigment. We have characterized the early receptor potential (ERP), a component of the electroretinogram arising from photoisomerization‐induced charge displacements in plasma membrane visual pigment, and used it to measure pigment bleaching and regeneration in living mice. The mouse ERP was characterized by an outward ‘R2’ charge displacement with a time constant of 215 μs that discharged through a membrane with an apparent time constant of ∼0.6 ms. After complete bleaching of rhodopsin, the ERP recovered in two phases. The initial, faster phase had a time constant of ∼1 min, accounted for ∼20% of the total, and was not dependent on the level of expression of the retinal pigment epithelium isomerase, Rpe65. The slower, complementary phase had a time constant of 23 min in wild‐type (WT) mice (C57Bl/6) and was substantially slowed in Rpe65+/− mice. Comparison of the ERPs of a mouse line expressing 150% of the normal level of cone M‐opsin with those of WT mice revealed that M‐opsin contributed 26% of the total WT ERP in these experiments, with the remaining 74% arising from rhodopsin. Thus, the fast regenerating fraction (20%) corresponds approximately to the fraction of the total ERP independently estimated to arise from M‐opsin. Because both phases of the ERP recover substantially faster than previous measurements of bulk rhodopsin regeneration in living mice, we conclude that delivery of the highly hydrophobic 11‐cis retinal to the interior of rod photoreceptors appears to be retarded by transit across the cytoplasmic gap between plasma and disc membranes.

  May 28, 2014   doi: 10.1113/jphysiol.2014.272518   open full text
• Wave reflections in the pulmonary arteries analysed with the reservoir–wave model.
  J. Christopher Bouwmeester, Israel Belenkie, Nigel G. Shrive, John V. Tyberg.
  The Journal of Physiology. May 27, 2014
  Key points In the pulmonary artery, we use the reservoir–wave model to separate the effects of a charging and discharging, elastic arterial reservoir from the effects of waves created by the contracting and relaxing heart. Wave intensity analysis quantifies the effects of waves that cause changes in pressure and flow and precisely identifies when waves created by the heart and reflections of these waves start and end. We show that negative wave reflections arise from the junction of lobar arteries stemming from the left and right pulmonary arteries. When blood volume is increased and pulmonary arteries become distended, the strength of negative wave reflections increases when 100% O2 is used for ventilation. Negative reflections suck blood downstream and, as they arrive when the heart is developing maximal pressure, negative reflections help to lower the back pressure the heart must pump against and, thus, they tend to increase the forward flow of blood. Abstract Conventional haemodynamic analysis of pressure and flow in the pulmonary circulation yields incident and reflected waves throughout the cardiac cycle, even during diastole. The reservoir–wave model provides an alternative haemodynamic analysis consistent with minimal wave activity during diastole. Pressure and flow in the main pulmonary artery were measured in anaesthetized dogs and the effects of hypoxia and nitric oxide, volume loading and positive end‐expiratory pressure were observed. The reservoir–wave model was used to determine the reservoir contribution to pressure and flow and once subtracted, resulted in ‘excess’ quantities, which were treated as wave‐related. Wave intensity analysis quantified the contributions of waves originating upstream (forward‐going waves) and downstream (backward‐going waves). In the pulmonary artery, negative reflections of incident waves created by the right ventricle were observed. Overall, the distance from the pulmonary artery valve to this reflection site was calculated to be 5.7 ± 0.2 cm. During 100% O2 ventilation, the strength of these reflections increased 10% with volume loading and decreased 4% with 10 cmH2O positive end‐expiratory pressure. In the pulmonary arterial circulation, negative reflections arise from the junction of lobar arteries from the left and right pulmonary arteries. This mechanism serves to reduce peak systolic pressure, while increasing blood flow.

  May 27, 2014   doi: 10.1113/jphysiol.2014.273094   open full text
• Mid‐ to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex‐specific manner.
  J. S. M. Cuffe, S. L. Walton, R. R. Singh, J. G. Spiers, H. Bielefeldt‐Ohmann, L. Wilkinson, M. H. Little, K. M. Moritz.
  The Journal of Physiology. May 27, 2014
  Maternal hypoxia is a common perturbation that may impair fetal development and programme sex specific disease outcomes in offspring. There is growing interest in the role of the placenta in mediating the effects of maternal hypoxia on fetal development, particularly in late gestation during maximal fetal growth. Multiple mechanisms have been proposed to play a role in hypoxia induced impairment of placental development. Here we investigated the role of glucocorticoids and glucose regulation. This study shows that fetal sex determines placental adaptations to maternal hypoxia: while maternal hypoxia increased maternal glucose and corticosterone levels in both sexes, placental adaptations to impaired maternal physiology were more evident in female fetuses, in which factors responsible for the regulation of glucocorticoids and nutrient transport were most severely affected by maternal hypoxia. Abstract Maternal hypoxia is a common perturbation that can disrupt placental and thus fetal development, contributing to neonatal impairments. Recently, evidence has suggested that physiological outcomes are dependent upon the sex of the fetus, with males more susceptible to hypoxic insults than females. This study investigated the effects of maternal hypoxia during mid‐ to late gestation on fetal growth and placental development and determined if responses were sex specific. CD1 mice were housed under 21% or 12% oxygen from embryonic day (E) 14.5 until tissue collection at E18.5. Fetuses and placentas were weighed before collection for gene and protein expression and morphological analysis. Hypoxia reduced fetal weight in both sexes at E18.5 by 7% but did not affect placental weight. Hypoxia reduced placental mRNA levels of the mineralocorticoid and glucocorticoid receptors and reduced the gene and protein expression of the glucocorticoid metabolizing enzyme HSD11B2. However, placentas of female fetuses responded differently to maternal hypoxia than did placentas of male fetuses. Notably, morphology was significantly altered in placentas from hypoxic female fetuses, with a reduction in placental labyrinth blood spaces. In addition mRNA expression of Glut1, Igf2 and Igf1r were reduced in placentas of female fetuses only. In summary, maternal hypoxia altered placental formation in a sex specific manner through mechanisms involving placental vascular development, growth factor and nutrient transporter expression and placental glucocorticoid signalling. This study provides insight into how sex differences in offspring disease development may be due to sex specific placental adaptations to maternal insults.

  May 27, 2014   doi: 10.1113/jphysiol.2014.272856   open full text
• Diet‐dependent modulation of gastro‐oesphageal vagal afferent mechanosensitivity by endogenous nitric oxide.
  Stephen J Kentish, Tracey A O’Donnell, Gary A Wittert, Amanda J Page.
  The Journal of Physiology. May 27, 2014
  Neuronal nitric oxide (NO) plays an important role in gastric motor activity and modulates the mechanosensitivity of gastro‐oesophageal vagal afferents. Effects of NO on food intake are dependent on feeding status. We sought to determine the effect of NO on gastro‐oesophageal vagal afferent activity in the normally fed, and food restricted state and the second messenger pathways mediating these effects. Eight week old female C56BL/6 mice were fed ad libitum or food restricted for 14 hr. An in vitro preparation was used to determine the functional effects of NO and the second messenger pathways involved. Expression of NO signal transduction molecules in vagal afferents was determined by reverse‐transcription polymerase chain reaction (RT‐PCR). Endogenous NO and the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) inhibited vagal mucosal afferent responses to tactile stimuli in mice fed ad libitum. After a 14 hr fast endogenous NO and SNAP potentiated tension and mucosal afferent responses to mechanical stimulation. The excitatory effect of NO was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. After a 14 hr fast expression of NADPH oxidase 2 (NOX2) mRNA in whole nodose ganglia was significantly reduced and the excitatory effect of NO on gastro‐oesophageal vagal afferents was lost. Under fasting conditions the inhibitory effect of NO was blocked with the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel blocker ivabradine and mRNA expression of HCN3 in the nodose ganglia was elevated. In conclusion, the role of NO in the peripheral modulation of gastro‐oesophageal vagal afferents is dynamic and dependent on feeding status. This article is protected by copyright. All rights reserved

  May 27, 2014   doi: 10.1113/jphysiol.2014.272674   open full text
• P2Y1 receptor‐mediated potentiation of inspiratory motor output in neonatal rat in vitro.
  TS Alvares, AL Revill, AG Huxtable, CD Lorenz, GD Funk.
  The Journal of Physiology. May 26, 2014
  PreBötzinger Complex (preBötC) inspiratory rhythm generating networks are excited by P2Y1R activation. Despite this, and the fact that inspiratory motoneurons (MNs) express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically‐active brainstem‐spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signaling on inspiratory output of phrenic and XII MNs that innervate pump and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mM) potentiated XII inspiratory burst amplitude by 60 ± 9%; ten‐fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole‐cell voltage‐clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca2+‐dependent non‐selective cation current, ICAN; high intracellular concentrations of BAPTA, which buffers Ca2+ increases necessary for activation of ICAN; and 9‐Phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real‐time PCR analysis of mRNA extracted from XII punches and laser‐microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4‐mediated ICAN, which amplifies inspiratory inputs. This article is protected by copyright. All rights reserved

  May 26, 2014   doi: 10.1113/jphysiol.2013.268136   open full text
• Maternal alcohol consumption in pregnancy enhances arterial stiffness and alters vasodilator function that varies between vascular beds in fetal sheep.
  Helena C. Parkington, Kelly R. Kenna, Foula Sozo, Harold A. Coleman, Alan Bocking, James F. Brien, Richard Harding, David W. Walker, Ruth Morley, Marianne Tare.
  The Journal of Physiology. May 23, 2014
  Key points To date, the effects of maternal alcohol consumption in pregnancy have focused on neurodevelopmental outcomes, with the impact on the arterial system poorly understood. In this study we investigated the effects of moderate maternal alcohol consumption (∼3 standard drinks per day) in late pregnancy on the ability of arteries in the fetus to relax, and hence deliver blood, and on arterial stiffness in five important organs. Maternal alcohol consumption in late pregnancy resulted in a marked increase in stiffness in arteries of the heart, kidney, gut, leg muscle and brain in the fetus, and this could slow contraction and relaxation and overall blood delivery. Coronary artery endothelial vasodilator function was severely blunted as a result of alcohol exposure, while in contrast endothelium‐dependent relaxation in renal and mesenteric arteries was enhanced. Together, the results of this study provide a warning for pregnant women and their carers that maternal consumption of ∼3 standard drinks per day, levels that do not evoke physical abnormalities or growth restriction, can dramatically alter arterial function in the fetus. Abstract While the impact of alcohol consumption by pregnant women on fetal neurodevelopment has received much attention, the effects on the cardiovascular system are not well understood. We hypothesised that repeated exposure to alcohol (ethanol) in utero would alter fetal arterial reactivity and wall stiffness, key mechanisms leading to cardiovascular disease in adulthood. Ethanol (0.75 g (kg body weight)–1) was infused intravenously into ewes over 1 h daily for 39 days in late pregnancy (days 95–133 of pregnancy, term ∼147 days). Maternal and fetal plasma ethanol concentrations at the end of the hour were ∼115 mg dl−1, and then declined to apparent zero over 8 h. At necropsy (day 134), fetal body weight and fetal brain–body weight ratio were not affected by alcohol infusion. Small arteries (250–300 μm outside diameter) from coronary, renal, mesenteric, femoral (psoas) and cerebral beds were isolated. Endothelium‐dependent vasodilatation sensitivity was reduced 10‐fold in coronary resistance arteries, associated with a reduction in endothelial nitric oxide synthase mRNA (P = 0.008). Conversely, vasodilatation sensitivity was enhanced 10‐fold in mesenteric and renal resistance arteries. Arterial stiffness was markedly increased (P = 0.0001) in all five vascular beds associated with an increase in elastic modulus and, in cerebral vessels, with an increase in collagen Iα mRNA. Thus, we show for the first time that fetal arteries undergo marked and regionally variable adaptations as a consequence of repeated alcohol exposure. These alcohol‐induced vascular effects occurred in the apparent absence of fetal physical abnormalities or fetal growth restriction.

  May 23, 2014   doi: 10.1113/jphysiol.2013.262873   open full text
• Response of the human detrusor to stretch is regulated by TREK‐1, a two‐pore‐domain (K2P) mechano‐gated potassium channel.
  Qi Lei, Xiao‐Qing Pan, Shaohua Chang, S. Bruce Malkowicz, Thomas J. Guzzo, Anna P. Malykhina.
  The Journal of Physiology. May 21, 2014
  Key points Mechano‐gated two‐pore‐domain potassium (K2P) channels are expressed in the human bladder, with TREK‐1 being the predominant functional subunit. TREK‐1 channels in bladder smooth muscle are activated by membrane stretch and negative pressure applied to the patch pipette. Inhibition of TREK‐1 channels in the human detrusor significantly delays relaxation of bladder smooth muscle and triggers small‐amplitude spontaneous contractions in response to stretch. Application of negative pressure to cell‐attached patches (–20 mmHg) causes a 19‐fold increase in the open probability (NPo) of human TREK‐1 channels. l‐Methionine (1 mm) dramatically decreases the NPo of TREK‐1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Addition of arachidonic acid (10 μm) increases the open probability of methionine‐inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). TREK‐1 channels may serve as a promising pharmacological target for bladder dysfunction in humans. Abstract The mechanisms of mechanosensitivity underlying the response of the human bladder to stretch are poorly understood. Animal data suggest that stretch‐activated two‐pore‐domain (K2P) K+ channels play a critical role in bladder relaxation during the filling phase. The objective of this study was to characterize the expression and function of stretch‐activated K2P channels in the human bladder and to clarify their physiological role in bladder mechanosensitivity. Gene and protein analysis of the K2P channels TREK‐1, TREK‐2 and TRAAK in the human bladder revealed that TREK‐1 is the predominantly expressed member of the mechano‐gated subfamily of K2P channels. Immunohistochemical labelling of bladder wall identified higher levels of expression of TREK‐1 in detrusor smooth muscle cells in comparison to bladder mucosa. Functional characterization and biophysical properties of the predominantly expressed member of the K2P family, the TREK‐1 channel, were evaluated by in vitro organ bath studies and the patch‐clamp technique. Electrophysiological recordings from single smooth muscle cells confirmed direct activation of TREK‐1 channels by mechanical stretch and negative pressure applied to the cell membrane. Inhibition of TREK‐1 channels in the human detrusor significantly delayed relaxation of the stretched bladder smooth muscle strips and triggered small‐amplitude spontaneous contractions. Application of negative pressure to cell‐attached patches (–20 mmHg) caused a 19‐fold increase in the open probability (NPo) of human TREK‐1 channels. l‐Methionine (1 mm), a specific TREK‐1 inhibitor, dramatically decreased the NPo of TREK‐1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Subsequent addition of arachidonic acid (10 μm), a channel opener, increased the open probability of methionine‐inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). The results of our study provide direct evidence that the response of the human detrusor to mechanical stretch is regulated by activation of mechano‐gated TREK‐1 channels. Impaired mechanosensation and mechanotransduction associated with the changes in stretch‐activated K2P channels may underlie myogenic bladder dysfunction in humans.

  May 21, 2014   doi: 10.1113/jphysiol.2014.271718   open full text
• Insulin modulates network activity in olfactory bulb slices: impact on odour processing.
  Nicola Kuczewski, Nicolas Fourcaud‐Trocmé, Agnès Savigner, Marc Thevenet, Pascaline Aimé, Samuel Garcia, Patricia Duchamp‐Viret, Brigitte Palouzier‐Paulignan.
  The Journal of Physiology. May 21, 2014
  Key points Olfactory function is largely under metabolic influence. Insulin, one of the major players between food intake and energy balance, is known to act at both central and peripheral levels. The present study assesses the action of insulin in olfactory bulb slices by using patch‐clamp recordings in young rats. The results show that insulin can alter both spontaneous and olfactory nerve‐induced firing activities in most of the main ouput neurons, this action being differentially exerted in two opposite directions. A mathematical model demonstrates that insulin, by acting in this way, could impact odour detection and discrimination mechanisms. Such an impact could be hypothesized as being exerted according to pertinent ecological characteristics, such as the alimentary/ethological valence of odour. Abstract Odour perception depends closely on nutritional status, in animals as in humans. Insulin, the principal anorectic hormone, appears to be one of the major candidates for ensuring the link between olfactory abilities and nutritional status, by modifying processing in the olfactory bulb (OB), one of its main central targets. The present study investigates whether and how insulin can act in OB, by evaluating its action on the main output neurons activities, mitral cells (MCs), in acute rat OB slices. Insulin was found to act at two OB network levels: (1) on MCs, by increasing their excitability, probably by inhibiting two voltage‐gated potassium (K+) channels; (2) on interneurons by modifying the GABAergic and on glutamatergic synaptic activity impinging on MCs, mainly reducing them. Insulin also altered the olfactory nerve (ON)‐evoked excitatory postsynaptic currents in 60% of MCs. Insulin decreased or increased the ON‐evoked responses in equal proportion and the direction of its effect depended on the initial neuron ON‐evoked firing rate. Indeed, insulin tended to decrease the high and to increase the low ON‐evoked firing rates, thereby reducing inter‐MC response firing variability. Therefore, the effects of insulin on the evoked firing rates were not carried out indiscriminately in the MC population. By constructing a mathematical model, the impact of insulin complex effects on OB was assessed at the population activity level. The model shows that the reduction of variability across cells could affect MC detection and discrimination abilities, mainly by decreasing and, less frequently, increasing them, depending on odour quality. Thus, as previously proposed, this differential action of insulin on MCs across odours would allow this hormone to put the olfactory function under feeding signal control, given the discerning valence of an odour as a function of nutritional status.

  May 21, 2014   doi: 10.1113/jphysiol.2013.269639   open full text
• Chronic intermittent hypoxia–hypercapnia blunts heart rate responses and alters neurotransmission to cardiac vagal neurons.
  Jhansi Dyavanapalli, Heather Jameson, Olga Dergacheva, Vivek Jain, Mona Alhusayyen, David Mendelowitz.
  The Journal of Physiology. May 19, 2014
  Key points Chronic intermittent hypoxia–hypercapnia (CIHH) in adult rats evoked hypertension and blunted the heart rate responses to acute hypoxia–hypercapnia (H–H). CIHH induced an increase in spontaneous inhibitory and decreased excitatory neurotransmission to cardiac vagal neurons. CIHH completely abolished acute H–H evoked inhibition of GABAergic while facilitating glycinergic neurotransmission to cardiac vagal neurons of nucleus ambiguus. These changes with CIHH inhibit cardiac vagal neurons to result in diminished cardioprotective vagal activity to the heart, characteristic of obstructive sleep apnoea. Abstract Patients with obstructive sleep apnoea experience chronic intermittent hypoxia–hypercapnia (CIHH) during sleep that elicit sympathetic overactivity and diminished parasympathetic activity to the heart, leading to hypertension and depressed baroreflex sensitivity. The parasympathetic control of heart rate arises from pre‐motor cardiac vagal neurons (CVNs) located in nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMNX). The mechanisms underlying diminished vagal control of heart rate were investigated by studying the changes in blood pressure, heart rate, and neurotransmission to CVNs evoked by acute hypoxia–hypercapnia (H–H) and CIHH. In vivo telemetry recordings of blood pressure and heart rate were obtained in adult rats during 4 weeks of CIHH exposure. Retrogradely labelled CVNs were identified in an in vitro brainstem slice preparation obtained from adult rats exposed either to air or CIHH for 4 weeks. Postsynaptic inhibitory or excitatory currents were recorded using whole cell voltage clamp techniques. Rats exposed to CIHH had increases in blood pressure, leading to hypertension, and blunted heart rate responses to acute H–H. CIHH induced an increase in GABAergic and glycinergic neurotransmission to CVNs in NA and DMNX, respectively; and a reduction in glutamatergic neurotransmission to CVNs in both nuclei. CIHH blunted the bradycardia evoked by acute H–H and abolished the acute H–H evoked inhibition of GABAergic transmission while enhancing glycinergic neurotransmission to CVNs in NA. These changes with CIHH inhibit CVNs and vagal outflow to the heart, both in acute and chronic exposures to H–H, resulting in diminished levels of cardioprotective parasympathetic activity to the heart as seen in OSA patients.

  May 19, 2014   doi: 10.1113/jphysiol.2014.273482   open full text
• Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices.
  Takuya Sasaki, Tomoe Ishikawa, Reimi Abe, Ryota Nakayama, Akiko Asada, Norio Matsuki, Yuji Ikegaya.
  The Journal of Physiology. May 19, 2014
  Key points In the brain, astrocytes detect neuronal activity and regulate neuronal excitability and synaptic transmission. Recent studies show that calcium elevations that are localized within astrocyte processes upregulate endogenous neurotransmission at nearby synapses. We demonstrated that at the network level calcium buffering in astrocytes caused a significant reduction in the correlated activity of neurons in cultured hippocampal slices. In contrast, the uncaging of calcium in astrocytes triggered synchronized activity in neuronal populations. This study provides experimental support for the functional relevance of astrocyte signalling to the maintenance of collective neuronal dynamics. Abstract Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level.

  May 19, 2014   doi: 10.1113/jphysiol.2014.272864   open full text
• Muscle Specific Kinase autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.
  Nazanin Ghazanfari, Marco Morsch, Stephen W. Reddel, Simon X. Liang, William D. Phillips.
  The Journal of Physiology. May 17, 2014
  Muscle Specific Kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti‐MuSK‐positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse labelling of AChRs revealed an accelerated loss of pre‐existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly‐synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β‐subunit‐Y390 was reduced at endplates. In contrast, endplate staining for β‐dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti‐MuSK IgG suppresses the endplate density of MuSK, thereby down‐regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2013.270207   open full text
• Myoblasts from intrauterine growth‐restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibers.
  Dustin T. Yates, Derek S. Clarke, Antoni R. Macko, Miranda J. Anderson, Leslie A. Shelton, Marie Nearing, Ronald E. Allen, Robert P. Rhoads, Sean W. Limesand.
  The Journal of Physiology. May 17, 2014
  Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofiber hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofiber areas. IUGR semitendinosus muscles had similar percentages of pax7‐positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin‐positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA‐positive/pax7‐positive; 44 ± 2% and 52 ± 1%) than controls. Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD‐positive cells than controls and replicated 20% less (P < 0.05) during a 2‐hour BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS‐supplemented media compared to control FSS‐supplemented media. In differentiation‐promoting media (2%FBS), IUGR and control myoblasts had similar percentages of myogenin‐positive nuclei after 5 days and formed similar‐sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofiber size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR‐born offspring. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2014.272591   open full text
• Noradrenergic modulation of masseter muscle activity during natural rapid eye movement sleep requires glutamatergic signalling at the trigeminal motor nucleus.
  Peter B. Schwarz, Saba Mir, John H. Peever.
  The Journal of Physiology. May 17, 2014
  Noradrenergic neurotransmission in the brainstem is closely coupled to changes in muscle activity across the sleep‐wake cycle, and noradrenaline is considered to be a key excitatory neuromodulator that reinforces arousal‐related stimulus on motoneurons to drive movement. However, it is unknown if α‐1 noradrenoceptor activation increases motoneuron responsiveness to excitatory glutamate (AMPA) receptor‐mediated inputs during natural behaviour. Here, we studied the effects of noradrenaline on AMPA receptor‐mediated motor activity at the motoneuron level in freely‐behaving rats, particularly during rapid eye movement (REM) sleep, a period exhibiting both AMPA receptor‐triggered muscle twitches and periods of muscle quiescence during which AMPA drive is silent. Male rats were instrumented for electromyography and electroencephalography recording to monitor sleep and waking behaviour, while implantation of a cannula into the trigeminal motor nucleus of the brainstem allowed us to perfuse noradrenergic and glutamatergic drugs by reverse microdialysis, using masseter muscle activity as an index of motoneuronal output. We found that endogenous excitation of both α‐1 noradrenoceptor and AMPA receptors during waking are coupled to motor activity; however, REM sleep exhibits an absence of endogenous α‐1 noradrenoceptor activity. Importantly, exogenous α‐1 noradrenoceptor stimulation cannot reverse the muscle twitch suppression induced by AMPA receptor blockade, nor can it elevate muscle activity during quiet REM, a phase when endogenous AMPA receptor activity is subthreshold. We conclude that the presence of an endogenous glutamatergic drive is necessary for noradrenaline to trigger muscle activity at the level of the motoneuron in a naturally‐behaving animal. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2014.272633   open full text
• The effective neural drive to muscles is the common synaptic input to motor neurons.
  Dario Farina, Francesco Negro, Jakob Lund Dideriksen.
  The Journal of Physiology. May 17, 2014
  We analyzed the transformation of synaptic input to the pool of motor neurons into the neural drive to the muscle. The aim was to explain the relations between common oscillatory signals sent to motor neurons and the effective component of the neural signal sent to muscles as output of the spinal cord circuitries. The approach is based on theoretical derivations, computer simulations, and experiments. It is shown theoretically that for frequencies smaller than the average discharge rates of the motor neurons, the pool of motor neurons determines a pure amplification of the frequency components common to all motor neurons, so that the common input is transmitted almost undistorted and the non‐common components are strongly attenuated. The effective neural drive to the muscle thus mirrors the common synaptic input to motor neurons. The simulations with three models of motor neuron confirmed the theoretical results by showing that the coherence function between common input components and the neural drive to the muscle tends to one when increasing the number of active motor neurons. This result, which was relatively insensitive to the type of model used, was also supported experimentally by observing that, in the low‐pass signal bandwidth, the peak in coherence between groups of motor units of the abductor digiti minimi muscle of 5 healthy subjects tended to one when increasing the number of motor units. These results have implications for our understanding of the neural control of muscles as well as for methods used for estimating the strength of common input to populations of motor neurons. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2014.273581   open full text
• Exercise training modulates functional sympatholysis and alpha‐adrenergic vasoconstrictor responsiveness in hypertensive and normotensive individuals.
  Stefan P. Mortensen, Michael Nyberg, Lasse Gliemann, Pia Thaning, Bengt Saltin, Ylva Hellsten.
  The Journal of Physiology. May 17, 2014
  Essential hypertension is linked to an increased sympathetic vasoconstrictor activity and reduced tissue perfusion. We investigated the role of exercise training on functional sympatholysis and postjunctional α‐adrenergic responsiveness in individuals with essential hypertension. Leg haemodynamics were measured before and after 8 weeks of aerobic training (3–4 times/week) in 8 hypertensive (47 ± 2 years) and 8 normotensive untrained individuals (46 ± 1 years) during arterial tyramine infusion, arterial ATP infusion and/or one‐legged knee extensions. Before training, exercise hypaeremia and leg vascular conductance (LVC) were lower in the hypertensive individuals (P < 0.05) and tyramine lowered exercise hypaeremia and VC in both groups (P < 0.05). Training lowered blood pressure in the hypertensive individuals (P < 0.05) and exercise hypaeremia was similar to the normotensive individuals in the trained state. After training, tyramine did not reduce exercise hyperaemia or LVC in either group. When tyramine was infused at rest, the reduction in blood flow and LVC was similar between groups, but exercise training lowered the magnitude of the reduction in blood flow and LVC (P < 0.05). There was no difference in the vasodilatory response to infused ATP or in muscle P2Y2 receptor content between the groups before and after training. However, training lowered the vasodilatory response to ATP and increased skeletal muscle P2Y2 receptor content in both groups (P < 0.05). These results demonstrate that exercise training improves functional sympatholysis and reduces postjunctional α‐adrenergic responsiveness in both normo‐ and hypertensive individuals. The ability for functional sympatholysis and the vasodilator and sympatholytic effect of intravascular ATP appears not to be altered in essential hypertension. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2014.273722   open full text
• Native store‐operated calcium channels are functionally expressed in mouse spinal cord dorsal horn neurons and regulate resting calcium homeostasis.
  Jingsheng Xia, Rong Pan, Xinghua Gao, Olimpia Meucci, Huijuan Hu.
  The Journal of Physiology. May 17, 2014
  Store‐operated calcium channels (SOCs) are calcium‐selective cation channels that mediate calcium entry in many different cell types. Store‐operated calcium entry (SOCE) is involved in various cellular functions. Increasing evidence suggests that impairment of SOCE is responsible for numerous disorders. Our previous study demonstrated that YM‐58483, a potent SOC inhibitor, strongly attenuates chronic pain by systemic or intrathecal injection and completely blocks the second phase of formalin‐induced spontaneous nocifensive behaviour, suggesting a potential role of SOCs in central sensitization. However, the expression of SOCs, their molecular identity and function in spinal cord dorsal horn neurons remain elusive. Here, we demonstrated that SOCs are expressed in dorsal horn neurons. Depletion of calcium stores from the endoplasmic reticulum (ER) induced large sustained calcium entry, which was blocked by SOC inhibitors, but not by voltage‐gated calcium channel blockers. Depletion of ER calcium stores activated inward calcium selective currents, which was reduced by replacing Ca2+ with Ba2+ and reversed by SOC inhibitors. Using the siRNA knockdown approach, we identified both STIM1 and STIM2 as important mediators of SOCE and SOC current, and Orai1 as a key component of the Ca2+ release‐activated Ca2+ (CRAC) channels in dorsal horn neurons. Knockdown of STIM1, STIM2 or Orai1 decreased resting Ca2+ levels. We also found that activation of NK1 receptors leaded to SOCE and activation of SOCs produced an excitatory action in dorsal horn neurons. Our findings reveal that a novel SOC signal is present in dorsal horn neurons and may play an important role in pain transmission. This article is protected by copyright. All rights reserved

  May 17, 2014   doi: 10.1113/jphysiol.2014.275065   open full text
• Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice.
  Gregor Sachse, Jörg Faulhaber, Anika Seniuk, Heimo Ehmke, Olaf Pongs.
  The Journal of Physiology. May 16, 2014
  Key points Large conductance voltage‐ and Ca2+‐activated K+ channels (BK channels) require the ancillary subunit BKβ1 for normal function in smooth muscle, renal and adrenal tissues. BK channels influence vascular tone and blood pressure in mice, and a gain‐of‐function BKβ1 polymorphism has been associated with low prevalence of diastolic hypertension in human population studies. In this study, we genetically removed the BKβ1 gene in three different strains of mice and then restored BKβ1 expression selectively in smooth muscle to determine its tissue‐specific contribution to blood pressure. We show that loss of BKβ1 in smooth muscle cells robustly increases vascular tone, but blood pressure of mice lacking BKβ1 was increased, unaltered or decreased depending on the genetic background. The results clarify the contested view that BK channel activity influences blood pressure by setting vascular tone and they shed light on the relative contribution of vascular and renal/adrenal BK channel activity to blood pressure levels. Abstract The large conductance voltage‐ and Ca2+‐activated K+ (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1−/−) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1−/− mice independent of genetic background, BKβ1−/− strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1+/+ controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1−/− strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1+/+ strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle‐restricted restoration of BKβ1 expression increased blood pressure in BKβ1−/− strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways.

  May 16, 2014   doi: 10.1113/jphysiol.2014.272880   open full text
• Maternal melatonin administration mitigates coronary stiffness and endothelial dysfunction, and improves heart resilience to insult in growth restricted lambs.
  Marianne Tare, Helena C. Parkington, Euan M. Wallace, Amy E. Sutherland, Rebecca Lim, Tamara Yawno, Harold A. Coleman, Graham Jenkin, Suzanne L. Miller.
  The Journal of Physiology. May 16, 2014
  Key points Failure of the placenta to develop and perform gives rise to intrauterine growth restriction (IUGR) in the fetus. IUGR is associated with impaired heart function in childhood and can even persist long term. Oxidative stress is increased in IUGR and we asked if an antioxidant could reduce this. Melatonin is a well‐known and well‐studied hormone, present in all of us, and it has potent antioxidant properties. In this study we administered melatonin to pregnant ewes carrying twins, one with IUGR. Maternal melatonin improved oxygen delivery to the IUGR fetus and strengthened and protected its heart against infarct. After birth, the poor function and stiffness in the coronary arteries of IUGR lambs were entirely prevented by melatonin. Our results demonstrate that administration of melatonin to a mother carrying an IUGR fetus can markedly dampen the adverse heart and artery effects in the offspring following birth. Abstract Intrauterine growth restriction (IUGR) is associated with impaired cardiac function in childhood and is linked to short‐ and long‐term morbidities. Placental dysfunction underlies most IUGR, and causes fetal oxidative stress which may impact on cardiac development. Accordingly, we investigated whether antenatal melatonin treatment, which possesses antioxidant properties, may afford cardiovascular protection in these vulnerable fetuses. IUGR was induced in sheep fetuses using single umbilical artery ligation on day 105–110 of pregnancy (term 147). Study 1: melatonin (2 mg h−1) was administered i.v. to ewes on days 5 and 6 after surgery. On day 7 fetal heart function was assessed using a Langendorff apparatus. Study 2: a lower dose of melatonin (0.25 mg h−1) was administered continuously following IUGR induction and the ewes gave birth normally at term. Lambs were killed when 24 h old and coronary vessels studied. Melatonin significantly improved fetal oxygenation in vivo. Contractile function in the right ventricle and coronary flow were enhanced by melatonin. Ischaemia–reperfusion‐induced infarct area was 3‐fold greater in IUGR hearts than in controls and this increase was prevented by melatonin. In isolated neonatal coronary arteries, endothelium‐dependent nitric oxide (NO) bioavailability was reduced in IUGR, and was rescued by modest melatonin treatment. Melatonin exposure also induced the emergence of an indomethacin‐sensitive vasodilation. IUGR caused marked stiffening of the coronary artery and this was prevented by melatonin. Maternal melatonin treatment reduces fetal hypoxaemia, improves heart function and coronary blood flow and rescues cardio‐coronary deficit induced by IUGR.

  May 16, 2014   doi: 10.1113/jphysiol.2014.270934   open full text
• Nucleus accumbens neuronal maturation differences in young rats bred for low versus high voluntary running behaviour.
  Michael D. Roberts, Ryan G. Toedebusch, Kevin D. Wells, Joseph M. Company, Jacob D. Brown, Clayton L. Cruthirds, Alexander J. Heese, Conan Zhu, George E. Rottinghaus, Thomas E. Childs, Frank W. Booth.
  The Journal of Physiology. May 15, 2014
  Key points Selective breeding experiments with laboratory rodents have demonstrated the heritability of voluntary exercise. We performed RNA sequencing and bioinformatics analyses of the reward and pleasure hub in the brain – the nucleus accumbens – in rats selectively bred for low voluntary running (LVR) versus high voluntary running (HVR). The discovery of unique genes and ‘cell cycle’‐related gene pathways between lines guided our hypothesis that neuron maturation may be lower in LVR rats. Testing of this hypothesis revealed that the LVR line inherently possessed fewer mature medium spiny neurons and fewer immature neurons than their HVR counterparts. However, minimal running in LVR rats appeared to rescue and/or reverse these effects. Neuron maturation in the nucleus accumbens is related to low running voluntary behaviour in our model; this allows researchers to understand the potential neural mechanisms that underlie the motivations for low physical activity behaviour. Abstract We compared the nucleus accumbens (NAc) transcriptomes of generation 8 (G8), 34‐day‐old rats selectively bred for low (LVR) versus high voluntary running (HVR) behaviours in rats that never ran (LVRnon‐run and HVRnon‐run), as well as in rats after 6 days of voluntary wheel running (LVRrun and HVRrun). In addition, the NAc transcriptome of wild‐type Wistar rats was compared. The purpose of this transcriptomics approach was to generate testable hypotheses as to possible NAc features that may be contributing to running motivation differences between lines. Ingenuity Pathway Analysis and Gene Ontology analyses suggested that ‘cell cycle’‐related transcripts and the running‐induced plasticity of dopamine‐related transcripts were lower in LVR versus HVR rats. From these data, a hypothesis was generated that LVR rats might have less NAc neuron maturation than HVR rats. Follow‐up immunohistochemistry in G9–10 LVRnon‐run rats suggested that the LVR line inherently possessed fewer mature medium spiny (Darpp‐32‐positive) neurons (P < 0.001) and fewer immature (Dcx‐positive) neurons (P < 0.001) than their G9–10 HVR counterparts. However, voluntary running wheel access in our G9–10 LVRs uniquely increased their Darpp‐32‐positive and Dcx‐positive neuron densities. In summary, NAc cellularity differences and/or the lack of running‐induced plasticity in dopamine signalling‐related transcripts may contribute to low voluntary running motivation in LVR rats.

  May 15, 2014   doi: 10.1113/jphysiol.2013.268805   open full text
• Mg2+ block properties of triheteromeric GluN1–GluN2B–GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones.
  Zhuo Huang, Alasdair J. Gibb.
  The Journal of Physiology. May 15, 2014
  Key points NMDAR receptors (NMDARs) are tetrameric cation channels permeable to calcium and blocked by Mg2+. Voltage‐dependent Mg2+ block of NMDARs is crucial to several forms of synaptic plasticity and to the integration of synaptic activity with neuronal activity. Although diheteromeric GluN1–GluN2A or GluN1–GluN2B NMDARs display stronger voltage‐dependent Mg2+ block than GluN1–GluN2C or GluN1–GluN2D NMDARs, the extracellular Mg2+ block properties for triheteromeric NMDARs are still elusive. Here, we show that in dopaminergic neurones the voltage dependence of Mg2+ block is less steep than previously observed in hippocampus or cortex, consistent with the presence of triheteromeric GluN1–GluN2B–GluN2D NMDARs. These results may help to understand the role of triheteromeric NMDARs in dopaminergic neurone synaptic plasticity and to inform simulations of dopaminergic neurone physiology. Abstract Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg2+ is an important regulator of NMDARs, and particularly the voltage dependence of Mg2+ block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg2+ block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1–GluN2B–GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage‐dependent Mg2+ block properties of triheteromeric NMDARs. In control conditions, external Mg2+ significantly inhibits the whole cell NMDA‐evoked current in a voltage‐dependent manner with IC50 values of 20.9 μm, 53.3 μm and 173 μm at −90 mV, −70 mV and −50 mV, respectively. When the GluN2B‐selective antagonist ifenprodil was applied, the Mg2+ sensitivity of the residual NMDA‐mediated currents (which is mainly carried by GluN1–GluN2B–GluN2D NMDARs) is reduced to IC50 values of 45.9 μm (−90 mV), 104 μm (−70 mV) and 276 μm (−50 mV), suggesting that triheteromeric GluN1–GluN2B–GluN2D NMDARs have less affinity for external Mg2+ than GluN1–GluN2B receptors. In addition, fitting INMDA–V curves with a trapping Mg2+ block model shows the triheteromeric GluN1–GluN2B–GluN2D NMDARs have weaker voltage‐dependent Mg2+ block (δ = 0.56) than GluN1–GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1–GluN2B–GluN2D rather than GluN1–GluN2D NMDARs are present. These data provide information relevant to Mg2+ block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.

  May 15, 2014   doi: 10.1113/jphysiol.2013.267864   open full text
• It's a gut feeling: How the gut microbiota affects the state of mind.
  Adam D. Farmer, Holly A. Randall, Qasim Aziz.
  The Journal of Physiology. May 13, 2014
  Common human experience shows that stress and anxiety may modulate gut function. Such observations have been combined with an increasing evidence base that has culminated in the concept of the brain–gut axis. Nevertheless, it has not been until recently that the gut and its attendant components have been considered to influence higher cerebral function and behaviour per se. Moreover, the proposal that the gut and the bacteria contained therein (collectively referred to as the microbiota) can modulate mood and behaviours, has an increasing body of supporting evidence, albeit largely derived from animal studies. The gut microbiota is a dynamic and diverse ecosystem and forms a symbiotic relationship with the host. Herein we describe the components of the gut microbiota and mechanisms by which it can influence neural development, complex behaviours and nociception. Furthermore, we propose the novel concept of a ‘state of gut’ rather than a state of mind, particularly in relation to functional bowel disorders. Finally, we address the exciting possibility that the gut microbiota may offer a novel area of therapeutic intervention across a diverse array of both affective and gastrointestinal disorders.

  May 13, 2014   doi: 10.1113/jphysiol.2013.270389   open full text
• Pre‐exposure to adenosine, acting via A2A receptors on endothelial cells, alters the protein kinase A dependence of adenosine‐induced dilation in skeletal muscle resistance arterioles.
  Nir Maimon, Patricia A. Titus, Ingrid H. Sarelius.
  The Journal of Physiology. May 13, 2014
  Key points The adenosine‐dependent component of functional dilation of small resistance arterioles acts via A2A receptors located on endothelium to activate KATP channels on associated vascular smooth muscle. A2A receptors are Gs‐coupled, hence receptor occupancy should activate cAMP/protein kinase A (PKA) cell signalling pathways However, pre‐exposure to adenosine alters the PKA dependence of the response and renders the vessel insensitive to PKA inhibition. The adenosine pre‐exposure effect is mimicked by pre‐activation of PKA and is specific to adenosine, as the PKA dependence of dilation to isoproterenol (another Gs‐coupled agonist) is not affected by pre‐exposure. Activation of PKA alone does not induce dilation. An additional signalling mechanism, dependent on increased EC Ca2+ via activation of cyclic nucleotide gated channels, is required together with PKA activation to produce A2A‐dependent vasodilation. This novel identification of variability in signalling downstream from a single receptor to produce the same response may reflect a mechanism for integration of key homeostatic responses. Abstract Adenosine (ADO) is an endogenous vasodilatory purine widely recognized to be a significant contributor to functional hyperaemia. Despite this, many aspects of the mechanisms by which ADO induces dilation in small resistance arterioles are not established, or appear contradictory. These include: identification of the primary receptor subtype; its location on endothelial (EC) or vascular smooth muscle cells; whether ADO acts on KATP channels in these resistance vessels; and the contribution of cAMP/protein kinase A (PKA) signalling to the response. In intravital microscopy studies of intact or EC‐denuded skeletal muscle arterioles, we show that ADO acts via A2A receptors located on ECs to produce vasodilation via activation of KATP channels located on vascular smooth muscle cells. Importantly, we found that the signalling pathway involves cAMP as expected, but that a requirement for PKA activation is demonstrable only if the vessel is not pre‐exposed to ADO. That is, PKA‐dependent signalling varies with pre‐exposure to ADO. Further, we show that PKA activation alone is not sufficient to dilate these arterioles; an additional EC calcium‐dependent signalling mechanism is required for vasodilation to ADO. The ability of arterioles in situ to respond to occupancy of a specific receptor by utilizing different cell signalling pathways under different conditions to produce the same response allows the arteriole to respond to key homeostatic requirements using more than a single signalling mechanism. Clearly, this is likely to be physiologically advantageous, but the role for this signalling flexibility in the integrated arteriolar response that underlies functional hyperaemia will require further exploration.

  May 13, 2014   doi: 10.1113/jphysiol.2013.265835   open full text
• Transient impairment of the axolemma following regional anaesthesia by lidocaine in humans.
  Mihai Moldovan, Kai Henrik Wiborg Lange, Niels Jacob Aachmann‐Andersen, Troels Wesenberg Kjær, Niels Vidiendal Olsen, Christian Krarup.
  The Journal of Physiology. May 13, 2014
  Key points We tested the recovery of motor axon conduction and multiple measures of excitability by ‘threshold‐tracking’ after ultrasound‐guided distal median nerve regional anaesthesia by lidocaine. Lidocaine caused a transient conduction failure that recovered completely by 3 h, whereas excitability recovered only partially by 6 h and fully by 24 h. The up to 7‐fold increase in threshold after complete recovery of conduction was associated with excitability changes that could only partially be explained by block of the voltage‐gated Na+ channel (VGSC). Mathematical modelling indicated that, apart from a reduction in the number of functioning VGSCs, lidocaine also caused a decrease of passive membrane resistance and an increase of capacitance. Our data suggest that lidocaine, even at clinical ‘sub‐blocking’ concentrations, could cause a reversible structural impairment of the axolemma. Abstract The local anaesthetic lidocaine is known to block voltage‐gated Na+ channels (VGSCs), although at high concentration it was also reported to block other ion channel currents as well as to alter lipid membranes. The aim of this study was to investigate whether the clinical regional anaesthetic action of lidocaine could be accounted for solely by the block of VGSCs or whether other mechanisms are also relevant. We tested the recovery of motor axon conduction and multiple measures of excitability by ‘threshold‐tracking’ after ultrasound‐guided distal median nerve regional anaesthesia in 13 healthy volunteers. Lidocaine caused rapid complete motor axon conduction block localized at the wrist. Within 3 h, the force of the abductor pollicis brevis muscle and median motor nerve conduction studies returned to normal. In contrast, the excitability of the motor axons at the wrist remained markedly impaired as indicated by a 7‐fold shift of the stimulus–response curves to higher currents with partial recovery by 6 h and full recovery by 24 h. The strength–duration properties were abnormal with markedly increased rheobase and reduced strength–duration time constant. The changes in threshold during electrotonus, especially during depolarization, were markedly reduced. The recovery cycle showed increased refractoriness and reduced superexcitability. The excitability changes were only partly similar to those previously observed after poisoning with the VGSC blocker tetrodotoxin. Assuming an unaltered ion‐channel gating, modelling indicated that, apart from up to a 4‐fold reduction in the number of functioning VGSCs, lidocaine also caused a decrease of passive membrane resistance and an increase of capacitance. Our data suggest that the lidocaine effects, even at clinical ‘sub‐blocking’ concentrations, could reflect, at least in part, a reversible structural impairment of the axolemma.

  May 13, 2014   doi: 10.1113/jphysiol.2014.270827   open full text
• Changes in spinal inhibitory networks induced by furosemide in humans.
  Wanalee Klomjai, Alexandra Lackmy‐Vallée, Rose Katz, Bernard Bussel, Djamel Bensmail, Jean‐Charles Lamy, Nicolas Roche.
  The Journal of Physiology. May 09, 2014
  During neural development in animals, GABAergic and glycinergic neurons are first excitatory, and then become inhibitory in the mature state. This developmental shift is mainly due to strong expression of the cation‐chloride KCC2 and down‐regulation of co‐transporters NKCC1 during maturation. The down‐regulation of co‐transporter KCC2 after spinal cord transection in animals leads to the depolarising (excitatory) action of GABA and glycine and thus results in a reduction of inhibitory synaptic efficiency. Furosemide, a loop diuretic, has been shown to selectively and reversibly block inhibitory postsynaptic potentials (IPSPs) without affecting excitatory postsynaptic potentials (EPSPs) in animal spinal neurons. Moreover, this diuretic has been also demonstrated to block the cation‐chloride co‐transporters. Here, we used furosemide to demonstrate changes in spinal inhibitory networks in healthy human subjects. Non‐invasive electrophysiological techniques were used to assess presynaptic inhibition, postsynaptic inhibition and the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons in the spinal cord. Orally‐administered furosemide, at doses commonly used in the clinic (40 mg), significantly reduced spinal inhibitory interneuronal activity for at least 70 min from intake compared to control experiments in the same subjects while no changes were observed in the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons. The reduction of inhibition was dose‐dependent. Our results provide indirect evidence that reversible changes in the cation‐chloride transport system induce modulations of inhibitory neuronal activity at spinal cord level in humans. This article is protected by copyright. All rights reserved

  May 09, 2014   doi: 10.1113/jphysiol.2013.265314   open full text
• A functional coupling between extrasynaptic NMDA receptors and A‐Type K+ channels under astrocyte control regulates hypothalamic neurosecretory neuronal activity.
  Krishna Naskar, Javier E. Stern.
  The Journal of Physiology. May 09, 2014
  Neuronal activity is controlled by a fine‐tuned balance between intrinsic properties and extrinsic synaptic inputs. Moreover, neighboring astrocytes are now recognized to influence a wide spectrum of neuronal functions. Yet, how these three key factors act in concert to modulate and fine tune neuronal output is not well understood. Here, we show that in rat hypothalamic magnocellular neurosecretory cells (MNCs), glutamate NMDA receptors (NMDARs) are negatively coupled to the transient, voltage‐gated A‐type K+ current (IA). We found that activation of NMDARs by extracellular glutamate levels influenced by astrocyte glutamate transporters, resulted in a significant inhibition of IA. The NMDAR‐IA functional coupling resulted from activation of extrasynaptic NMDARs, was calcium‐ and PKC‐dependent, and involved enhanced steady state, voltage‐dependent inactivation of IA. The NMDAR‐IA coupling diminished the latency to the first evoked spike in response to membrane depolarization and increased the total number of evoked action potentials, strengthening thus the neuronal input/output function. Finally, we found a blunted NMDA‐mediated inhibition of IA in dehydrated rats. Together, our findings support a novel signalling mechanism that involves a functional coupling between extrasynaptic NMDARs and A‐type K+ channels, which is influenced by local astrocytes. We show this signalling complex to play an important role in modulating hypothalamic neuronal excitability, which may contribute to adaptive responses during a sustained osmotic challenge such as dehydration. This article is protected by copyright. All rights reserved

  May 09, 2014   doi: 10.1113/jphysiol.2014.270793   open full text
• Dehydration affects cerebral blood flow but not its metabolic rate for oxygen during maximal exercise in trained humans.
  Steven J. Trangmar, Scott T. Chiesa, Christopher G. Stock, Kameljit K. Kalsi, Niels H. Secher, José González‐Alonso.
  The Journal of Physiology. May 09, 2014
  Intense exercise is associated with a reduction in cerebral blood flow (CBF), but regulation of CBF during strenuous exercise in the heat with dehydration is unclear. We assessed internal‐(ICA) and common‐carotid artery (CCA) haemodynamics (indicative of CBF and extra‐cranial blood flow), middle cerebral artery velocity (MCA Vmean), a‐v differences, and blood temperature in 10 trained males during incremental cycling to exhaustion in the heat (35 °C) in control, dehydrated and rehydrated states. Dehydration reduced body mass (75.8 ± 3 vs. 78.2 ± 3 kg), increased internal temperature (38.3 ± 0.1 vs. 36.8 ± 0.1 °C), impaired exercise capacity (269 ± 11 vs. 336 ± 14 W), and lowered ICA and MCA Vmean by 12–23% without compromising CCA blood flow. During euhydrated incremental exercise on a separate day, however, exercise capacity and ICA, MCA Vmean and CCA dynamics were preserved. The fast decline in cerebral perfusion with dehydration was accompanied by increased O2 extraction (P < 0.05), resulting in a maintained cerebral metabolic rate for oxygen (CMRO2). In all conditions, reductions in ICA and MCA Vmean were associated with declining cerebral vascular conductance, increasing jugular venous noradrenaline, and falling PaCO2 (R2 ≥ 0.41, P ≤ 0.01) whereas CCA flow and conductance were related to elevated blood temperature. In conclusion, dehydration accelerated the decline in CBF by decreasing PaCO2 and enhancing vasoconstrictor activity. However, the circulatory strain on the human brain during maximal exercise does not compromise CMRO2 because of compensatory increases in O2 extraction. This article is protected by copyright. All rights reserved

  May 09, 2014   doi: 10.1113/jphysiol.2014.272104   open full text
• Constitutive phosphorylation of myosin phosphatase targeting subunit‐1 in smooth muscle.
  Ming‐Ho Tsai, Audrey N. Chang, Jian Huang, Weiqi He, H. Lee Sweeney, Minsheng Zhu, Kristine E. Kamm, James T. Stull.
  The Journal of Physiology. May 09, 2014
  Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle‐specific deletion of MYPT1 in adult mice. Deep‐sequencing analyses of mRNA and immunoblotting revealed MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wildtype or MYPT1‐deficient tissues. Isolated MYPT1‐deficient tissues from MYPT1SM−/− mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5‐fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wildtype tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1‐deficient tissues may be similar to the attenuated MLCP activity in wildtype tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist‐induced MYPT1 phosphorylation to inhibit MLCP activity. In summary, MYPT1‐deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed. This article is protected by copyright. All rights reserved

  May 09, 2014   doi: 10.1113/jphysiol.2014.273011   open full text
• Balance between the proximal dendritic compartment and the soma determines spontaneous firing rate in midbrain dopamine neurons.
  Jinyoung Jang, Ki Bum Um, Miae Jang, Shin Hye Kim, Hana Cho, Sungkwon Chung, Hyun Jin Kim, Myoung Kyu Park.
  The Journal of Physiology. May 09, 2014
  Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that require the elaborate composition of many ion channels in the somatodendritic compartments. Understanding the major determinants of the spontaneous firing rate (SFR) of the midbrain DA neurons is important because they determine the basal DA levels in target areas, including the striatum. Since spontaneous firing occurs synchronously at the soma and dendrites, the electrical coupling between the soma and dendritic compartments has been regarded as a key determinant for the SFR. However, it is not known whether this somatodendritic coupling is served by the whole dendritic compartments or only parts of them. In the rat substantia nigra pars compacta (SNc) DA neurons, here we demonstrate that the balance between the proximal dendritic compartment and the soma determines the SFR. Isolated SNc DA neurons showed a wide range of soma size and a variable number of primary dendrites but preserved a quite consistent SFR. The SFR was not correlated with the soma size or with the number of primary dendrites, but it was strongly correlated with the area ratios of the proximal dendritic compartments to the somatic compartment. Tetrodotoxin puff and local Ca2+ perturbation experiments, computer simulation, and local glutamate uncaging experiments suggest the importance of the proximal dendritic compartments in pacemaker activity. These data indicate that the proximal dendritic compartments, not the whole dendritic compartments, play a key role in the somatodendritic balance that determines the SFR in the DA neurons. This article is protected by copyright. All rights reserved

  May 09, 2014   doi: 10.1113/jphysiol.2014.275032   open full text
• Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma.
  Maurizio Vitadello, Elena Germinario, Barbara Ravara, Luciano Dalla Libera, Daniela Danieli‐Betto, Luisa Gorza.
  The Journal of Physiology. May 08, 2014
  Key points Attenuation of disuse muscle atrophy by means of a pharmacological approach represents the wanted solution for patients who cannot exercise. Nutraceutics, e.g. the vegetal polyphenol curcumin, are relatively safe substances. By lowering oxidative stress, curcumin counteracted the loss of muscle mass and force of soleus muscles, reproduced in the laboratory rat by hindlimb unloading Curcumin effects are mediated by the chaperone protein Grp94, which maintains active neuronal nitric oxide synthase molecules at their physiological site in the skeletal myofibre. The systemic administration of very low doses of curcumin appears promising for counteracting muscle atrophy in bedridden patients. Abstract Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose‐regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco‐endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross‐sectional area by approximately 30% (P ≤ 0.02) and maintained a force–frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin‐induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug‐positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro‐atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.

  May 08, 2014   doi: 10.1113/jphysiol.2013.268672   open full text
• Adiponectin is sufficient, but not required, for exercise‐induced increases in the expression of skeletal muscle mitochondrial enzymes.
  Ian R. W. Ritchie, Tara L. MacDonald, David C. Wright, David J. Dyck.
  The Journal of Physiology. May 08, 2014
  Key points Adiponectin is a regulator of skeletal muscle mitochondrial biogenesis. Previous research using the ob/ob mouse (leptin deficient, low adiponectin) has suggested that the presence of adipokines, including adiponectin, is necessary for exercise‐induced increases in mitochondrial content. In the current study, we examined the importance of adiponectin as a regulator of skeletal muscle mitochondrial content in response to exercise by comparing wildtype and adiponectin deficient mice. Adiponectin deficient mice showed no differences in resting VO2, RER, or time to exhaustion during exercise when compared to wildtype mice. There were no differences in various protein markers of mitochondrial content. A single bout of treadmill running increased the mRNA expression of mitochondrial proteins similarly in wildtype and adiponectin deficient mice. Chronic exercise (8 weeks) also increased the protein content of mitochondrial markers similarly in wildtype and adiponectin deficient mice. We conclude that Ad is not required for exercise‐induced increases in muscle mitochondrial proteins. Abstract Adiponectin (Ad) has been proposed to be a regulator of mitochondrial biogenesis in skeletal muscle, and necessary for exercise‐induced increases in mitochondrial content. We first confirmed that Ad could acutely increase the expression of mitochondrial proteins during a 10 h incubation in isolated soleus and extensor digitorum longus (EDL) muscles. Next, we further examined the role of Ad as a regulator of mitochondrial content using Ad knockout (AdKO) mice. The AdKO animals showed no differences in resting VO2, respiratory exchange ratio, or in time to exhaustion during exercise when compared to wild‐type (WT) mice. There was a reduction in resting palmitate oxidation in isolated soleus from AdKO animals (−23%, P < 0.05) but not EDL, and 5‐aminoimidazole‐4‐carboxamide (AICAR)‐stimulated palmitate oxidation was similar in both genotypes regardless of muscle. There were no differences in protein markers of mitochondrial content (COX4, CORE1, CS, PDHE1α) in red and white gastrocnemius between WT and AdKO animals. A single bout of treadmill running increased the phosphorylation of AMP‐activated protein kinase (AMPK) and the mRNA expression of mitochondrial proteins in red and white gastrocnemius in both WT and AdKO animals, with no differences between genotypes. Finally, 8 weeks of chronic exercise training increased the protein content of mitochondrial markers similarly (∼25–35%) in red gastrocnemius from both WT and AdKO mice. Collectively, our results demonstrate that the absence of Ad is not accompanied by reductions in mitochondrial protein content, or a reduction in aerobic exercise capacity. We conclude that Ad is not required for the maintenance of mitochondrial content, or for exercise‐induced increases in skeletal muscle mitochondrial proteins.

  May 08, 2014   doi: 10.1113/jphysiol.2014.273680   open full text
• Agonists binding nicotinic receptors elicit specific channel‐opening patterns at αγ and αδ sites.
  Patrick Stock, Dmitrij Ljaschenko, Manfred Heckmann, Josef Dudel.
  The Journal of Physiology. May 06, 2014
  Key points High‐resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh) were compared. Ebd binds with 75‐fold higher affinity at αγ than at αδ sites, whereas CCh and ACh prefer αδ sites of nicotinic ACh receptor (nAChR) channels. Similar short (τO1), intermediate (τO2) and long (τO3) types of opening were observed. τO2 openings were maximally prevalent at low Ebd concentrations, binding at αγ sites, whereas τO1 openings appear to be generated at αδ sites. Short (∼0.75 ms) bursts of openings (τB1) arise from the αγ site, and long (>10 ms) bursts (τB2) arise from double liganded receptors. The duration of bursts and of openings within bursts depended on the agonist. Limited by the temporal resolution, the closings within bursts were invariant at 3 μs. Blocking αδ sites with α‐conotoxin M1 (CTx) eliminated both τO1 and τB2 and left only τO2 and the short τB1 bursts, as expected. Abstract ‘Embryonic’ muscle‐type nicotinic acetylcholine receptor channels (nAChRs) bind ligands at interfaces of α‐ and γ‐ or δ‐subunits. αγ and αδ sites differ in affinity, but their contributions to opening the channel have remained elusive. We compared high‐resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh). Ebd binds with 75‐fold higher affinity at αγ than at αδ sites, whereas CCh and ACh prefer αδ sites. Similar short (τO1), intermediate (τO2) and long (τO3) types of opening were observed with all three agonists. τO2 openings were maximally prevalent at low Ebd concentrations, binding at αγ sites. By contrast, τO1 openings appear to be generated at αδ sites. In addition, two types of burst appeared: short bursts of an average of 0.75 ms (τB1) that should arise from the αγ site, and long bursts of 12–25 ms (τB2) in duration arising from double liganded receptors. Limited by the temporal resolution, the closings within bursts were invariant at 3 μs. Corrected for missed closings, in the case of ACh the openings within long bursts lasted 170 μs and those in short bursts about 30 μs. Blocking αδ sites with α‐conotoxin M1 (CTx) eliminated both τO1 and τB2 and left only τO2 and the short τB1 bursts, as expected. Furthermore we found desensitization when the receptors bound ACh only at the αγ site. When CTx was applied to ‘embryonic’ mouse endplates, monoquantal current rise times were increased, and amplitude and decay time constants were reduced, as expected. Thus the αγ and αδ sites of nAChRs elicit specific channel‐opening patterns.

  May 06, 2014   doi: 10.1113/jphysiol.2013.267781   open full text
• Contrasting actions of group I metabotropic glutamate receptors in distinct mouse striatal neurones.
  John G. Partridge, Amanda E. Lewin, Jessica R. Yasko, Stefano Vicini.
  The Journal of Physiology. May 01, 2014
  Key points Pharmacological activation of striatal Group I metabotropic glutamate receptors (mGluRs) increases the occurrence of GABAA‐mediated currents in striatal spiny projection neurones (SPNs). Genetically identified striatal interneurones are depolarized by Group I mGluR activation. Group I mGluR activation elevates intracellular calcium in genetically identified striatal interneurones expressing a genetically encoded calcium indicator. Group I mGluR activation results in increased intracellular calcium in SPNs only after priming with calcium influx. Combined electrophysiology and calcium imaging reveals that mGluR activation is not accompanied by depolarization in SPNs. Abstract In mouse striatum, metabotropic glutamate receptor (mGluR) activation leads to several modulatory effects in synaptic transmission. These effects range from dampening of glutamate release from excitatory terminals to depolarization of divergent classes of interneurones. We compared the action of group I mGluR activation on several populations of striatal neurones using a combination of genetic identification, electrophysiology, and Ca2+ imaging techniques. Patch‐clamp recordings from spiny projection neurones (SPNs) and various interneurone populations demonstrated that the group I mGluR agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG) robustly depolarizes several interneurone classes that form GABAergic synapses onto SPNs. We further utilized the genetic reporter mouse strain Ai38, which expresses the calcium indicator protein GCaMP3 in a Cre‐dependent manner. Breeding Ai38 mice with various neurone selective, promoter‐driven Cre recombinase mice resulted in GCaMP3 expression in defined cell populations in striatum. Consistent with our electrophysiological findings, group I agonist applications increased intracellular levels of calcium ([Ca2+]i) in all interneurone populations tested. We also found that acute DHPG application evoked a transient, rapid increase in [Ca2+]i from only a small percentage of identifiable SPNs. Surprisingly, this fast [Ca2+]i response exhibited a robust enhancement or sensitization, in a calcium‐dependent fashion. Following several procedures to increase [Ca2+]i, the vast majority of SPNs responded with rapid changes in [Ca2+]i to mGluR agonists in a time‐dependent fashion. These findings extend our understanding on group I mGluR influence of striatal output via powerful, local GABAergic connections in addition to [Ca2+]i dynamics that impact on activity or spike‐timing‐dependent forms of synaptic plasticity.

  May 01, 2014   doi: 10.1113/jphysiol.2014.272773   open full text
• The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio).
  Cosima S. Porteus, Sara J. Abdallah, Jacob Pollack, Yusuke Kumai, Raymond W.M. Kwong, Hong M. Yew, William K. Milsom, Steve F. Perry.
  The Journal of Physiology. April 24, 2014
  The current study investigated the role of hydrogen sulphide (H2S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish, Danio rerio. Both larval and adult zebrafish exhibited a dose‐dependent increase in ventilation to sodium sulphide (Na2S), a H2S donor. In vertebrates, cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CSE) are enzymes that catalyze the endogenous production of H2S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation; addition of Na2S to the water partially rescued the effects of inhibiting endogenous H2S production. In zebrafish larvae (4 days post fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs; putative oxygen chemoreceptors) increased significantly when these cells were exposed to 50 μm Na2S, supporting a role for H2S in Ca2+‐evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, while cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H2S can be produced in the putative oxygen sensing cells of zebrafish, the NECs, where it would appear to play a pivotal role in promoting the hypoxic ventilatory response. This article is protected by copyright. All rights reserved

  April 24, 2014   doi: 10.1113/jphysiol.2014.271098   open full text
• A novel short‐term plasticity of intrinsic excitability in the hippocampal ca1 pyramidal cells.
  A. Sánchez‐Aguilera, J. L. Sánchez‐Alonso, M. A. Vicente‐Torres, A. Colino.
  The Journal of Physiology. April 24, 2014
  Changes in neuronal activity often trigger compensatory mechanisms aimed at regulating network activity homeostatically. Here we have identified and characterized a novel form of compensatory short‐term plasticity of membrane excitability, which develops early after the eye‐opening period in rats (P16–19 days) but not before that developmental stage (P9–12 days old). Holding the membrane potential of CA1 neurons right below the firing threshold from 15 s to several minutes induced a potentiation of the repolarizing phase of the action potentials that contributed to a decrease in the firing rate of CA1 pyramidal neurons in vitro. Furthermore, the mechanism for inducing of this plasticity required the action of intracellular calcium entering through T‐type calcium channels. This increase in calcium subsequently activated the calcium‐sensor K+ channel interacting protein 3 which led to the increase of an A‐type potassium current. These results suggest that calcium modulation of somatic A‐current represents a new form of homeostatic regulation that provides CA1 pyramidal neurons with the ability to preserve their firing abilities in response to membrane potential variations on a scale from tens of seconds to several minutes. This article is protected by copyright. All rights reserved

  April 24, 2014   doi: 10.1113/jphysiol.2014.273185   open full text
• Ponto‐medullary nuclei involved in the generation of sequential pharyngeal swallowing and concomitant protective laryngeal adduction in situ.
  Tara G. Bautista, Mathias Dutschmann.
  The Journal of Physiology. April 23, 2014
  Key points Laryngeal adduction is a major mechanism in sealing off entry to the trachea to prevent aspiration during swallowing. An experimental protocol to reliably elicit sequential swallowing by oral injection of small volumes of water was developed in the in situ perfused brainstem preparation of juvenile rats. The sequential swallowing motor pattern consists of two distinct components: (i) phasic swallowing, indicated by rhythmic sequential vagal nerve bursting, and (ii) protective laryngeal adduction, indicated by background tonic vagal discharge. Pharmacological manipulation of the Kölliker–Fuse nucleus revealed that it specifically mediates the protective tonic laryngeal adduction, while GABAergic neurotransmission is needed in the nucleus of the solitary tract for the generation of the sequential swallowing motor pattern. We conclude that sequential swallow motor patterning, including effective airway protection, requires balanced excitatory–inhibitory synaptic interaction within the nucleus of the solitary tract and the Kölliker–Fuse nucleus, as well as between the two nuclei. Abstract Both swallowing and respiration involve postinspiratory laryngeal adduction. Swallowing‐related postinspiratory neurons are likely to be located in the nucleus of the solitary tract (NTS) and those involved in respiration are found in the Kölliker–Fuse nucleus (KF). The function of KF and NTS in the generation of swallowing and its coordination with respiration was investigated in perfused brainstem preparations of juvenile rats (n = 41). Orally injected water evoked sequential pharyngeal swallowing (s‐PSW) seen as phasic, spindle‐shaped bursting of vagal nerve activity (VNA) against tonic postinspiratory discharge. KF inhibition by microinjecting isoguvacine (GABAA receptor agonist) selectively attenuated tonic postinspiratory VNA (n = 10, P < 0.001) but had no effect on frequency or timing of s‐PSW. KF disinhibition after bicuculline (GABAA receptor antagonist) microinjections caused an increase of the tonic VNA (n = 8, P < 0.01) resulting in obscured and delayed phasic s‐PSW. Occurrence of spontaneous PSW significantly increased after KF inhibition (P < 0.0001) but not after KF disinhibition (P = 0.14). NTS isoguvacine microinjections attenuated the occurrence of all PSW (n = 5, P < 0.01). NTS bicuculline microinjections (n = 6) resulted in spontaneous activation of a disordered PSW pattern and long‐lasting suppression of respiratory activity. Pharmacological manipulation of either KF or NTS also triggered profound changes in respiratory postinspiratory VNA. Our results indicate that the s‐PSW comprises two functionally distinct components. While the primary s‐PSW is generated within the NTS, a KF‐mediated laryngeal adductor reflex safeguards the lower airways from aspiration. Synaptic interaction between KF and NTS is required for s‐PSW coordination with respiration as well as for proper gating and timing of s‐PSW.

  April 23, 2014   doi: 10.1113/jphysiol.2014.272468   open full text
• Mitochondria‐targeted antioxidant (MitoQ) ameliorates age‐related arterial endothelial dysfunction in mice.
  Rachel A. Gioscia‐Ryan, Thomas J. LaRocca, Amy L. Sindler, Melanie C. Zigler, Michael P. Murphy, Douglas R. Seals.
  The Journal of Physiology. April 23, 2014
  Key points The development of age‐related arterial endothelial dysfunction, a key antecedent of increased cardiovascular disease (CVD) risk, is mediated largely by reduced nitric oxide bioavailability as a consequence of oxidative stress. Mitochondria are critical signalling organelles in the vasculature, which, when dysregulated, become a source of excessive reactive oxygen species; the role of mitochondria‐derived oxidative stress in age‐related vascular dysfunction is unknown. We show that a mitochondria‐targeted antioxidant, MitoQ, ameliorates vascular endothelial dysfunction in old mice and that these improvements are associated with the normalization of mitochondria‐derived oxidative stress and markers of arterial mitochondrial health. These results indicate that mitochondria‐derived oxidative stress is an important mechanism underlying the development of age‐related vascular endothelial dysfunction and therefore may be a promising therapeutic target. Mitochondria‐targeted antioxidants represent a novel strategy for preserving healthy vascular endothelial function in primary ageing and preventing age‐related CVD in humans. Abstract Age‐related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age‐related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria‐targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium‐dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium‐independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age‐related increases in total and mitochondria‐derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age‐related increase in endothelial susceptibility to acute mitochondrial damage (rotenone‐induced impairment in EDD). Our results suggest that mitochondria‐derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria‐targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age‐related CVD.

  April 23, 2014   doi: 10.1113/jphysiol.2013.268680   open full text
• Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus.
  Rongqing Chen, Akihito Okabe, Haiyan Sun, Salim Sharopov, Ileana L. Hanganu‐Opatz, Sergei N. Kolbaev, Atsuo Fukuda, Heiko J. Luhmann, Werner Kilb.
  The Journal of Physiology. April 23, 2014
  Key points Taurine has a pro‐ and anticonvulsive effect on the immature hippocampus, depending on the dose. The taurine effect is mediated by GABAA and glycine receptors. The taurine effect can be partially mimicked by glycine. Inhibition of glycine receptors has a weak proconvulsive effect on the immature hippocampus. We conclude that an endogenous activation of glycine receptors by glycine or taurine contributed to the control of neuronal excitability in the immature hippocampus. Abstract While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4–7) rat using field potential recordings. Bath application of 100 μm taurine or 10 μm glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μm 4‐aminopyridine in low Mg2+ solution. This proconvulsive effect was prevented by 3 μm strychnine or after incubation with the loop diuretic bumetanide (10 μm), suggesting that it required glycine receptors and an active NKCC1‐dependent Cl− accumulation. Application of higher doses of taurine (≥1 mm) or glycine (100 μm) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μm) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro‐ and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

  April 23, 2014   doi: 10.1113/jphysiol.2014.271700   open full text
• Mapping the cellular electrophysiology of rat sympathetic preganglionic neurones to their roles in cardiorespiratory reflex integration: a whole cell recording study in situ.
  Alexey O. Stalbovskiy, Linford J. B. Briant, Julian F. R. Paton, Anthony E. Pickering.
  The Journal of Physiology. April 23, 2014
  Key points Sympathetic preganglionic neurones (SPNs) gatekeep the activity flowing from the CNS to the periphery and their intrinsic properties are believed to play an important integrative role in determining the firing patterns. Previous cell recording studies have explored the electrophysiological characteristics of SPNs but until now it has not been possible to link this knowledge to their roles in cardiorespiratory integration. We used the working heart–brainstem preparation to make whole‐cell patch clamp recordings from thoracic SPNs (n = 98). The SPNs were classified into muscle vasoconstrictor‐like (MVClike, 39%) and cutaneous vasoconstrictor‐like (CVClike, 28%) on the basis of their dichotomous responses to cardiorespiratory reflex activation. The MVClike SPNs have higher baseline firing frequencies and distinctive intrinsic properties. Their firing is driven by a barrage of excitatory synaptic potentials with both tonic and respiratory modulated components. The CVClike SPNs show stereotyped rhythmical membrane potential oscillations that underpin their action potential discharge. We propose that these striking differences in the intrinsic properties of the classes of SPNs are likely to play an important role in patterning the sympathetic outflow. Abstract Sympathetic preganglionic neurones (SPNs) convey sympathetic activity flowing from the CNS to the periphery to reach the target organs. Although previous in vivo and in vitro cell recording studies have explored their electrophysiological characteristics, it has not been possible to relate these characteristics to their roles in cardiorespiratory reflex integration. We used the working heart–brainstem preparation to make whole cell patch clamp recordings from T3–4 SPNs (n = 98). These SPNs were classified by their distinct responses to activation of the peripheral chemoreflex, diving response and arterial baroreflex, allowing the discrimination of muscle vasoconstrictor‐like (MVClike, 39%) from cutaneous vasoconstrictor‐like (CVClike, 28%) SPNs. The MVClike SPNs have higher baseline firing frequencies (2.52 ± 0.33 Hz vs. CVClike 1.34 ± 0.17 Hz, P = 0.007). The CVClike have longer after‐hyperpolarisations (314 ± 36 ms vs. MVClike 191 ± 13 ms, P < 0.001) and lower input resistance (346 ± 49  MΩ vs. MVClike 496 ± 41 MΩ, P < 0.05). MVClike firing was respiratory‐modulated with peak discharge in the late inspiratory/early expiratory phase and this activity was generated by both a tonic and respiratory‐modulated barrage of synaptic events that were blocked by intrathecal kynurenate. In contrast, the activity of CVClike SPNs was underpinned by rhythmical membrane potential oscillations suggestive of gap junctional coupling. Thus, we have related the intrinsic electrophysiological properties of two classes of SPNs in situ to their roles in cardiorespiratory reflex integration and have shown that they deploy different cellular mechanisms that are likely to influence how they integrate and shape the distinctive sympathetic outputs.

  April 23, 2014   doi: 10.1113/jphysiol.2014.270769   open full text
• CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression.
  Oleg S. Matusovsky, Emily M. Nakada, Linda Kachmar, Elizabeth D. Fixman, Anne‐Marie Lauzon.
  The Journal of Physiology. April 23, 2014
  Key points Activated CD4+ T cells enhance the contractility of airway smooth muscle. In order to enhance contractility, contact between CD4+ T cells and smooth muscle is required. The enhanced contractility is correlated with increased levels of fast myosin isoform. Our data suggest that inflammatory cells promote airway smooth muscle hypercontractility in airway hyper‐responsiveness and asthma. Abstract Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T‐helper 2 (Th2) inflammatory response. CD4+ T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4+ T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen‐stimulated CD4+ T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co‐incubation with antigen‐stimulated CD4+ T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4+ T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co‐incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper‐responsiveness and asthma.

  April 23, 2014   doi: 10.1113/jphysiol.2014.270843   open full text
• Fibre type‐specific satellite cell response to aerobic training in sedentary adults.
  Christopher S. Fry, Brian Noehren, Jyothi Mula, Margo F. Ubele, Philip M. Westgate, Philip A. Kern, Charlotte A. Peterson.
  The Journal of Physiology. April 23, 2014
  Key points Satellite cell activation and fusion accompany resistance exercise training. Aerobic exercise training is capable of inducing subtle muscle fibre hypertrophy; however, the role of satellite cell activation during aerobic exercise‐induced muscle adaptation is unknown. Twelve weeks of aerobic training in sedentary subjects yielded an increase in myosin heavy chain type I and type II muscle fibre cross‐sectional area. Satellite cell activation and myonuclear addition occurred only in myosin heavy chain type I fibres, with no change in myosin heavy chain type II fibres. These results help us better understand the role of satellite cells in muscle fibre adaptation to aerobic exercise, and suggest differential fibre type regulation of the myonuclear domain. Abstract In the present study, we sought to determine the effect of a traditional, 12 week aerobic training protocol on skeletal muscle fibre type distribution and satellite cell content in sedentary subjects. Muscle biopsies were obtained from the vastus lateralis [n = 23 subjects (six male and 17 female); body mass index 30.7 ± 1.2 kg m−2] before and after 12 weeks of aerobic training performed on a cycle ergometer. Immunohistochemical analyses were used to quantify myosin heavy chain (MyHC) isoform expression, cross‐sectional area and satellite cell and myonuclear content. Following training, a decrease in MyHC hybrid type IIa/IIx fibre frequency occurred, with a concomitant increase in pure MyHC type IIa fibres. Pretraining fibre type correlated with body mass index, and the change in fibre type following training was associated with improvements in maximal oxygen consumption. Twelve weeks of aerobic training also induced increases in mean cross‐sectional area in both MyHC type I and type IIa fibres. Satellite cell content was also increased following training, specifically in MyHC type I fibres, with no change in the number of satellite cells associated with MyHC type II fibres. With the increased satellite cell content following training, an increase in myonuclear number per fibre also occurred in MyHC type I fibres. Hypertrophy of MyHC type II fibres occurred without detectable myonuclear addition, suggesting that the mechanisms underlying growth in fast and slow fibres differ. These data provide intriguing evidence for a fibre type‐specific role of satellite cells in muscle adaptation following aerobic training.

  April 23, 2014   doi: 10.1113/jphysiol.2014.271288   open full text
• Neural substrates underlying fear‐evoked freezing: the periaqueductal grey–cerebellar link.
  Stella Koutsikou, Jonathan J. Crook, Emma V. Earl, J. Lianne Leith, Thomas C. Watson, Bridget M. Lumb, Richard Apps.
  The Journal of Physiology. April 22, 2014
  Key points At the heart of the brain circuitry underlying fear behaviour is the periaqueductal grey (PAG). We address an important gap in understanding regarding the neural pathways and mechanisms that link the PAG to distinct patterns of motor response associated with survival behaviours. We identify a highly localised part of the cerebellum (lateral vermal lobule VIII, pyramis) as a key supraspinal node within a chain of connections that links the PAG to the spinal cord to elicit fear‐evoked freezing behaviour. Expression of fear‐evoked freezing behaviour, both conditioned and innate, is dependent on cerebellar pyramis neural input–output pathways. We also address an important controversy in the literature, namely whether or not ventrolateral PAG (vlPAG) increases muscle tone. We provide evidence that activation of the vlPAG causes an increase in α‐motoneurone excitability, consistent with a role in generating muscle tone associated with fear‐evoked freezing. Abstract The central neural pathways involved in fear‐evoked behaviour are highly conserved across mammalian species, and there is a consensus that understanding them is a fundamental step towards developing effective treatments for emotional disorders in man. The ventrolateral periaqueductal grey (vlPAG) has a well‐established role in fear‐evoked freezing behaviour. The neural pathways underlying autonomic and sensory consequences of vlPAG activation in fearful situations are well understood, but much less is known about the pathways that link vlPAG activity to distinct fear‐evoked motor patterns essential for survival. In adult rats, we have identified a pathway linking the vlPAG to cerebellar cortex, which terminates as climbing fibres in lateral vermal lobule VIII (pyramis). Lesion of pyramis input–output pathways disrupted innate and fear‐conditioned freezing behaviour. The disruption in freezing behaviour was strongly correlated to the reduction in the vlPAG‐induced facilitation of α‐motoneurone excitability observed after lesions of the pyramis. The increased excitability of α‐motoneurones during vlPAG activation may therefore drive the increase in muscle tone that underlies expression of freezing behaviour. By identifying the cerebellar pyramis as a critical component of the neural network subserving emotionally related freezing behaviour, the present study identifies novel neural pathways that link the PAG to fear‐evoked motor responses.

  April 22, 2014   doi: 10.1113/jphysiol.2013.268714   open full text
• Excitability and responsiveness of rat barrel cortex neurons in the presence and absence of spontaneous synaptic activity in vivo.
  Tristan Altwegg‐Boussac, Mario Chavez, Séverine Mahon, Stéphane Charpier.
  The Journal of Physiology. April 18, 2014
  The amplitude and temporal dynamics of spontaneous synaptic activity in the cerebral cortex vary as a function of brain states. To directly assess the impact of different ongoing synaptic activities on neocortical function, we performed in vivo intracellular recordings from barrel cortex neurons in rats under two pharmacological conditions generating either oscillatory or tonic synaptic drive. Cortical neurons membrane excitability and firing responses were compared, in the same neurons, before and after complete suppression of background synaptic drive following systemic injection of a high dose of anaesthetic. Compared to the oscillatory state, the tonic pattern resulted in a more depolarized and less fluctuating membrane potential (Vm), a lower input resistance (Rm) and steeper relations of firing frequency versus injected current (F‐I). Whatever their temporal dynamics, suppression of background synaptic activities increased mean Vm, without affecting Rm, and induced a rightward shift of F‐I curves. Both types of synaptic drive generated a high variability in current‐induced firing rate and patterns in cortical neurons, which was much reduced after removal of spontaneous activity. These findings suggest that oscillatory and tonic synaptic patterns differentially facilitate the input‐output function of cortical neurons but result in a similar moment‐to‐moment variability in spike responses to incoming depolarizing inputs. This article is protected by copyright. All rights reserved

  April 18, 2014   doi: 10.1113/jphysiol.2013.270561   open full text
• Histamine inhibits the melanin‐concentrating hormone system: implications for sleep and arousal.
  Gregory S. Parks, Nicholas D. Olivas, Taruna Ikrar, Nayna M. Sanathara, Lien Wang, Zhiwei Wang, Olivier Civelli, Xiangmin Xu.
  The Journal of Physiology. April 17, 2014
  Key points Melanin‐concentrating hormone (MCH) neurons express histamine‐3 receptor (H3R) mRNA but not histamine‐1 (H1R) or histamine‐2 (H2R) receptor mRNA. Histamine inhibits MCH neurons by activating postsynaptic H3R. This effect is mediated through G protein‐dependent inwardly rectifying potassium (GIRK) channels. Histamine may function to silence MCH neurons during wakefulness. Abstract Melanin‐concentrating hormone (MCH)‐producing neurons are known to regulate a wide variety of physiological functions such as feeding, metabolism, anxiety and depression, and reward. Recent studies have revealed that MCH neurons receive projections from several wake‐promoting brain regions and are integral to the regulation of rapid eye movement (REM) sleep. Here, we provide evidence in both rats and mice that MCH neurons express histamine‐3 receptors (H3R), but not histamine‐1 (H1R) or histamine‐2 (H2R) receptors. Electrophysiological recordings in brain slices from a novel line of transgenic mice that specifically express the reporter ZsGreen in MCH neurons show that histamine strongly inhibits MCH neurons, an effect which is TTX insensitive, and blocked by the intracellular presence of GDP‐β‐S. A specific H3R agonist, α‐methylhistamine, mimicks the inhibitory effects of histamine, and a specific neutral H3R antagonist, VUF 5681, blocks this effect. Tertiapin Q (TPQ), a G protein‐dependent inwardly rectifying potassium (GIRK) channel inhibitor, abolishes histaminergic inhibition of MCH neurons. These results indicate that histamine directly inhibits MCH neurons through H3R by activating GIRK channels and suggest that that inhibition of the MCH system by wake‐active histaminergic neurons may be responsible for silencing MCH neurons during wakefulness and thus may be directly involved in the regulation of sleep and arousal.

  April 17, 2014   doi: 10.1113/jphysiol.2013.268771   open full text
• Limb position sense, proprioceptive drift and muscle thixotropy at the human elbow joint.
  A. Tsay, G. Savage, T. J. Allen, U. Proske.
  The Journal of Physiology. April 17, 2014
  Key points When a blindfolded subject holds his or her arm at a particular angle, its reported position shifts over time; this is known as proprioceptive drift. Here, we show that in relation to position sense at the elbow, the direction of perceived shifts is consistent with adaptation in discharge levels of sensory receptors in elbow muscles. Raising or lowering receptor discharge levels by similar amounts in opposing muscles at the elbow using muscle conditioning abolishes proprioceptive drift, but large position errors may result. The present experiments provide an explanation for proprioceptive drift and indicate that, in a forearm position‐matching task, the brain is not concerned with actual discharge levels from arm muscles, but with their difference. Abstract These experiments on the human forearm are based on the hypothesis that drift in the perceived position of a limb over time can be explained by receptor adaptation. Limb position sense was measured in 39 blindfolded subjects using a forearm‐matching task. A property of muscle, its thixotropy, a contraction history‐dependent passive stiffness, was exploited to place muscle receptors of elbow muscles in a defined state. After the arm had been held flexed and elbow flexors contracted, we observed time‐dependent changes in the perceived position of the reference arm by an average of 2.8° in the direction of elbow flexion over 30 s (Experiment 1). The direction of the drift reversed after the arm had been extended and elbow extensors contracted, with a mean shift of 3.5° over 30 s in the direction of elbow extension (Experiment 2). The time‐dependent changes could be abolished by conditioning elbow flexors and extensors in the reference arm at the test angle, although this led to large position errors during matching (±10°), depending on how the indicator arm had been conditioned (Experiments 3 and 4). When slack was introduced in the elbow muscles of both arms, by shortening muscles after the conditioning contraction, matching errors became small and there was no drift in position sense (Experiments 5 and 6). These experiments argue for a receptor‐based mechanism for proprioceptive drift and suggest that to align the two forearms, the brain monitors the difference between the afferent signals from the two arms.

  April 17, 2014   doi: 10.1113/jphysiol.2013.269365   open full text
• Impairments in mitochondrial palmitoyl‐CoA respiratory kinetics that precede development of diabetic cardiomyopathy are prevented by resveratrol in ZDF rats.
  Marie‐Soleil Beaudoin, Christopher G. R. Perry, Alicia M. Arkell, Adrian Chabowski, Jeremy A. Simpson, David C. Wright, Graham P. Holloway.
  The Journal of Physiology. April 14, 2014
  Key points Dysfunctional mitochondrial respiration may contribute to the establishment of diabetic cardiomyopathy, but this remains controversial; resveratrol, a polyphenol compound, has been shown to recover heart contractile function in rodent models of high‐fat‐diet‐induced cardiac dysfunction. Therefore, we studied mitochondrial respiratory kinetic function in ZDF rats before overt diabetes and cardiac dysfunction manifested, and determined the efficacy of resveratrol to recover potential derangements in mitochondrial bioenergetics. We show that the electron transport chain functions normally in ZDF rats, as pyruvate and ADP respiratory kinetics were normal. In contrast, in ZDF rats, we show impairments in the sensitivity of mitochondria to lipids (palmitoyl‐CoA) as well as the accumulation of reactive lipids and increased mitochondrial reactive oxygen species (ROS) emission rates. Supplementation with resveratrol improved palmitoyl‐CoA respiratory kinetics and reactive lipid profiles, and normalized mitochondrial ROS emission rates. Abstract Alterations in lipid metabolism within the heart may have a causal role in the establishment of diabetic cardiomyopathy; however, this remains equivocal. Therefore, in the current study we determined cardiac mitochondrial bioenergetics in ZDF rats before overt type 2 diabetes and diabetic cardiomyopathy developed. In addition, we utilized resveratrol, a compound previously shown to improve, prevent or reverse cardiac dysfunction in high‐fat‐fed rodents, as a tool to potentially recover dysfunctions within mitochondria. Fasting blood glucose and invasive left ventricular haemodynamic analysis confirmed the absence of type 2 diabetes and diabetic cardiomyopathy. However, fibrosis was already increased (P < 0.05) ∼70% in ZDF rats at this early stage in disease progression. Assessments of mitochondrial ADP and pyruvate respiratory kinetics in permeabilized fibres from the left ventricle revealed normal electron transport chain function and content. In contrast, the apparent Km to palmitoyl‐CoA (P‐CoA) was increased (P < 0.05) ∼60%, which was associated with an accumulation of intracellular triacylgycerol, diacylglycerol and ceramide species. In addition, the capacity for mitochondrial reactive oxygen species emission was increased (P < 0.05) ∼3‐fold in ZDF rats. The provision of resveratrol reduced fibrosis, P‐CoA respiratory sensitivity, reactive lipid accumulation and mitochondrial reactive oxygen species emission rates. Altogether the current data support the supposition that a chronic dysfunction within mitochondrial lipid‐supported bioenergetics contributes to the development of diabetic cardiomyopathy, as this was present before overt diabetes or cardiac dysfunction. In addition, we show that resveratrol supplementation prevents these changes, supporting the belief that resveratrol is a potent therapeutic approach for preventing diabetic cardiomyopathy.

  April 14, 2014   doi: 10.1113/jphysiol.2013.270538   open full text
• Modulating effect of SIRT1 activation induced by resveratrol on Foxo1‐associated apoptotic signalling in senescent heart.
  Thomas K. Sin, Angus P. Yu, Benjamin Y. Yung, Shea Ping Yip, Lawrence W. Chan, Cesar S. Wong, Michael Ying, John A. Rudd, Parco M. Siu.
  The Journal of Physiology. April 14, 2014
  Key points Cardiac function is impaired and Foxo1/Bim‐related apoptotic signalling is up‐regulated in senescent heart Activation of SIRT1 deacetylase activity by resveratrol attenuates the Foxo1/Bim signalling axis in senescent heart Abstract Elevations of cardiomyocyte apoptosis and fibrotic deposition are major characteristics of the ageing heart. Resveratrol, a polyphenol in grapes and red wine, is known to improve insulin resistance and increase mitochondrial biogenesis through the SIRT1–PGC‐1α signalling axis. Recent studies attempted to relate SIRT1 activation by resveratrol to the regulation of apoptosis in various disease models of cardiac muscle. In the present study, we tested the hypothesis that long‐term (8‐month) treatment of resveratrol would activate SIRT1 and improve the cardiac function of senescent mice through suppression of Foxo1‐associated pro‐apoptotic signalling. Our echocardiographic measurements indicated that the cardiac systolic function measured as fractional shortening and ejection fraction was significantly reduced in aged mice when compared with the young mice. These reductions, however, were not observed in resveratrol‐treated hearts. Ageing significantly reduced the deacetylase activity, but not the protein abundance of SIRT1 in the heart. This reduction was accompanied by increased acetylation of the Foxo1 transcription factor and transactivation of its target, pro‐apoptotic Bim. Subsequent analyses indicated that pro‐apoptotic signalling measured as p53, Bax and apoptotic DNA fragmentation was up‐regulated in the heart of aged mice. In contrast, resveratrol restored SIRT1 activity and suppressed elevations of Foxo1 acetylation, Bim and pro‐apoptotic signalling in the aged heart. In parallel, resveratrol also attenuated the ageing‐induced elevations of fibrotic collagen deposition and markers of oxidative damage including 4HNE and nitrotyrosine. In conclusion, these novel data demonstrate that resveratrol mitigates pro‐apoptotic signalling in senescent heart through a deacetylation mechanism of SIRT1 that represses the Foxo1–Bim‐associated pro‐apoptotic signalling axis.

  April 14, 2014   doi: 10.1113/jphysiol.2014.271387   open full text
• Computational analysis of Ca2+ dynamics in isolated cardiac mitochondria predicts two distinct modes of Ca2+ uptake.
  Shivendra G. Tewari, Amadou K. S. Camara, David F. Stowe, Ranjan K. Dash.
  The Journal of Physiology. April 10, 2014
  Key points Cytosolic, but not matrix, Mg2+ inhibits mitochondrial Ca2+ uptake through the Ca2+ uniporter (CU). The majority of mitochondrial Ca2+ uptake under physiological levels of cytosolic Ca2+ and Mg2+ is through a fast uptake pathway, namely the ryanodine receptor (RyR)‐type channel (RTC), that has characteristics similar to the ryanodine receptor. Modulation of mitochondrial RTC adaptation and opening probability by cytosolic Mg2+ is not robust, in contrast to that of cardiac sarcoplasmic reticulum RyR. Model analysis of the mitochondrial Ca2+ sequestration system further validates the existence of two different classes of Ca2+ buffering proteins, i.e. Class 1 and Class 2. The Ca2+ buffering capacity of Class 1 protein is auto‐regulated by the rate at which Ca2+ is taken up by cardiac mitochondria. The quantitative framework suggests differential roles for the two modes of Ca2+ uptake pathways: CU–Ca2+ buffering, and RTC–Ca2+ modulated bioenergetics. Abstract Cardiac mitochondria can act as a significant Ca2+ sink and shape cytosolic Ca2+ signals affecting various cellular processes, such as energy metabolism and excitation–contraction coupling. However, different mitochondrial Ca2+ uptake mechanisms are still not well understood. In this study, we analysed recently published Ca2+ uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca2+ uptake, at physiological levels of cytosolic Ca2+ and Mg2+, occurs through a fast Ca2+ uptake pathway, which is neither the Ca2+ uniporter nor the rapid mode of Ca2+ uptake. This fast Ca2+ uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg2+‐dependent enhancement of the RyR adaptation was not evident in this RyR‐type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca2+ uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca2+ buffers that comprise the mitochondrial Ca2+ sequestration system. The modelling study further indicates that the Ca2+ buffers respond differentially depending on the source of Ca2+ uptake. In particular, it suggests that the Class 1 Ca2+ buffering capacity is auto‐regulated by the rate at which Ca2+ is taken up by mitochondria.

  April 10, 2014   doi: 10.1113/jphysiol.2013.268847   open full text
• Modulation of ionotropic glutamate receptor function by vertebrate galectins.
  Bryan A. Copits, Claire G. Vernon, Ryuichi Sakai, Geoffrey T. Swanson.
  The Journal of Physiology. April 07, 2014
  Key points Lectins are a family of small evolutionarily conserved sugar‐binding proteins that regulate a diverse array of physiological processes ranging from immune system activation to cancer cell metastasis. Ionotropic glutamate receptor function can be modulated by plant‐derived lectins, but the physiological relevance of this activity is unclear as no analogous function has been identified in animal lectins. We found that a variety of vertebrate lectins, including human brain‐expressed galectin‐1, modulated glutamate receptor kinetics in a subunit and lectin‐dependent manner, which critically depended on complex oligosaccharide processing. Galectin application slowed neuronal kainate receptor currents from nociceptive dorsal root ganglion neurons. We propose that brain‐expressed galectins are potential endogenous modulators of neuronal glutamate receptors, which may play important roles in diseases of altered cellular excitability, such as epilepsy or chronic pain. Abstract AMPA and kainate receptors are glutamate‐gated ion channels whose function is known to be altered by a variety of plant oligosaccharide‐binding proteins, or lectins, but the physiological relevance of this activity has been uncertain because no lectins with analogous allosteric modulatory effects have been identified in animals. We report here that members of the prototype galectin family, which are β‐galactoside‐binding lectins, exhibit subunit‐specific allosteric modulation of desensitization of recombinant homomeric and heteromeric AMPA and kainate receptors. Galectin modulation of GluK2 kainate receptors was dependent upon complex oligosaccharide processing of N‐glycosylation sites in the amino‐terminal domain and downstream linker region. The sensitivity of GluA4 AMPA receptors to human galectin‐1 could be enhanced by supplementation of culture media with uridine and N‐acetylglucosamine (GlcNAc), precursors for the hexosamine pathway that supplies UDP‐GlcNAc for synthesis of complex oligosaccharides. Neuronal kainate receptors in dorsal root ganglia were sensitive to galectin modulation, whereas AMPA receptors in cultured hippocampal neurons were insensitive, which could be a reflection of differential N‐glycan processing or receptor subunit selectivity. Because glycan content of integral proteins can be modified dynamically, we postulate that physiological or pathological conditions in the CNS could arise in which galectins alter excitatory neurotransmission or neuronal excitability through their actions on AMPA or kainate receptors.

  April 07, 2014   doi: 10.1113/jphysiol.2013.269597   open full text
• Greater excitability and firing irregularity of tufted cells underlies distinct afferent‐evoked activity of olfactory bulb mitral and tufted cells.
  Shawn D. Burton, Nathaniel N. Urban.
  The Journal of Physiology. April 07, 2014
  Key points The two classes of principal neurons in the mammalian main olfactory bulb, mitral and tufted cells, respond with different firing latencies and rates to afferent‐evoked input; how these differences in activity arise is incompletely understood. Tufted cells receive stronger afferent‐evoked excitation than mitral cells, but this difference alone is insufficient to account for the greater afferent‐evoked firing in tufted versus mitral cells. Mitral and tufted cells exhibit significant intrinsic functional differences; compared to mitral cells, tufted cells fire action potentials with shorter durations and faster afterhyperpolarizations and exhibit twofold greater firing rate–current curve gains and peak rates. Tufted cells exhibit diverse firing modes, including tonic firing and irregular stuttering, and on average fire more irregularly than mitral cells. Collectively, stronger afferent excitation, greater intrinsic excitability and more irregular firing in tufted cells combine to drive distinct responses of mitral and tufted cells to sensory input. Abstract Mitral and tufted cells, the two classes of principal neurons in the mammalian main olfactory bulb, exhibit morphological differences but remain widely viewed as functionally equivalent. Results from several recent studies, however, suggest that these two cell classes may encode complementary olfactory information in their distinct patterns of afferent‐evoked activity. To understand how these differences in activity arise, we have performed the first systematic comparison of synaptic and intrinsic properties between mitral and tufted cells. Consistent with previous studies, we found that tufted cells fire with higher probability and rates and shorter latencies than mitral cells in response to physiological afferent stimulation. This stronger response of tufted cells could be partially attributed to synaptic differences, as tufted cells received stronger afferent‐evoked excitation than mitral cells. However, differences in intrinsic excitability also contributed to the differences between mitral and tufted cell activity. Compared to mitral cells, tufted cells exhibited twofold greater excitability and peak instantaneous firing rates. These differences in excitability probably arise from differential expression of voltage‐gated potassium currents, as tufted cells exhibited faster action potential repolarization and afterhyperpolarizations than mitral cells. Surprisingly, mitral and tufted cells also showed firing mode differences. While both cell classes exhibited regular firing and irregular stuttering of action potential clusters, tufted cells demonstrated a greater propensity to stutter than mitral cells. Collectively, stronger afferent‐evoked excitation, greater intrinsic excitability and more irregular firing in tufted cells can combine to drive distinct responses of mitral and tufted cells to afferent‐evoked input.

  April 07, 2014   doi: 10.1113/jphysiol.2013.269886   open full text
• Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.
  Alexey V. Dvornikov, Sukriti Dewan, Olga V. Alekhina, F. Bryan Pickett, Pieter P. Tombe.
  The Journal of Physiology. April 04, 2014
  Key points The zebrafish is emerging as an attractive cost‐effective model for the study of structure–function relationships. However, cardiac contractile function in the zebrafish remains to be investigated. We applied novel approaches used to study contractile function at the cellular level in mammalian models to zebrafish. We found that contractile force regulation in the adult zebrafish shares many similarities with that in the mammalian myocardium as previously determined by others and ourselves, indicating that the zebrafish is an appropriate model system for the study of cardiac contractile biology. Abstract The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost‐effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure–function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca2+ were measured. We observed the modulation of twitch force, but not of intracellular Ca2+, by both extracellular [Ca2+] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length‐dependent activation. Moreover, modulation of myofilament activation–relaxation and force redevelopment kinetics by varied Ca2+ activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure–function relationships.

  April 04, 2014   doi: 10.1113/jphysiol.2014.270678   open full text
• Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres.
  Joachim Nielsen, Arthur J. Cheng, Niels Ørtenblad, Håkan Westerblad.
  The Journal of Physiology. April 04, 2014
  Key points Muscle glycogen (the storage form of glucose) is consumed during muscle work and the depletion of glycogen is thought to be a main contributor to muscle fatigue. In this study, we used a novel approach to first measure fatigue‐induced reductions in force and tetanic Ca2+ in isolated single mouse muscle fibres following repeated contractions and subsequently quantify the subcellular distribution of glycogen in the same fibre. Using this approach, we investigated whether the decreased tetanic Ca2+ induced by repeated contractions was associated with glycogen depletion in certain subcellular regions. The results show a positive correlation between depletion of glycogen located within the myofibrils and low tetanic Ca2+ after repetitive stimulation. We conclude that subcellular glycogen depletion has a central role in the decrease in tetanic Ca2+ that occurs during repetitive contractions. Abstract In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz, 350 ms tetani given at 2 s (high‐intensity fatigue, HIF) or 10 s (low‐intensity fatigue, LIF) intervals, while force and [Ca2+]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P < 0.01, n = 9) and LIF (P < 0.01, n = 5) fibres, respectively. At fatigue, the mean inter‐ and intramyofibrillar glycogen content was 60–75% lower than in rested control fibres (P < 0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue‐induced decrease in tetanic [Ca2+]i and the reduction in intermyofibrillar (P = 0.051) and intramyofibrillar (P = 0.0008) glycogen. In conclusion, the fatigue‐induced decrease in tetanic [Ca2+]i, and hence force, is accompanied by major reductions in inter‐ and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool.

  April 04, 2014   doi: 10.1113/jphysiol.2014.271528   open full text
• Short‐term sustained hypoxia induces changes in the coupling of sympathetic and respiratory activities in rats.
  Davi J. A. Moraes, Leni G. H. Bonagamba, Kauê M. Costa, João H. Costa‐Silva, Daniel B. Zoccal, Benedito H. Machado.
  The Journal of Physiology. April 02, 2014
  Key points Hypoxia activates peripheral chemoreceptors producing an increase in breathing and arterial pressure. In conditions of sustained hypoxia, an increase in ventilation and arterial blood pressure is observed that persists after the return to normoxia. We show in rats that sustained hypoxia for 24 h produces glutamate‐dependent changes in the activity of expiratory and sympathetic neurones of the rostral ventrolateral medulla, which are essential for the control of respiratory and sympathetic activities. These neuronal changes induced by sustained hypoxia are critical for the emergence of coupled active expiration and augmented sympathetic activity. These findings contribute to a better understanding of cardiorespiratory adjustments associated with sustained hypoxia in individuals experiencing high altitudes. Abstract Individuals experiencing sustained hypoxia (SH) exhibit adjustments in the respiratory and autonomic functions by neural mechanisms not yet elucidated. In the present study we evaluated the central mechanisms underpinning the SH‐induced changes in the respiratory pattern and their impact on the sympathetic outflow. Using a decerebrated arterially perfused in situ preparation, we verified that juvenile rats exposed to SH (10% O2) for 24 h presented an active expiratory pattern, with increased abdominal, hypoglossal and vagal activities during late‐expiration (late‐E). SH also enhanced the activity of augmenting‐expiratory neurones and depressed the activity of post‐inspiratory neurones of the Bötzinger complex (BötC) by mechanisms not related to changes in their intrinsic electrophysiological properties. SH rats exhibited high thoracic sympathetic activity and arterial pressure levels associated with an augmented firing frequency of pre‐sympathetic neurones of the rostral ventrolateral medulla (RVLM) during the late‐E phase. The antagonism of ionotropic glutamatergic receptors in the BötC/RVLM abolished the late‐E bursts in expiratory and sympathetic outputs of SH rats, indicating that glutamatergic inputs to the BötC/RVLM are essential for the changes in the expiratory and sympathetic coupling observed in SH rats. We also observed that the usually silent late‐E neurones of the retrotrapezoid nucleus/parafacial respiratory group became active in SH rats, suggesting that this neuronal population may provide the excitatory drive essential to the emergence of active expiration and sympathetic overactivity. We conclude that short‐term SH induces the activation of medullary expiratory neurones, which affects the pattern of expiratory motor activity and its coupling with sympathetic activity.

  April 02, 2014   doi: 10.1113/jphysiol.2013.262212   open full text
• Reduction in maternal Polycomb levels contributes to transgenerational inheritance of a response to toxic stress in flies.
  Shay Stern, Orli Snir, Eran Mizrachi, Matana Galili, Inbal Zaltsman, Yoav Soen.
  The Journal of Physiology. March 31, 2014
  Key points Previous work on epigenetic transgenerational phenomena focused on chromatin modifications or small RNAs as potential carriers of non‐genetic transgenerational influence. We describe a hitherto non‐appreciated mode of trans‐generational influence by which physiological stress in one generation can impact multiple generations of non‐exposed offspring. This mode of transfer involves persistent changes in the composition of maternal RNA in the early offspring embryos. In particular we show that reduction in maternal Polycomb gene levels have a functional contribution to trans‐generational inheritance of induced gene expression. Our findings extend the mechanistic repertoire of epigenetic inheritance by providing evidence connecting changes in maternal RNA with trans‐generational inheritance of induced phenotypes. Abstract Transgenerational persistence of parental responses to environmental stimuli has been reported in various organisms, but the underlying mechanisms remain underexplored. In one of these reported examples, we have shown that exposure of fly larvae to G418 antibiotic leads to non‐Mendelian inheritance of ectopic induction of certain developmental genes. Here we investigate if this inheritance involves changes in mRNA composition within the early, maternal‐stage offspring embryos of exposed flies. Exposure to G418 in F1 modified the maternal RNA levels of many genes in their early (F2) embryos. This includes reduction of maternal Polycomb group genes which persisted in the following generation of embryos (F3). To investigate the functional meaning of this reduction, we compared genetically normal embryos of Polycomb mutant females to normal embryos of normal females. Analysis with two different alleles of Polycomb, Pc1 and Pc3, revealed that maternal reduction in Polycomb gene dosage has a positive influence on the inheritance of induced expression. Together, this shows that exposure to G418 stress reduces the maternal levels of Polycomb in the offspring embryos and this reduction contributes to the inheritance of induced expression.

  March 31, 2014   doi: 10.1113/jphysiol.2014.271445   open full text
• Increase in cytosolic Ca2+ produced by hypoxia and other depolarizing stimuli activates a non‐selective cation channel in chemoreceptor cells of rat carotid body.
  Dawon Kang, Jiaju Wang, James O. Hogan, Rudi Vennekens, Marc Freichel, Carl White, Donghee Kim.
  The Journal of Physiology. March 27, 2014
  Key points Hypoxia is thought to depolarize glomus cells by inhibiting the outward K+ current, which sets in motion a cascade of ionic events that lead to transmitter secretion, increased afferent carotid sinus nerve activity and increased ventilation. Our study of Na+‐permeable channels in glomus cells has revealed that hypoxia not only inhibits TASK background K+ channels but also indirectly activates a non‐selective cation channel with a single channel conductance of 20 pS. Under physiological conditions, the reversal potential of the cation channel is ∼ –28 mV, indicating that Na+ influx is also involved in hypoxia‐induced excitation of glomus cells. Activation of the 20 pS cation channel is present when the O2 content is 5% or less, indicating that Na+ influx occurs during moderate to severe hypoxia (<5% O2), but not mild hypoxia (>5% O2). The 20 pS cation channel is directly activated by a rise in intracellular Ca2+. Thus, factors that elevate intracellular Ca2+ such as hypoxia, extracellular acidosis and high external KCl all activate the cation channel. A feed‐forward mechanism may be present in which an initial depolarization‐induced rise in intracellular Ca2+ opens the Na+‐permeable cation channel, and the Na+ influx causes additional depolarization and influx of Ca2+ into glomus cells. Abstract The current model of O2 sensing by carotid body chemoreceptor (glomus) cells is that hypoxia inhibits the outward K+ current and causes cell depolarization, Ca2+ influx via voltage‐dependent Ca2+ channels and a rise in intracellular [Ca2+] ([Ca2+]i). Here we show that hypoxia (<5% O2), in addition to inhibiting the two‐pore domain K+ channels TASK‐1/3 (TASK), indirectly activates an ∼20 pS channel in isolated glomus cells. The 20 pS channel was permeable to K+, Na+ and Cs+ but not to Cl− or Ca2+. The 20 pS channel was not sensitive to voltage. Inhibition of TASK by external acid, depolarization of glomus cells with high external KCl (20 mm) or opening of the Ca2+ channel with FPL64176 activated the 20 pS channel when 1 mm Ca2+ was present in the external solution. Ca2+ (10 μm) applied to the cytosolic side of inside‐out patches activated the 20 pS channel. The threshold [Ca2+]i for activation of the 20 pS channel in cell‐attached patches was ∼200 nm. The reversal potential of the 20 pS channel was estimated to be −28 mV. Our results reveal a sequential mechanism in which hypoxia (<5% O2) first inhibits the K+ conductance and then activates a Na+‐permeable, non‐selective cation channel via depolarization‐induced rise in [Ca2+]i. Our results suggest that inhibition of K+ efflux and stimulation of Na+ influx both contribute to the depolarization of glomus cells during moderate to severe hypoxia.

  March 27, 2014   doi: 10.1113/jphysiol.2013.266957   open full text
• Prophylactic erythropoietin exacerbates ventilation‐induced lung inflammation and injury in preterm lambs.
  Graeme R. Polglase, Samantha K. Barton, Jacqueline M. Melville, Valerie Zahra, Megan J. Wallace, Melissa L. Siew, Mary Tolcos, Timothy J. M. Moss.
  The Journal of Physiology. March 27, 2014
  Key points Erythropoietin (EPO) has been suggested as a potential treatment for bronchopulmonary dysplasia (BPD) in preterm infants. Ventilation‐induced lung injury (VILI) is a major cause of BPD in preterm neonates. We investigated whether early high‐dose EPO (i.v. 5000 IU kg−1) administration can reduce lung inflammation and injury resultant from VILI in ventilated preterm lambs. Early high‐dose EPO administration increased mRNA expression of early markers of lung inflammation and injury and systemic injury controls. Early high‐dose EPO worsened histological assessment of inflammation, airway wall thickness, haemorrhage and total injury compared to controls. Early high‐dose EPO may increase the incidence and severity of respiratory disease in ventilated, preterm neonates. Abstract Ventilation‐induced lung injury (VILI) of preterm neonates probably contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Erythropoietin (EPO) has been suggested as a therapy for BPD. The aim of this study was to determine whether prophylactic administration of EPO reduces VILI in preterm newborn lambs. Lambs at 126 days of gestation (term is 147 days) were delivered and ventilated with a high tidal volume strategy for 15 min to cause lung injury, then received gentle ventilation until 2 h of age. Lambs were randomized to receive intravenous EPO (5000 IU kg−1: Vent+EPO; n = 6) or phosphate‐buffered saline (Vent; n = 7) soon after birth: unventilated controls (UVC; n = 8) did not receive ventilation or any treatment. Physiological parameters were recorded throughout the experimental procedure. Samples of lung were collected for histological and molecular assessment of inflammation and injury. Samples of liver were collected to assess the systemic acute phase response. Vent+EPO lambs received higher F IO 2, P aO 2 and oxygenation during the first 10 min than Vent lambs. There were no differences in physiological indices beyond this time. Total lung injury score, airway wall thickness, inflammation and haemorrhage were higher in Vent+EPO lambs than in Vent lambs. Lung inflammation and early markers of lung and systemic injury were elevated in ventilated lambs relative to unventilated lambs; EPO administration further increased lung inflammation and markers of lung and systemic injury. Prophylactic EPO exacerbates VILI, which may increase the incidence and severity of long‐term respiratory disease. More studies are required before EPO can be used for lung protection in preterm infants.

  March 27, 2014   doi: 10.1113/jphysiol.2013.270348   open full text
• In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.
  Jennifer Olt, Stuart L. Johnson, Walter Marcotti.
  The Journal of Physiology. March 27, 2014
  Key points Sound and balance information is detected and processed by sensory hair cells in the auditory and vestibular organs, respectively. The zebrafish represents a potentially powerful model organism in which to investigate sensory encoding by hair cells because of its accessibility for in vivo studies and its pliable genetics. Our current understanding of hair cell biophysics in the developing zebrafish is very limited. In this study, we used in vivo and near‐physiological in vitro recordings to measure basolateral membrane currents, voltage changes and synaptic activity in hair cells in the developing and mature zebrafish. We found that the biophysical profile of lateral line hair cells in the zebrafish changes from the larval to the juvenile stage, and that juvenile neuromasts contain a much higher proportion of mature cells. These results demonstrate the potential of the zebrafish for investigating the mechanisms of signal encoding and transmission by hair cells. Abstract Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near‐physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature‐like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature‐type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching.

  March 27, 2014   doi: 10.1113/jphysiol.2013.265108   open full text
• Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D‐CFTR.
  Zhe Xu, Luísa S. Pissarra, Carlos M. Farinha, Jia Liu, Zhiwei Cai, Patrick H. Thibodeau, Margarida D. Amaral, David N. Sheppard.
  The Journal of Physiology. March 27, 2014
  Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life‐shortening genetic disease cystic fibrosis (CF). Gene changes (called second‐site mutations or revertants) that restore function to F508del, the most common CF mutation, also alter the behaviour of the CF mutant G551D. Revertants have direct impact on the structure of CFTR, but they exert their effects in a mutation‐specific way. Information about the action of revertants assists the development of new therapies that target the root cause of CF. Abstract Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine‐framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α‐helix as G550 and R555. Both G550E and 4RK augmented strongly CFTR‐mediated iodide efflux from BHK cells expressing G551D‐CFTR. To learn how revertant mutations influence G551D‐CFTR function, we studied protein processing and single‐channel behaviour. Neither G550E nor 4RK altered the expression and maturation of G551D‐CFTR protein. By contrast, both revertants had marked effects on G551D‐CFTR channel gating, increasing strongly opening frequency, while 4RK also diminished noticeably the duration of channel openings. Because G551D‐CFTR channel gating is ATP independent, we investigated whether revertant mutations restore ATP dependence to G551D‐CFTR. Like wild‐type CFTR, the activity of 4RK‐G551D‐CFTR varied with ATP concentration, suggesting that 4RK confers some ATP dependence on the G551D‐CFTR channel. Thus, the revertant mutations G550E and 4RK alter the gating pattern and ATP dependence of G551D‐CFTR without restoring single‐channel activity to wild‐type levels. Based on their impact on the CF mutants F508del and G551D, we conclude that G550E and 4RK have direct effects on CFTR structure, but that their action on CFTR processing and channel function is CF mutation specific.

  March 27, 2014   doi: 10.1113/jphysiol.2014.271817   open full text
• Adaptive changes in the motor cortex during and after longterm forelimb immobilization in adult rats.
  Riccardo Viaro, Mirco Budri, Pierantonio Parmiani, Gianfranco Franchi.
  The Journal of Physiology. March 24, 2014
  Key points To shed light on the controversial issue of how chronic immobilization affects cortical output, adult rats were subjected to intracortical microstimulation at different time‐points during and after unilateral forelimb casting. After cast application, cortical hypoexcitability appeared bilateral, specific for forelimb area, but stronger in the contralateral‐to‐cast hemisphere. Cortical excitability progressively decreased over 30 days of immobilization and, after cast removal, steadily increased, but remained partial at 15 days. Cortical application of the GABAA‐receptor antagonist bicuculline revealed an impairment of intracortical synaptic connectivity in the forelimb area during the cast period and for up to 15 days after cast removal. Rehabilitation using a rotarod performance protocol did not advance the normalization of normal forelimb map extension and enabled cortical output towards the distal forelimb only in sites that had maintained their excitability. Cortical hypoexcitability following immobilization is caused by reversible impairment of intracortical synaptic connectivity. This may suggest new approaches in conditions that require longterm limb immobilization. Abstract Experimental and clinical studies have attempted to evaluate the changes in cortical activity seen after immobilization‐induced longterm sensorimotor restriction, although results remain controversial. We used intracortical microstimulation (ICMS), which provides topographic movement representations of the motor areas in both hemispheres with optimal spatial characterization, combined with behavioural testing to unravel the effects of limb immobilization on movement representations in the rat primary motor cortex (M1). Unilateral forelimb immobilization in rats was achieved by casting the entire limb and leaving the cast in place for 15 or 30 days. Changes in M1 were bilateral and specific for the forelimb area, but were stronger in the contralateral‐to‐cast hemisphere. The threshold current required to evoke forelimb movement increased progressively over the period in cast, whereas the forelimb area size decreased and the non‐excitable area size increased. Casting resulted in a redistribution of proximal/distal movement representations: proximal forelimb representation increased, whereas distal representation decreased in size. ICMS after cast removal showed a reversal of changes, which remained partial at 15 days. Local application of the GABAA‐antagonist bicuculline revealed the impairment of cortical synaptic connectivity in the forelimb area during the period of cast and for up to 15 days after cast removal. Six days of rehabilitation using a rotarod performance protocol after cast removal did not advance map size normalization in the contralateral‐to‐cast M1 and enabled the cortical output towards the distal forelimb only in sites that had maintained their excitability. These results are relevant to our understanding of adult M1 plasticity during and after sensorimotor deprivation, and to new approaches to conditions that require longterm limb immobilization.

  March 24, 2014   doi: 10.1113/jphysiol.2013.268821   open full text
• Emergence of sigh rhythmogenesis in the embryonic mouse.
  Coralie Chapuis, Sandra Autran, Gilles Fortin, John Simmers, Muriel Thoby‐Brisson.
  The Journal of Physiology. March 21, 2014
  Key points The respiratory oscillator of the pre‐Bötzinger complex (preBötC) can generate distinct inspiratory motor patterns underlying eupnoeic and sigh‐related rhythmic activities. The preBötC can generate ‘fictive’ eupnoea at embryonic stages, but its ability to also generate sigh‐like activity remains unexplored at prenatal stages. Here, using mouse brainstem slice preparations, we show that sigh‐like activity emerges during embryonic development but later than eupnoeic rhythmogenesis. Inspiratory cells active during the latter are also active during fictive sighing, although a small subset of neurons was found to fire exclusively during sighs. Effective glycinergic inhibitory signalling is also required for sigh generation. We conclude that the developmental emergence of a sigh‐generating capability occurs after the onset of eupnoeic rhythmogenesis and requires an appropriate maturational state of chloride‐mediated glycinergic synaptic transmission. Abstract In mammals, eupnoeic breathing is periodically interrupted by spontaneous augmented breaths (sighs) that include a larger‐amplitude inspiratory effort, typically followed by a post‐sigh apnoea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre‐Bötzinger complex (preBötC) can generate the distinct inspiratory motor patterns for both eupnoea‐ and sigh‐related behaviour. During mouse embryonic development, the preBötC begins to generate eupnoeic rhythmicity at embryonic day (E) 15.5, but the network's ability to also generate sigh‐like activity remains unexplored at prenatal stages. Using transverse brainstem slice preparations we monitored the neuronal population activity of the preBötC at different embryonic ages. Spontaneous sigh‐like rhythmicity was found to emerge progressively, being expressed in 0/32 slices at E15.5, 7/30 at E16.5, 9/22 at E17.5 and 23/26 at E18.5. Calcium imaging showed that the preBötC cell population that participates in eupnoeic‐like discharge was also active during fictive sighs. However, patch‐clamp recordings revealed the existence of an additional small subset of neurons that fired exclusively during sigh activity. Changes in glycinergic inhibitory synaptic signalling, either by pharmacological blockade, functional perturbation or natural maturation of the chloride co‐transporters KCC2 or NKCC1 selectively, and in an age‐dependent manner, altered the bi‐phasic nature of sigh bursts and their coordination with eupnoeic bursting, leading to the generation of an atypical monophasic sigh‐related event. Together our results demonstrate that the developmental emergence of a sigh‐generating capability occurs after the onset of eupnoeic rhythmogenesis and requires the proper maturation of chloride‐mediated glycinergic synaptic transmission.

  March 21, 2014   doi: 10.1113/jphysiol.2013.268730   open full text
• Dietary pre‐exposure of rats to fish oil does not enhance myocardial efficiency of isolated working hearts or their left ventricular trabeculae.
  Soyeon Goo, June‐Chiew Han, Linley A. Nisbet, Ian J. LeGrice, Andrew J. Taberner, Denis S. Loiselle.
  The Journal of Physiology. March 17, 2014
  Key points Dietary fish oil has been found to have protective effects against cardiovascular disease, particularly in its role as an anti‐arrhythmic agent. An additional mechanism proposed for the putative cardio‐protective effect is enhanced efficiency of metabolic energy usage by the heart. We tested whether dietary supplementation of fish oil enhances cardiac efficiency or otherwise improves mechano‐energetic performance. Experiments were performed at two distinct physiological levels (whole organ and isolated tissues) employing two independent metabolic indices (oxygen consumption and heat production), respectively. Feeding rats with either a fish oil‐enriched or a saturated fatty acid‐enriched diet did not alter the efficiency of either the isolated whole heart or its left ventricular trabeculae. Abstract Numerous epidemiological studies, supported by clinical and experimental findings, have suggested beneficial effects of dietary fish or fish oil supplementation on cardiovascular health. One such experimental study showed a profound (100%) increase in myocardial efficiency (i.e. the ratio of work output to metabolic energy input) of the isolated whole heart, achieved by a corresponding decrease in the rate of myocardial oxygen consumption. However, a number of other investigations have returned null results on the latter energetic index. Such conflicting findings have motivated us to undertake a re‐examination. To that effect, we investigated the effects of dietary fatty acid supplementation on myocardial mechano‑energetics, with our primary focus on cardiac efficiency. We used both isolated hearts and isolated left ventricular trabeculae of rats fed with one of three distinct diets: reference (REF), fish oil‐supplemented (FO) or saturated fat‐supplemented (SFA). For all three groups, and at both spatial levels, we supplied 10 mm glucose as the exogenous metabolic substrate. In the working heart experiments, we found no difference in the average mechanical efficiency among the three dietary groups: 14.8 ± 1.1% (REF), 13.9 ± 0.6% (FO) and 13.6 ± 0.7% (SFA). Likewise, we observed no difference in peak mechanical efficiency of left ventricular trabeculae among the REF, FO and SFA groups: 13.3 ± 1.4, 11.2 ± 2.2 and 12.5 ± 1.5%, respectively. We conclude that there is no effect of a period of pre‐exposure to a diet supplemented with either fish oil or saturated fatty acids on the efficiency of the myocardium at either spatial level: tissue or whole heart.

  March 17, 2014   doi: 10.1113/jphysiol.2013.269977   open full text
• Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double‐blind, randomised, controlled trial.
  Gøran Paulsen, Kristoffer T. Cumming, Geir Holden, Jostein Hallén, Bent Ronny Rønnestad, Ole Sveen, Arne Skaug, Ingvild Paur, Nasser E. Bastani, Hege Nymo Østgaard, Charlotte Buer, Magnus Midttun, Fredrik Freuchen, Håvard Wiig, Elisabeth Tallaksen Ulseth, Ina Garthe, Rune Blomhoff, Haakon B. Benestad, Truls Raastad.
  The Journal of Physiology. March 12, 2014
  Key points Recent studies have indicated that antioxidant supplementation may blunt adaptations to exercise, such as mitochondrial biogenesis induced by endurance training. However, studies in humans are sparse and results are conflicting. Isolated vitamin C and E supplements are widely used, and unravelling the interference of these vitamins in cellular and physiological adaptations to exercise is of interest to those who exercise for health purposes and to athletes. Our results show that vitamin C and E supplements blunted the endurance training‐induced increase of mitochondrial proteins (COX4), which is important for improving muscular endurance. Training‐induced increases in V̇O2 max and running performance were not detectably affected by the supplementation. The present study contributes to understanding of how antioxidants may interfere with adaptations to exercise in humans, and the results indicate that high dosages of vitamins C and E should be used with caution. Abstract In this double‐blind, randomised, controlled trial, we investigated the effects of vitamin C and E supplementation on endurance training adaptations in humans. Fifty‐four young men and women were randomly allocated to receive either 1000 mg of vitamin C and 235 mg of vitamin E or a placebo daily for 11 weeks. During supplementation, the participants completed an endurance training programme consisting of three to four sessions per week (primarily of running), divided into high‐intensity interval sessions [4–6 × 4–6 min; >90% of maximal heart rate (HRmax)] and steady state continuous sessions (30–60 min; 70–90% of HRmax). Maximal oxygen uptake (V̇O2 max ), submaximal running and a 20 m shuttle run test were assessed and blood samples and muscle biopsies were collected, before and after the intervention. Participants in the vitamin C and E group increased their V̇O2 max (mean ± s.d.: 8 ± 5%) and performance in the 20 m shuttle test (10 ± 11%) to the same degree as those in the placebo group (mean ± s.d.: 8 ± 5% and 14 ± 17%, respectively). However, the mitochondrial marker cytochrome c oxidase subunit IV (COX4) and cytosolic peroxisome proliferator‐activated receptor‐γ coactivator 1 α (PGC‐1α) increased in the m. vastus lateralis in the placebo group by 59 ± 97% and 19 ± 51%, respectively, but not in the vitamin C and E group (COX4: −13 ± 54%; PGC‐1α: −13 ± 29%; P ≤ 0.03, between groups). Furthermore, mRNA levels of CDC42 and mitogen‐activated protein kinase 1 (MAPK1) in the trained muscle were lower in the vitamin C and E group than in the placebo group (P ≤ 0.05). Daily vitamin C and E supplementation attenuated increases in markers of mitochondrial biogenesis following endurance training. However, no clear interactions were detected for improvements in V̇O2 max and running performance. Consequently, vitamin C and E supplementation hampered cellular adaptations in the exercised muscles, and although this did not translate to the performance tests applied in this study, we advocate caution when considering antioxidant supplementation combined with endurance exercise.

  March 12, 2014   doi: 10.1113/jphysiol.2013.267419   open full text
• Intrinsic vascular dopamine – a key modulator of hypoxia‐induced vasodilatation in splanchnic vessels.
  Uwe Pfeil, Jitka Kuncova, Doerthe Brüggmann, Renate Paddenberg, Amir Rafiq, Michael Henrich, Markus A. Weigand, Klaus‐Dieter Schlüter, Marco Mewe, Ralf Middendorff, Jana Slavikova, Wolfgang Kummer.
  The Journal of Physiology. March 10, 2014
  Key points Dopamine is a member of the catecholamine family and a precursor in the biosynthetic pathway of adrenaline and noradrenaline, which acts as an independent neurotransmitter in the sympathetic nervous system and as a paracrine hormone. We found that the arterial wall of systemic vessels itself, i.e. the endothelial cells and the underlying tissue, produces a substantial pool of dopamine. This intrinsic vascular dopamine is released upon stimulation by decreasing oxygen concentrations, causing a dilatation of the blood vessel, thereby increasing blood flow and subsequently oxygenation of the tissue. This study identifies dopamine as a novel non‐neuronal intrinsic vasodilator in the arterial wall, crucially involved in PO2‐driven modulation of vascular tone and maintenance of tissue oxygenation under conditions where reduced oxygen supply may cause severe damage to body systems as in stroke, heart infarction and pulmonary hypertension. Abstract Dopamine not only is a precursor of the catecholamines noradrenaline and adrenaline but also serves as an independent neurotransmitter and paracrine hormone. It plays an important role in the pathogenesis of hypertension and is a potent vasodilator in many mammalian systemic arteries, strongly suggesting an endogenous source of dopamine in the vascular wall. Here we demonstrated dopamine, noradrenaline and adrenaline in rat aorta and superior mesenteric arteries (SMA) by radioimmunoassay. Chemical sympathectomy with 6‐hydroxydopamine showed a significant reduction of noradrenaline and adrenaline, while dopamine levels remained unaffected. Isolated endothelial cells were able to synthesize and release dopamine upon cAMP stimulation. Consistent with these data, mRNAs coding for catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic l‐amino acid decarboxylase, and dopamine‐β‐hydroxylase were detected by RT‐PCR in cultured endothelial cells from SMA. TH protein was detected by immunohistochemisty and Western blot. Exposure of endothelial cells to hypoxia (1% O2) increased TH mRNA. Vascular smooth muscle cells partially expressed catecholaminergic traits. A physiological role of endogenous vascular dopamine was shown in SMA, where D1 dopamine receptor blockade abrogated hypoxic vasodilatation. Experiments on SMA with endothelial denudation revealed a significant contribution of the endothelium, although subendothelial dopamine release dominated. From these results we conclude that endothelial cells and cells of the underlying vascular wall synthesize and release dopamine in an oxygen‐regulated manner. In the splanchnic vasculature, this intrinsic non‐neuronal dopamine is the dominating vasodilator released upon lowering of oxygen tension.

  March 10, 2014   doi: 10.1113/jphysiol.2013.262626   open full text
• Passive hind‐limb cycling improves cardiac function and reduces cardiovascular disease risk in experimental spinal cord injury.
  Christopher R. West, Mark A. Crawford, Malihe‐Sadat Poormasjedi‐Meibod, Katharine D. Currie, Andre Fallavollita, Violet Yuen, John H. McNeill, Andrei V. Krassioukov.
  The Journal of Physiology. March 10, 2014
  Key points Using a wide array of experimental approaches, we demonstrate for the first time that spinal cord injury is associated with a rapid and sustained impairment in cardiac structure and function that is present as early as 1 week post‐injury. We provide novel data demonstrating that spinal cord injury elicits an altered Starling curve and myocardial fibrosis. The latter of these may be secondary to an up‐regulation of transforming growth factor beta‐1 and mothers against decapentaplegic homolog 3 mRNA, both of which form part of a well‐known fibrotic signalling pathway. Passive hind‐limb cycling averts the spinal cord injury‐induced impairments in cardiac structure and function, prevents myocardial fibrosis and improves blood lipid profiles. Passive lower‐limb cycling represents an elegant, cost‐effective and widely accessible therapeutic strategy that may reduce the clinical cardiovascular burden imposed by spinal cord injury and other neurological disorders. Abstract Spinal cord injury (SCI) causes altered autonomic control and severe physical deconditioning that converge to drive maladaptive cardiac remodelling. We used a clinically relevant experimental model to investigate the cardio‐metabolic responses to SCI and to establish whether passive hind‐limb cycling elicits a cardio‐protective effect. Initially, 21 male Wistar rats were evenly assigned to three groups: uninjured control (CON), T3 complete SCI (SCI) or T3 complete SCI plus passive hind‐limb cycling (SCI‐EX; 2 × 30 min day−1, 5 days week−1 for 4 weeks beginning 6 days post‐SCI). On day 32, cardio‐metabolic function was assessed using in vivo echocardiography, ex vivo working heart assessments, cardiac histology/molecular biology and blood lipid profiles. Twelve additional rats (n = 6 SCI and n = 6 SCI‐EX) underwent in vivo echocardiography and basal haemodynamic assessments pre‐SCI and at days 7, 14 and 32 post‐SCI to track temporal cardiovascular changes. Compared with CON, SCI exhibited a rapid and sustained reduction in left ventricular dimensions and function that ultimately manifested as reduced contractility, increased myocardial collagen deposition and an up‐regulation of transforming growth factor beta‐1 (TGFβ1) and mothers against decapentaplegic homolog 3 (Smad3) mRNA. For SCI‐EX, the initial reduction in left ventricular dimensions and function at day 7 post‐SCI was completely reversed by day 32 post‐SCI, and there were no differences in myocardial contractility between SCI‐EX and CON. Collagen deposition was similar between SCI‐EX and CON. TGFβ1 and Smad3 were down‐regulated in SCI‐EX. Blood lipid profiles were improved in SCI‐EX versus SCI. We provide compelling novel evidence that passive hind‐limb cycling prevents cardiac dysfunction and reduces cardiovascular disease risk in experimental SCI.

  March 10, 2014   doi: 10.1113/jphysiol.2013.268367   open full text
• Role of nitrite in regulation of fetal cephalic circulation in sheep.
  Giang T. Truong, Hobe J. Schröder, Taiming Liu, Meijuan Zhang, Eriko Kanda, Shannon Bragg, Gordon G. Power, Arlin B. Blood.
  The Journal of Physiology. March 05, 2014
  Key points Recent evidence in adult humans demonstrates that nitrite, at physiological concentrations, can be converted into vasodilating amounts of NO, thus constituting an alternative to NO production by NO synthases. Nitrite reacts with deoxyhaemoglobin to produce NO, a reaction proposed to mediate the vasodilating effects of nitrite. We have demonstrated previously that the rate of this reaction is ∼2‐fold faster with fetal haemoglobin than adult haemoglobin. Thus, we hypothesized that nitrite would be a potent vasodilator in the cephalic vasculature served by the carotid artery in the fetal sheep. In conflict with human adult forearm studies, we find that nitrite is not a vasodilator in the fetal sheep cephalic vasculature, despite the fact that nitrite is converted to NO more efficiently by fetal haemoglobin. The results suggest that the vasodilatory effects of nitrite are age‐ and species‐specific, and that the reaction of nitrite with deoxyhaemoglobin is not rate limiting with respect to its ability to decrease vascular tone. Abstract Nitrite has been postulated to provide a reservoir for conversion to nitric oxide (NO), especially in tissues with reduced oxygen levels as in the fetus. Nitrite would thus provide local vasodilatation and restore a balance between oxygen supply and need, a putative mechanism of importance especially in the brain. The current experiments test the hypothesis that exogenous nitrite acts as a vasodilator in the cephalic vasculature of the intact, near term fetal sheep. Fetuses were first instrumented to measure arterial blood pressure and carotid artery blood flow and then studied 4–5 days later while in utero without anaesthesia. Initially l‐nitro‐arginine (LNNA) was given to block endogenous NO production. Carotid resistance to flow increased 2‐fold from 0.54 ± 0.01 (SEM) to 1.20 ± 0.08 mmHg min ml−1 (in 13 fetuses, P < 0.001), indicating NO tonically reduces cerebral vascular tone. Sodium nitrite (or saline as control) was then infused in increasing step‐doses from 0.01 to 33 μm in half‐log increments over a period of 2 h. Carotid artery pressure, blood flow and vascular resistance did not change compared to fetuses receiving saline, even at plasma nitrite concentrations two orders of magnitude above the physiological range. The results indicate that while cephalic vascular tone is controlled by endogenous nitric oxide synthase activity, exogenously administered nitrite is not a vasodilator at physiological concentrations in the vasculature served by the carotid artery of fetal sheep.

  March 05, 2014   doi: 10.1113/jphysiol.2013.269340   open full text
• Glutamate receptors in the nucleus tractus solitarius contribute to ventilatory acclimatization to hypoxia in rat.
  Matthew E. Pamenter, J. Austin Carr, Ariel Go, Zhenxing Fu, Stephen G. Reid, Frank L. Powell.
  The Journal of Physiology. March 05, 2014
  Key points Ventilation increases more with chronic than acute hypoxia and does not return to control levels when normoxia is restored, indicating plasticity in the reflexes that control breathing. Glutamate is the primary excitatory neurotransmitter between arterial chemoreceptors that sense hypoxia and neural circuits that control breathing in the brainstem. We microinjected specific glutamate receptor antagonists into the brainstem of awake unrestrained rats and found NMDA‐type glutamate receptors explain increased ventilatory sensitivity to hypoxia after chronic hypoxia. AMPA‐type glutamate receptors mediate increased ventilatory drive in normoxia after chronic hypoxia, as well as increased ventilation in acute hypoxia after chronic hypoxia and in control conditions. Phosphorylation of AMPA and NMDA receptors is increased by chronic hypoxia. The results indicate that plasticity in different glutamate receptors have unique effects on the reflexes that control breathing in chronic hypoxia and may share cellular mechanisms with other models of neural plasticity. Abstract When exposed to a hypoxic environment the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time‐dependent increase in ventilation occurs, which increases the HVR. This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. Currently these mechanisms of HVR plasticity are unknown and we hypothesized that they involve glutamatergic synapses in the nucleus tractus solitarius (NTS), where afferent endings from arterial chemoreceptors terminate. To test this, we treated rats held in normoxia (CON) or 10% O2 (CSH) for 7 days and measured ventilation in conscious, unrestrained animals before and after microinjecting glutamate receptor agonists and antagonists into the NTS. In normoxia, AMPA increased ventilation 25% and 50% in CON and CSH, respectively, while NMDA doubled ventilation in both groups (P < 0.05). Specific AMPA and NMDA receptor antagonists (NBQX and MK801, respectively) abolished these effects. MK801 significantly decreased the HVR in CON rats, and completely blocked the acute HVR in CSH rats but had no effect on ventilation in normoxia. NBQX decreased ventilation whenever it was increased relative to normoxic controls; i.e. acute hypoxia in CON and CSH, and normoxia in CSH. These results support our hypothesis that glutamate receptors in the NTS contribute to plasticity in the HVR with CSH. The mechanism underlying this synaptic plasticity is probably glutamate receptor modification, as in CSH rats the expression of phosphorylated NR1 and GluR1 proteins in the NTS increased 35% and 70%, respectively, relative to that in CON rats.

  March 05, 2014   doi: 10.1113/jphysiol.2013.268706   open full text
• Slowed muscle oxygen uptake kinetics with raised metabolism are not dependent on blood flow or recruitment dynamics.
  Rob C. I. Wüst, James R. McDonald, Yi Sun, Brian S. Ferguson, Matthew J. Rogatzki, Jessica Spires, John M. Kowalchuk, L. Bruce Gladden, Harry B. Rossiter.
  The Journal of Physiology. February 27, 2014
  Key points A slow adjustment of skeletal muscle oxygen uptake (V̇O2) to produce energy during exercise predisposes to early fatigue. In human studies, V̇O2 kinetics are slow when exercise is initiated from an elevated baseline; this is proposed to reflect slow blood flow regulation and/or recruitment of muscle fibres containing few mitochondria. To investigate this, we measured V̇O2 kinetics in canine muscle, with experimental control over muscle activation and blood flow. We found that V̇O2 kinetics remained slow when contractions were initiated from an elevated baseline despite experimentally increased blood flow and uniform fibre activation. These data challenge our current understanding of the control of muscle V̇O2 and demand consideration of new alternative mediators for V̇O2 control. Abstract Oxygen uptake kinetics (τV̇O2) are slowed when exercise is initiated from a raised metabolic rate. Whether this reflects the recruitment of muscle fibres differing in oxidative capacity, or slowed blood flow (Q̇) kinetics is unclear. This study determined τV̇O2 in canine muscle in situ, with experimental control over muscle activation and Q̇ during contractions initiated from rest and a raised metabolic rate. The gastrocnemius complex of nine anaesthetised, ventilated dogs was isolated and attached to a force transducer. Isometric tetanic contractions (50 Hz; 200 ms duration) via supramaximal sciatic nerve stimulation were used to manipulate metabolic rate: 3 min stimulation at 0.33 Hz (S1), followed by 3 min at 0.67 Hz (S2). Circulation was initially intact (SPON), and subsequently isolated for pump‐perfusion (PUMP) above the greatest value in SPON. Muscle V̇O2 was determined contraction‐by‐contraction using an ultrasonic flowmeter and venous oximeter, and normalised to tension‐time integral (TTI). τV̇O2/TTI and τQ̇ were less in S1SPON (mean ± s.d.: 13 ± 3 s and 12 ± 4 s, respectively) than in S2SPON (29 ± 19 s and 31 ± 13 s, respectively; P < 0.05). τV̇O2/TTI was unchanged by pump‐perfusion (S1PUMP, 12 ± 4 s; S2PUMP, 24 ± 6 s; P < 0.001) despite increased O2 delivery; at S2 onset, venous O2 saturation was 21 ± 4% and 65 ± 5% in SPON and PUMP, respectively. V̇O2 kinetics remained slowed when contractions were initiated from a raised metabolic rate despite uniform muscle stimulation and increased O2 delivery. The intracellular mechanism may relate to a falling energy state, approaching saturating ADP concentration, and/or slowed mitochondrial activation; but further study is required. These data add to the evidence that muscle V̇O2 control is more complex than previously suggested.

  February 27, 2014   doi: 10.1113/jphysiol.2013.267476   open full text
• Exercise training, but not resveratrol, improves metabolic and inflammatory status in skeletal muscle of aged men.
  Jesper Olesen, Lasse Gliemann, Rasmus Biensø, Jakob Schmidt, Ylva Hellsten, Henriette Pilegaard.
  The Journal of Physiology. February 27, 2014
  Key points Ageing is associated with lifestyle‐related metabolic diseases, and exercise training has been suggested to counteract such metabolic deteriorations. The natural antioxidant resveratrol has been reported to exert ‘exercise‐like’ health beneficial metabolic and anti‐inflammatory effects in rodents, but little is known about the metabolic effects of resveratrol supplementation alone and in combination with exercise training in humans. The present findings showed that exercise training markedly improved muscle endurance, increased content and activity of oxidative proteins in skeletal muscle and reduced markers of oxidative stress and inflammation in skeletal muscle of aged men. Resveratrol alone did not elicit metabolic effects in healthy aged subjects, but even impaired the exercise training‐induced improvements in markers of oxidative stress and inflammation in skeletal muscle. Abstract The aim was to investigate the metabolic and anti‐inflammatory effects of resveratrol alone and when combined with exercise training in skeletal muscle of aged human subjects. Healthy, physically inactive men (60–72 years old) were randomized to either 8 weeks of daily intake of 250 mg resveratrol or placebo or to 8 weeks of high‐intensity exercise training with 250 mg resveratrol or placebo. Before and after the interventions, resting blood samples and muscle biopsies were obtained and a one‐legged knee‐extensor endurance exercise test was performed. Exercise training increased skeletal muscle peroxisome proliferator‐activated receptor‐γ co‐activator‐1α mRNA ∼1.5‐fold, cytochrome c protein ∼1.3‐fold, cytochrome c oxidase I protein ∼1.5‐fold, citrate synthase activity ∼1.3‐fold, 3‐hydroxyacyl‐CoA dehydrogenase activity ∼1.3‐fold, inhibitor of κB‐α and inhibitor of κB‐β protein content ∼1.3‐fold and time to exhaustion in the one‐legged knee‐extensor endurance exercise test by ∼1.2‐fold, with no significant additive or adverse effects of resveratrol on these parameters. Despite an overall ∼25% reduction in total acetylation level in skeletal muscle with resveratrol, no exclusive resveratrol‐mediated metabolic effects were observed on the investigated parameters. Notably, however, resveratrol blunted an exercise training‐induced decrease (∼20%) in protein carbonylation and decrease (∼40%) in tumour necrosis factor α mRNA content in skeletal muscle. In conclusion, resveratrol did not elicit metabolic improvements in healthy aged subjects; in fact, resveratrol even impaired the observed exercise training‐induced improvements in markers of oxidative stress and inflammation in skeletal muscle. Collectively, this highlights the metabolic efficacy of exercise training in aged subjects and does not support the contention that resveratrol is a potential exercise mimetic in healthy aged subjects.

  February 27, 2014   doi: 10.1113/jphysiol.2013.270256   open full text
• Shank2 mutant mice display a hypersecretory response to cholera toxin.
  Eun Suk Jung, Joonhee Park, Heon Yung Gee, Jinsei Jung, Shin Hye Noh, Jung‐Soo Lee, Wito Richter, Wan Namkung, Min Goo Lee.
  The Journal of Physiology. February 25, 2014
  Key points Among the three Shank proteins in human (Shank1–3), Shank2 is abundantly expressed in epithelial cells. However, the in vivo physiological role of Shank2 in epithelial transport remains elusive. The functional activity and expression of cystic fibrosis transmembrane conductance regulator (CFTR) and fluid secretion in the gastrointestinal epithelia were examined in the intestines of Shank2+/+ and Shank2−/− mice using an integrated molecular and physiological approach. Shank2 deletion augmented the CFTR‐mediated short‐circuit current in the mouse colon and profoundly increased the cholera toxin‐induced fluid accumulation in the mouse intestine. Shank2 appears to be a key molecule regulating epithelial fluid secretion. Modulation of Shank2‐mediated function would be helpful to treat diseases associated with hypersecretion or hyposecretion in epithelia. Abstract Shank2 is a PDZ (PSD‐95/discs large/ZO‐1)‐based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2−/−) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2+/+ and Shank2−/− mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent short‐circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR–Shank2–phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin‐induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.

  February 25, 2014   doi: 10.1113/jphysiol.2013.268631   open full text
• Genetic ablation of ryanodine receptor 2 phosphorylation at Ser‐2808 aggravates Ca2+‐dependent cardiomyopathy by exacerbating diastolic Ca2+ release.
  Bin Liu, Hsiang‐Ting Ho, Florencia Velez‐Cortes, Qing Lou, Carmen R. Valdivia, Bjorn C. Knollmann, Hector H. Valdivia, Sandor Gyorke.
  The Journal of Physiology. February 21, 2014
  Key points Phosphorylation at Ser‐2808 is suggested to result in RyR2 hyperactivity, i.e. ‘leakiness’, thus contributing to the pathology of cardiac diseases. We studied the effect of disabling phosphorylation at Ser‐2808 of RyR2 in a genetic model of Ca2+‐dependent cardiomyopathy, which was caused by leaky RyR2. RyR2 phosphorylation was high at Ser‐2808 in myocytes expressing wild‐type (WT) RyR2; protein phosphatase increased RyR2 leakiness in cells expressing WT, but not in mutant RyR2s with disabled Ser‐2808 phosphorylation sites. Rather than alleviating cardiac disease, ablation of the Ser‐2808 exacerbated the disease phenotype by reducing survival, impairing in vivo cardiac function and enhancing RyR2 Ca2+ leak and mitochondrial damage. These results suggest a novel mode of RyR2 regulation via dephosphorylation at Ser‐2808 in normal and diseased hearts. Abstract Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser‐2808 is suggested to mediate the physiological ‘fight or flight’ response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca2+. In the present study, we examined the potential role of RyR2 phosphorylation at Ser‐2808 in the progression of Ca2+‐dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca2+ leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser‐2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca2+ leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca2+ handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser‐2808 than in mice with complete ablation. Wild‐type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser‐2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca2+ leak attributable to partial dephosphorylation of RyR2 tetramers at Ser‐2808 from more fully phosphorylated state. Thus, exacerbated Ca2+ leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca2+‐dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca2+ signalling.

  February 21, 2014   doi: 10.1113/jphysiol.2013.264689   open full text
• Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling.
  Dóra Bodnár, Nikolett Geyer, Olga Ruzsnavszky, Tamás Oláh, Bence Hegyi, Mónika Sztretye, János Fodor, Beatrix Dienes, Ágnes Balogh, Zoltán Papp, László Szabó, Géza Müller, László Csernoch, Péter Szentesi.
  The Journal of Physiology. February 19, 2014
  Key points Hypermuscularity associated with naturally occurring mutations in the myostatin gene as found in Compact mice results in increased muscle mass but reduced specific force. The calcium sensitivity of the contractile apparatus as assessed on chemically skinned skeletal muscle fibres under isometric conditions is not altered in these animals. While the resting calcium concentration remains unaffected, depolarization‐evoked increases in intracellular calcium concentration are suppressed. Spontaneous calcium release events from sarcoplasmic reticulum are also decreased in frequency, amplitude and spatial spread. Our results suggest that mutations in the myostatin gene are accompanied by alterations in excitation contraction coupling, which manifest as a reduction in sarcoplasmic calcium release. Abstract Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin‐deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation–contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (MstnCmpt‐dl1Abc or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa–force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca2+ concentration ([Ca2+]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura‐2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nm, n = 38 and 45), the amplitude of KCl‐evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nm, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod‐2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mMs−1) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mm s−1) with no change in the peak‐to‐steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

  February 19, 2014   doi: 10.1113/jphysiol.2013.261958   open full text
• Reflex control of inflammation by sympathetic nerves, not the vagus.
  D. Martelli, S. T. Yao, M. J. McKinley, R. M. McAllen.
  The Journal of Physiology. February 19, 2014
  Key points It is believed that the CNS controls inflammation via the autonomic nervous system, but the strength of this action and the neural pathways responsible are unclear. In anaesthetized rats we measured the inflammatory response to lipopolysaccharide (LPS, 60 μg kg−1, i.v.) by plasma tumour necrosis factor α (TNFα) levels 90 min later. Bilateral section of the splanchnic sympathetic nerves before LPS treatment resulted in a 5‐fold increase in the plasma TNFα response, but bilateral vagotomy had no effect. LPS treatment strongly increased efferent activity in the splanchnic sympathetic nerve and its splenic branch; vagotomy did not affect this. These results show that, besides directly stimulating inflammation, LPS engages a powerful anti‐inflammatory reflex that can inhibit the plasma TNFα response by 80%. The reflex efferent arm is in the splanchnic sympathetic nerves; the vagi play no part. Abstract We investigated a neural reflex that controls the strength of inflammatory responses to immune challenge – the inflammatory reflex. In anaesthetized rats challenged with intravenous lipopolysaccharide (LPS, 60 μg kg−1), we found strong increases in plasma levels of the key inflammatory mediator tumour necrosis factor α (TNFα) 90 min later. Those levels were unaffected by previous bilateral cervical vagotomy, but were enhanced approximately 5‐fold if the greater splanchnic sympathetic nerves had been cut. Sham surgery had no effect, and plasma corticosterone levels were unaffected by nerve sections, so could not explain this result. Electrophysiological recordings demonstrated that efferent neural activity in the splanchnic nerve and its splenic branch was strongly increased by LPS treatment. Splenic nerve activity was dependent on inputs from the splanchnic nerves: vagotomy had no effect on the activity in either nerve. Together, these data demonstrate that immune challenge with this dose of LPS activates a neural reflex that is powerful enough to cause an 80% suppression of the acute systemic inflammatory response. The efferent arm of this reflex is in the splanchnic sympathetic nerves, not the vagi as previously proposed. As with other physiological responses to immune challenge, the afferent pathway is presumptively humoral: the present data show that vagal afferents play no measurable part. Because inflammation sits at the gateway to immune responses, this reflex could play an important role in immune function as well as inflammatory diseases.

  February 19, 2014   doi: 10.1113/jphysiol.2013.268573   open full text
• Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis.
  Nicolas Delestrée, Marin Manuel, Caroline Iglesias, Sherif M. Elbasiouny, C. J. Heckman, Daniel Zytnicki.
  The Journal of Physiology. February 19, 2014
  Key points Intrinsic hyperexcitability of spinal motoneurones is thought to contribute to excitotoxicity during amyotrophic lateral sclerosis (ALS), but it has never been demonstrated that adult motoneurones become hyperexcitable before disconnection from their muscle fibres. We found an increased input conductance in motoneurones recorded in a mouse model of ALS. Yet, most cells retained normal excitability as measured by current onset for firing and input–output gain. This indicates successful regulation of excitability, compensating for the increase in conductance. In contrast, some cells became hypoexcitable, losing their ability to fire repetitively to quasi‐stationary inputs before denervation. Hypoexcitability might therefore be an early marker of disease progression. We thereby demonstrate that, if excitotoxicity is indeed a mechanism leading to degeneration in ALS, it is not caused by changes in the intrinsic electrical properties of the motoneurones but most probably by extrinsic factors such as excessive synaptic excitation. Abstract In amyotrophic lateral sclerosis (ALS), an adult onset disease in which there is progressive degeneration of motoneurones, it has been suggested that an intrinsic hyperexcitability of motoneurones (i.e. an increase in their firing rates), contributes to excitotoxicity and to disease onset. Here we show that there is no such intrinsic hyperexcitability in spinal motoneurones. Our studies were carried out in an adult mouse model of ALS with a mutated form of superoxide dismutase 1 around the time of the first muscle fibre denervations. We showed that the recruitment current, the voltage threshold for spiking and the frequency–intensity gain in the primary range are all unchanged in most spinal motoneurones, despite an increased input conductance. On its own, increased input conductance would decrease excitability, but the homeostasis for excitability is maintained due to an upregulation of a depolarizing current that is activated just below the spiking threshold. However, this homeostasis failed in a substantial fraction of motoneurones, which became hypoexcitable and unable to produce sustained firing in response to ramps of current. We found similar results both in lumbar motoneurones recorded in anaesthetized mice, and in sacrocaudal motoneurones recorded in vitro, indicating that the lack of hyperexcitability is not caused by anaesthetics. Our results suggest that, if excitotoxicity is indeed a mechanism leading to degeneration in ALS, it is not caused by the intrinsic electrical properties of motoneurones but by extrinsic factors such as excessive synaptic excitation.

  February 19, 2014   doi: 10.1113/jphysiol.2013.265843   open full text
• GABAergic and glycinergic inputs modulate rhythmogenic mechanisms in the lamprey respiratory network.
  Elenia Cinelli, Donatella Mutolo, Brita Robertson, Sten Grillner, Massimo Contini, Tito Pantaleo, Fulvia Bongianni.
  The Journal of Physiology. February 19, 2014
  Key points In this study we investigated the role of GABA and glycine receptors within the respiratory central pattern generator, i.e. the paratrigeminal respiratory group (pTRG), and the vagal motoneuron region of the lamprey. Only GABA‐mediated inhibition modulates the pTRG both during apnoea induced by blockade of glutamatergic transmission and under basal conditions. Both GABA‐ and glycine‐mediated inhibition within the vagal region are involved in the regulation of respiratory frequency via ascending excitatory projections to the pTRG. Projecting neurons are retrogradely labelled from the pTRG, and intense GABA immunoreactivity is present within the pTRG and the vagal motoneuron region. Inhibitory mechanisms, which appear to be evolutionarily conserved, regulate network excitability and may provide an important contribution to rhythmic activities, such as respiration and locomotion. Abstract We have previously shown that GABA and glycine modulate respiratory activity in the in vitro brainstem preparations of the lamprey and that blockade of GABAA and glycine receptors restores the respiratory rhythm during apnoea caused by blockade of ionotropic glutamate receptors. However, the neural substrates involved in these effects are unknown. To address this issue, the role of GABAA, GABAB and glycine receptors within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, and the vagal motoneuron region was investigated both during apnoea induced by blockade of glutamatergic transmission and under basal conditions through microinjections of specific antagonists. The removal of GABAergic, but not glycinergic transmission within the pTRG, causes the resumption of rhythmic respiratory activity during apnoea, and reveals the presence of a modulatory control of the pTRG under basal conditions. A blockade of GABAA and glycine receptors within the vagal region strongly increases the respiratory frequency through disinhibition of neurons projecting to the pTRG from the vagal region. These neurons were retrogradely labelled (neurobiotin) from the pTRG. Intense GABA immunoreactivity is observed both within the pTRG and the vagal area, which corroborates present findings. The results confirm the pTRG as a primary site of respiratory rhythm generation, and suggest that inhibition modulates the activity of rhythm‐generating neurons, without any direct role in burst formation and termination mechanisms.

  February 19, 2014   doi: 10.1113/jphysiol.2013.268086   open full text
• Sarcoplasmic reticulum Ca2+ uptake and leak properties and SERCA protein expression in type I and type II fibres of human skeletal muscle.
  C.R. Lamboley, R.M. Murphy, M.J. McKenna, G.D. Lamb.
  The Journal of Physiology. February 19, 2014
  The Ca2+ uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically‐skinned muscle fibres were exposed to solutions with the free [Ca2+] heavily buffered in the range pCa (=−log10[Ca2+]) 7.3 to 6.0 for set times and the amount of net SR Ca2+ accumulation determined from the force response elicited upon emptying the SR of all Ca2+. Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half maximal Ca2+ uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half maximal Ca2+ uptake rate at ∼pCa 6.6. Maximal Ca2+ uptake rate was ∼0.18 and ∼0.21 mmol Ca2+ per litre fibre volume per second in type I and type II fibres respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg2+] from 1 to 3 mM had no significant effect on the net Ca2+ uptake rate at pCa 6.0, indicating that there was little or no calcium‐induced calcium release occurring through the Ca2+ release channels during uptake in either fibre type. Ca2+ leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2 fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca2+ leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca2+ uptake in the type I human fibres can account for the higher relative level of SR Ca2+ loading observed in type I compared to type II fibres, and the SR Ca2+ leakage characteristics of the human fibres suggest that the SERCA are regulated differently than in rat and contribute comparatively less to resting metabolic rate. This article is protected by copyright. All rights reserved

  February 19, 2014   doi: 10.1113/jphysiol.2013.269373   open full text
• Purinergic inhibitory regulation of murine detrusor muscles mediated by PDGFRα+ interstitial cells.
  Haeyeong Lee, Byoung H. Koh, Lauren E. Peri, Kenton M. Sanders, Sang Don Koh.
  The Journal of Physiology. February 17, 2014
  Key points Platelet‐derived growth factor receptor‐α‐positive (PDGFRα+) interstitial cells in detrusor muscles may participate in post‐junctional responses to neurotransmitters. PDGFRα+ interstitial cells express purinergic receptors (P2Y) and small conductance Ca2+‐activated K+ channels (mainly SK3). ATP elicited large amplitude outward currents and hyperpolarization in PDGFRα+ cells. SK channel blockers and a P2Y1 receptor antagonist blocked responses to ATP. ATP elicited only minor responses in PDGFRα+ cells of P2ry1−/− mice. ATP elicited transient inward currents in smooth muscle cells and purinergic receptor (P2X) agonists had no effect on PDGFRα+ cells. A specific P2Y1 receptor blocker decreased electrical field stimulation‐induced relaxation. Our findings provide an explanation for the purinergic relaxation of detrusor muscles and describe a novel mechanism for inhibitory regulation of bladder muscles that may control detrusor excitability during the filling phase. Abstract Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction could be due to activation of inward currents in smooth muscle cells, but the mechanism of purinergic relaxation has not been determined. We recently reported a new class of interstitial cells in detrusor muscles and showed that these cells could be identified with antibodies against platelet‐derived growth factor receptor‐α (PDGFRα+ cells). The current density of small conductance Ca2+‐activated K+ (SK) channels in these cells is far higher (∼100 times) than in smooth muscle cells. Thus, we examined purinergic receptor (P2Y) mediated SK channel activation as a mechanism for purinergic relaxation. P2Y receptors (mainly P2ry1 gene) were highly expressed in PDGFRα+ cells. Under voltage clamp conditions, ATP activated large outward currents in PDGFRα+ cells that were inhibited by blockers of SK channels. ATP also induced significant hyperpolarization under current clamp conditions. A P2Y1 agonist, MRS2365, mimicked the effects of ATP, and a P2Y1 antagonist, MRS2500, inhibited ATP‐activated SK currents. Responses to ATP were largely abolished in PDGFRα+ cells of P2ry1−/− mice, and no response was elicited by MRS2365 in these cells. A P2X receptor agonist had no effect on PDGFRα+ cells but, like ATP, activated transient inward currents in smooth muscle cells (SMCs). A P2Y1 antagonist decreased nerve‐evoked relaxation. These data suggest that purines activate SK currents via mainly P2Y1 receptors in PDGFRα+ cells. Our findings provide an explanation for purinergic relaxation in detrusor muscles and show that there are no discrete inhibitory nerve fibres. A dual receptive field for purines provides the basis for inhibitory neural regulation of excitability.

  February 17, 2014   doi: 10.1113/jphysiol.2013.267989   open full text
• Muscle contraction increases interstitial nitric oxide as predicted by a new model of local blood flow regulation.
  Aleksander S. Golub, Bjorn K. Song, Roland N. Pittman.
  The Journal of Physiology. February 17, 2014
  Key points The metabolic theory of blood flow regulation suggests that, when tissue cells experience a reduction of oxygen supply, they produce metabolic vasodilators, which increase the lumen of arterioles and hence local blood flow. A century of intensive research has not found a single metabolic vasodilator to account for observed flow changes, therefore current thought is that there are many vasodilators. We have proposed an alternative hypothesis based on the interaction of two well‐known molecular mechanisms for generation and removal of the intercellular signalling radicals nitric oxide (NO) and superoxide. The proposed mechanism of regulation predicts a sharp increase in NO concentration in the intercellular space at the onset of muscle contraction. Experiments with the NO‐sensitive fluorescent indicator DAF‐FM, loaded into the intercellular space, confirmed the rapid response of the NO‐related signal at the beginning of contractions and rapid washout of the indicator after their termination. Abstract The prevailing metabolic theory of local blood flow regulation suggests the dilatation of arterioles in response to tissue hypoxia via the emission of multiple metabolic vasodilators by parenchymal cells. We have proposed a mechanism of regulation, built from well‐known components, which assumes that arterioles are normally dilated in metabolically active tissues, due to the emission of NO by the endothelium of microvessels. Regulation of local blood flow aims at preventing an excessive supply of oxygen (O2) and glucose to the tissue and thus provides an adequate supply, in contrast to the metabolic regulation theory which requires permanent hypoxia to generate the metabolic vasodilators. The mediator of the restrictive signal is superoxide anion (O2−) released by membrane NAD(P)H oxidases into the interstitial space, where it neutralizes NO at a diffusion‐limited rate. This model predicts that the onset of muscle contraction will lead to the cessation of O2− production, which will cause an elevation of interstitial NO concentration and an increase in fluorescence of the NO probe DAF‐FM after its conversion to DAF‐T. The time course of DAF‐T fluorescence in contracting muscle is predicted by also considering the washout from the muscle of the interstitially loaded NO indicator. Experiments using pulse fluorimetry confirmed an increase in the interstitial concentration of NO available for reaction with DAF‐FM during bouts of muscle contraction. The sharp increase in interstitial [NO] is consistent with the hypothesis that the termination of the neutralizing superoxide flow into the interstitium is associated with the activation of mitochondria and a reduction of the interstitial oxygen tension. The advantage of the new model is its ability to explain the interaction of metabolic activity and local blood flow through the adequate delivery of glucose and oxygen.

  February 17, 2014   doi: 10.1113/jphysiol.2013.267302   open full text
• Huntingtin‐associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells.
  Kimberly D. Mackenzie, Michael D. Duffield, Heshan Peiris, Lucy Phillips, Mark P. Zanin, Ee Hiok Teo, Xin‐Fu Zhou, Damien J. Keating.
  The Journal of Physiology. February 17, 2014
  Key points Huntingtin‐associated protein 1 (HAP1) is expressed in neurons and endocrine cells, in which it is thought to regulate vesicle trafficking. HAP1 is a binding partner of the Huntington's disease (HD)‐causing protein huntingtin, and binding is stronger in HD. Whether HAP1 regulates a significant end‐point of vesicle transport, exocytosis, and what stage of exocytosis HAP1 may regulate, is unknown. We use mouse chromaffin cells to demonstrate that HAP1 regulates exocytosis via two potentially interlinked mechanisms: control of vesicle docking and the readily releasable vesicle pool, and regulation of fusion pore stabilization. These results establish HAP1 as a significant player in exocytosis control with potential relevance for HD and for a number of neuronal and homeostatic pathways. Abstract Huntingtin‐associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon‐fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1−/− and HAP1+/+ littermate mice. Numbers of Ca2+‐dependent and Ca2+‐independent full fusion events in HAP1−/− cells are significantly decreased compared with those in HAP1+/+ cells. We observed no change in the frequency of ‘kiss‐and‐run’ fusion events or in Ca2+ entry. Whereas release per full fusion event is unchanged in HAP1−/− cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre‐spike foot signals. Kiss‐and‐run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1−/− cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.

  February 17, 2014   doi: 10.1113/jphysiol.2013.268342   open full text
• Acute pancreatitis decreases the sensitivity of pancreas‐projecting dorsal motor nucleus of the vagus neurones to group II metabotropic glutamate receptor agonists in rats.
  Tanja Babic, R. Alberto Travagli.
  The Journal of Physiology. February 17, 2014
  Key points Acute pancreatitis is one of the most severe disorders of the exocrine pancreas. Pancreatic exocrine secretions (PES) are under regulatory control of dorsal motor nucleus of the vagus (DMV) neurones and their activity is regulated by inhibitory GABAergic and excitatory glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas‐projecting DMV neurones and modulate PES. In this study, we show that acute pancreatitis induces a long‐lasting increase in excitatory synaptic transmission to pancreas‐projecting neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonists. These data suggest that changes in group II mGluR expression in the DMV may underlie short‐ and long‐term changes in PES in acute pancreatitis. Abstract Recent studies have shown that pancreatic exocrine secretions (PES) are modulated by dorsal motor nucleus of the vagus (DMV) neurones, whose activity is finely tuned by GABAergic and glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas‐projecting DMV neurones and increase PES. In the present study, we used a combination of in vivo and in vitro approaches aimed at characterising the effects of caerulein‐induced acute pancreatitis (AP) on the vagal neurocircuitry modulating pancreatic functions. In control rats, microinjection of bicuculline into the DMV increased PES, whereas microinjections of kynurenic acid had no effect. Conversely, in AP rats, microinjection of bicuculline had no effect, whereas kynurenic acid decreased PES. DMV microinjections of the group II mGluR agonist APDC and whole cell recordings of excitatory currents in identified pancreas‐projecting DMV neurones showed a reduced functional response in AP rats compared to controls. Moreover, these changes persisted up to 3 weeks following the induction of AP. These data demonstrate that AP increases the excitatory input to pancreas‐projecting DMV neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonist.

  February 17, 2014   doi: 10.1113/jphysiol.2013.270108   open full text
• Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine.
  Coral L. Murrant, Jason D. Dodd, Andrew J. Foster, Kristin A. Inch, Fiona R. Muckle, Della A. Ruiz, Jeremy A. Simpson, Jordan H.P. Scholl.
  The Journal of Physiology. February 17, 2014
  Key points The role of prostaglandins in the changes in blood flow and microvascular vasodilation associated with exercise and muscle contraction is controversial. Whether prostaglandins are produced independently during muscle contraction or whether their production is dependent on the production of adenosine is not well understood. We show that prostaglandins are an important component of the microvascular vasodilation associated with muscle contraction but only under specific contractile conditions. Further, we show that microvascular vasodilation in response to adenosine is not dependent on prostaglandins. Therefore, we conclude that there are specific contractile conditions under which prostaglandins are an important component of the vasodilation induced by muscle contraction and we propose that prostaglandin‐induced microvascular vasodilation during exercise is independent of adenosine. Abstract Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 μm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10−7–10−5 m) extraluminally, (to mimic muscle contraction) in the absence and presence of l‐NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and l‐NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). l‐NAME, INDO and l‐NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in response to muscle contraction but did not obtain evidence that extraluminal ADO causes vasodilatation through NO or PG or both. Thus, we propose that PG‐induced microvascular vasodilation during exercise is independent of ADO.

  February 17, 2014   doi: 10.1113/jphysiol.2013.264259   open full text
• Looping circuit: a novel mechanism for prolonged spontaneous [Ca2+]i increases in developing embryonic mouse brainstem.
  Hirofumi Watari, Amanda J. Tose, Martha M. Bosma.
  The Journal of Physiology. February 14, 2014
  Key points Calcium concentration is kept at extremely low levels inside brain cells; each episode of calcium entry is cleared within seconds. Changes in calcium entry are mediated by spontaneous activity in embryonic mouse brainstem from embryonic day 11.5 to 13.5. Transiently, at embryonic day 12.5, spontaneous events occur frequently such that calcium concentration stays above baseline levels for minutes. This unusual phenomenon, which we termed ‘bash bursts’, is caused by an event that propagates by looping along a defined path; the path gets modified a day later, ending it. The results help us to understand how prolonged increases in calcium concentration can occur in development and how the increases may influence the development of serotonin and dopamine circuits that are related to neurological diseases later in life, such as depression and Parkinson's disease. Abstract Most cells maintain [Ca2+]i at extremely low levels; calcium entry usually occurs briefly, and within seconds it is cleared. However, at embryonic day 12.5 in the mouse brainstem, trains of spontaneous events occur with [Ca2+]i staying close to peak value, well above baseline, for minutes; we termed this ‘bash bursts’. Here, we investigate the mechanism of this unusual activity using calcium imaging and electrophysiology. Bash bursts are triggered by an event originating at the mid‐line of the rostral hindbrain and are usually the result of that event propagating repeatedly along a defined circular path. The looping circuit can either encompass both the midbrain and hindbrain or remain in the hindbrain only, and the type of loop determines the duration of a single lap time, 5 or 3 s, respectively. Bash bursts are supported by high membrane excitability of mid‐line cells and are regulated by persistent inward ‘window current’ at rest, contributing to spontaneous activity. This looping circuit is an effective means for increasing [Ca2+]i at brief, regular intervals. Bash bursts disappear by embryonic day 13.5 via alteration of the looping circuit, curtailing the short epoch of bash bursts. The resulting sustained [Ca2+]i may influence development of raphe serotonergic and ventral tegmental dopaminergic neurons by modulating gene expression.

  February 14, 2014   doi: 10.1113/jphysiol.2013.265892   open full text
• Complementary functions of SK and Kv7/M potassium channels in excitability control and synaptic integration in rat hippocampal dentate granule cells.
  Pedro Mateos‐Aparicio, Ricardo Murphy, Johan F. Storm.
  The Journal of Physiology. February 14, 2014
  Key points Previous studies showed that different firing patterns of hippocampal dentate granule cells (DGCs) can trigger different network responses. However, the intrinsic DGC mechanisms controlling their excitability, spike patterns and synaptic integration in DGCs, remain poorly understood. SK and Kv7/M channels play important roles controlling neuronal integration and excitability, but their specific roles vary between cell types. Both channel types are expressed in DGCs, but their roles are unclear. We found that SK channels are the main generators of medium afterhyperpolarizations in DGCs, thus causing negative feedback regulation of spiking (spike frequency adaptation) and calcium influx. In contrast, Kv7/M perform subthreshold and ‘feed‐forward’ control of input resistance, postsynaptic integration, action potential threshold and excitability, thus weakening EPSP–spike coupling. Thus, in DGCs, the SK and Kv7/M channels seem to perform complementary functions in postsynaptic integration and excitability control. This may have important consequences for dentate network physiology. Abstract The dentate granule cells (DGCs) form the most numerous neuron population of the hippocampal memory system, and its gateway for cortical input. Yet, we have only limited knowledge of the intrinsic membrane properties that shape their responses. Since SK and Kv7/M potassium channels are key mechanisms of neuronal spiking and excitability control, afterhyperpolarizations (AHPs) and synaptic integration, we studied their functions in DGCs. The specific SK channel blockers apamin or scyllatoxin increased spike frequency (excitability), reduced early spike frequency adaptation, fully blocked the medium‐duration AHP (mAHP) after a single spike or spike train, and increased postsynaptic EPSP summation after spiking, but had no effect on input resistance (Rinput) or spike threshold. In contrast, blockade of Kv7/M channels by XE991 increased Rinput, lowered the spike threshold, and increased excitability, postsynaptic EPSP summation, and EPSP–spike coupling, but only slightly reduced mAHP after spike trains (and not after single spikes). The SK and Kv7/M channel openers 1‐EBIO and retigabine, respectively, had effects opposite to the blockers. Computational modelling reproduced many of these effects. We conclude that SK and Kv7/M channels have complementary roles in DGCs. These mechanisms may be important for the dentate network function, as CA3 neurons can be activated or inhibition recruited depending on DGC firing rate.

  February 14, 2014   doi: 10.1113/jphysiol.2013.267872   open full text
• Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.
  Helga Vitzthum, Anika Seniuk, Laura Helene Schulte, Maxie Luise Müller, Hannah Hetz, Heimo Ehmke.
  The Journal of Physiology. February 13, 2014
  Key points The adrenal hormone aldosterone can stimulate K+ secretion during hyperkalaemia and Na+ reabsorption during hypovolaemia in the kidney. Angiotensin II is thought to switch the physiological mode of action from K+ excretion towards Na+ retention, but how the regulation is achieved when angiotensin II levels are suppressed by high Na+ intake remains unknown. We report that both dietary K+ depletion and dietary K+ loading provoke renal Na+ retention and increase blood pressure in Na+ replete mice, but these occur through different renal kinase signalling and Na+ transport pathways. An angiotensin II‐ and aldosterone‐independent activation of the sodium‐chloride cotransporter NCC contributes to the blood pressure increase induced by K+ depletion, whereas the hypertensive response to K+ loading is dependent on neither aldosterone nor Na+ transport via the epithelial sodium channel ENaC. These findings imply a major impact of K+ homeostasis on renal Na+ handling in the Na+ replete state and suggest a mechanism for the hypertensive effect of the Western diet (high Na+ and low K+) in humans. Abstract A network of kinases, including WNKs, SPAK and Sgk1, is critical for the independent regulation of K+ and Na+ transport in the distal nephron. Angiotensin II is thought to act as a key hormone in orchestrating these kinases to switch from K+ secretion during hyperkalaemia to Na+ reabsorption during intravascular volume depletion, thus keeping disturbances in electrolyte and blood pressure homeostasis at a minimum. It remains unclear, however, how K+ and Na+ transport are regulated during a high Na+ intake, which is associated with suppressed angiotensin II levels and a high distal tubular Na+ load. We therefore investigated the integrated blood pressure, renal, hormonal and gene and protein expression responses to large changes of K+ intake in Na+ replete mice. Both low and high K+ intake increased blood pressure and caused Na+ retention. Low K+ intake was accompanied by an upregulation of the sodium‐chloride cotransporter (NCC) and its activating kinase SPAK, and inhibition of NCC normalized blood pressure. Renal responses were unaffected by angiotensin AT1 receptor antagonism, indicating that low K+ intake activates the distal nephron by an angiotensin‐independent mode of action. High K+ intake was associated with elevated plasma aldosterone concentrations and an upregulation of the epithelial sodium channel (ENaC) and its activating kinase Sgk1. Surprisingly, high K+ intake increased blood pressure even during ENaC or mineralocorticoid receptor antagonism, suggesting the contribution of aldosterone‐independent mechanisms. These findings show that in a Na+ replete state, changes in K+ intake induce specific molecular and functional adaptations in the distal nephron that cause a functional coupling of renal K+ and Na+ handling, resulting in Na+ retention and high blood pressure when K+ intake is either restricted or excessively increased.

  February 13, 2014   doi: 10.1113/jphysiol.2013.266924   open full text
• Reduced contribution of endothelin to the regulation of systemic and pulmonary vascular tone in severe familial hypercholesterolaemia.
  Shawn B. Bender, Vincent J. Beer, Darla L. Tharp, Elza D. Deel, Douglas K. Bowles, Dirk J. Duncker, M. Harold Laughlin, Daphne Merkus.
  The Journal of Physiology. February 13, 2014
  Key points Familial hypercholesterolaemia (FH) causes vascular dysfunction involving reduced nitric oxide (NO) bioavailability and limits exercise‐induced vasodilatation in the systemic, but not pulmonary, vasculature. The mechanism(s) underlying blunted exercise‐induced systemic vasodilatation in FH are unclear but may involve enhanced endothelin‐1 (ET‐1)‐mediated vasoconstriction resulting from lessened NO‐dependent inhibition. In a chronically instrumented swine model of FH, ET‐1 receptor inhibition in vivo did not restore systemic exercise‐induced vasodilatation but rather revealed a reduced role for ET‐1 in regulating systemic and pulmonary vascular tone at rest and during exercise associated with reduced circulating ET‐1 in FH swine. In contrast, isolated skeletal muscle arterioles from FH swine exhibited enhanced ET‐1 sensitivity due to reduced NO with no change in smooth muscle ET receptor expression. These results increase understanding of FH‐associated vascular dysfunction by revealing a novel reduction in ET production and resultant attenuation of ET‐dependent vascular tone in vivo in FH. Abstract Vascular dysfunction has been associated with familial hypercholesterolaemia (FH), a severe form of hyperlipidaemia. We recently demonstrated that swine with FH exhibit reduced exercise‐induced systemic, but not pulmonary, vasodilatation involving reduced nitric oxide (NO) bioavailability. Since NO normally limits endothelin (ET) action, we examined the hypothesis that reduced systemic vasodilatation during exercise in FH swine results from increased ET‐mediated vasoconstriction. Systemic and pulmonary vascular responses to exercise were examined in chronically instrumented normal and FH swine in the absence and presence of the ETA/B receptor antagonist tezosentan. Intrinsic reactivity to ET was further assessed in skeletal muscle arterioles. FH swine exhibited ∼9‐fold elevation in total plasma cholesterol versus normal swine. Similar to our recent findings, systemic, not pulmonary, vasodilatation during exercise was reduced in FH swine. Blockade of ET receptors caused marked systemic vasodilatation at rest and during exercise in normal swine that was significantly reduced in FH swine. The reduced role of ET in FH swine in vivo was not the result of decreased arteriolar ET responsiveness, as responsiveness was increased in isolated arterioles. Smooth muscle ET receptor protein content was unaltered by FH. However, circulating plasma ET levels were reduced in FH swine. ET receptor antagonism caused pulmonary vasodilatation at rest and during exercise in normal, but not FH, swine. Therefore, contrary to our hypothesis, FH swine exhibit a generalised reduction in the role of ET in regulating vascular tone in vivo probably resulting from reduced ET production. This may represent a unique vascular consequence of severe familial hypercholesterolaemia.

  February 13, 2014   doi: 10.1113/jphysiol.2013.267351   open full text
• Omega‐3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle.
  E. A. F. Herbst, S. Paglialunga, C. Gerling, J. Whitfield, K. Mukai, A. Chabowski, G. J. F. Heigenhauser, L. L. Spriet, G. P. Holloway.
  The Journal of Physiology. February 13, 2014
  Key points Following fish oil supplementation, omega‐3 fatty acids are incorporated into cellular membranes, which may affect lipid–protein interactions and therefore the function of embedded proteins. As the components of the electron transport chain required for oxidative phosphorylation are contained in the mitochondrial membrane, omega‐3 supplementation may alter metabolic function. We supplemented male participants for 12 weeks with fish oil [eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)] and analysed mitochondrial function and reactive oxygen species (ROS) emissions in permeabilized muscle fibres from the vastus lateralis muscle. Supplementation incorporated EPA and DHA into mitochondrial membranes, but did not result in changes in maximal mitochondrial respiratory function or pyruvate respiration kinetics. However, the apparent Km for ADP was decreased following supplementation, and was independent of creatine, changes in the protein content of ADP synthase or ANT transporters. The propensity for ROS emissions increased with omega‐3 supplementation, although there were no changes in markers of lipid or protein oxidative damage. These results demonstrate that omega‐3 supplementation improves mitochondrial ADP kinetics, suggesting post‐translational modification of existing proteins. Abstract Studies have shown increased incorporation of omega‐3 fatty acids into whole skeletal muscle following supplementation, although little has been done to investigate the potential impact on the fatty acid composition of mitochondrial membranes and the functional consequences on mitochondrial bioenergetics. Therefore, we supplemented young healthy male subjects (n = 18) with fish oils [2 g eicosapentaenoic acid (EPA) and 1 g docosahexanoic acid (DHA) per day] for 12 weeks and skeletal muscle biopsies were taken prior to (Pre) and following (Post) supplementation for the analysis of mitochondrial membrane phospholipid composition and various assessments of mitochondrial bioenergetics. Total EPA and DHA content in mitochondrial membranes increased (P < 0.05) ∼450 and ∼320%, respectively, and displaced some omega‐6 species in several phospholipid populations. Mitochondrial respiration, determined in permeabilized muscle fibres, demonstrated no change in maximal substrate‐supported respiration, or in the sensitivity (apparent Km) and maximal capacity for pyruvate‐supported respiration. In contrast, mitochondrial responses during ADP titrations demonstrated an enhanced ADP sensitivity (decreased apparent Km) that was independent of the creatine kinase shuttle. As the content of ANT1, ANT2, and subunits of the electron transport chain were unaltered by supplementation, these data suggest that prolonged omega‐3 intake improves ADP kinetics in human skeletal muscle mitochondria through alterations in membrane structure and/or post‐translational modification of ATP synthase and ANT isoforms. Omega‐3 supplementation also increased the capacity for mitochondrial reactive oxygen species emission without altering the content of oxidative products, suggesting the absence of oxidative damage. The current data strongly emphasize a role for omega‐3s in reorganizing the composition of mitochondrial membranes while promoting improvements in ADP sensitivity.

  February 13, 2014   doi: 10.1113/jphysiol.2013.267336   open full text
• Ectopic release of glutamate contributes to spillover at parallel fibre synapses in the cerebellum.
  Saju Balakrishnan, Katharine L. Dobson, Claire Jackson, Tomas C. Bellamy.
  The Journal of Physiology. February 13, 2014
  Key points Release of neurotransmitter can sometimes occur outside of the synaptic cleft, a process known as ectopic release. Spillover of the excitatory transmitter glutamate between synapses occurs when parallel fibres in the cerebellum are stimulated at high frequencies. We investigated the effect of activity‐dependent and pharmacological reduction of ectopic release on the time course of postsynaptic currents and found that the decay time is reduced, suggesting that ectopic release contributes to spillover at the synapses. This finding suggests that ectopic transmission can cause activation of extrasynaptic receptors even at low frequencies, and so may play a significant role in synaptic plasticity. The results help us understand how signalling in and around the synapse can alter network activity in the cerebellum, a brain region essential for fine motor coordination. Abstract In the rat cerebellar molecular layer, spillover of glutamate between parallel fibre synapses can lead to activation of perisynaptic receptors that mediate short‐ and long‐term plasticity. This effect is greatest when clusters of fibres are stimulated at high frequencies, suggesting that glutamate clearance mechanisms must be overwhelmed before spillover can occur. However, parallel fibres can also release transmitter directly into the extracellular space, from ‘ectopic’ release sites. Ectopic transmission activates AMPA receptors on the Bergmann glial cell processes that envelop parallel fibre synapses, but the possible contribution of this extrasynaptic release to intersynaptic communication has not been explored. We exploited long‐term depression of ectopic transmission, and selective pharmacology, to investigate the impact of these release sites on the time course of Purkinje neuron excitatory postsynaptic currents (EPSCs). Depletion of ectopic release pools by activity‐dependent long‐term depression decreased EPSC decay time, revealing a ‘late’ current that is present when fibres are stimulated at low frequencies. This effect was enhanced when glutamate transporters were inhibited, and reduced when extracellular diffusion was impeded. Blockade of N‐type Ca2+ channels inhibited ectopic transmission to Bergmann glia and decreased EPSC decay time. Similarly, perfusion of the Ca2+ chelator EGTA‐AM into the slice progressively eliminated ectopic transmission to glia and decreased EPSC decay time with closely similar time courses. Collectively, this evidence suggests that ectopically released glutamate contributes to spillover transmission, and that ectopic release therefore degrades the spatial precision of synapses that fire infrequently, and may make them more prone to exhibit plasticity.

  February 13, 2014   doi: 10.1113/jphysiol.2013.267039   open full text
• In vivo quantification of lymph viscosity and pressure in lymphatic vessels and draining lymph nodes of arthritic joints in mice.
  Echoe M. Bouta, Ronald W. Wood, Edward B. Brown, Homaira Rahimi, Christopher T. Ritchlin, Edward M. Schwarz.
  The Journal of Physiology. February 12, 2014
  Previously, it was found that the popliteal lymph node (PLN) enlarges during the pre‐arthritic ‘expanding’ phase, and then ‘collapses’ with adjacent knee flare and is associated with the loss of the intrinsic lymphatic pulse. However, the mechanisms responsible are unknown and we therefore developed in vivo methods to measure lymph viscosity, lymphatic pumping pressure (LPP) in the lymphatic vessels afferent to the PLN, and lymph node pressure (LNP). Multiphoton fluorescence recovery after photobleaching (MP‐FRAP) was used to calculate lymph viscosity and speed; no difference was found among mice with wild‐type (WT), expanding or collapsed PLN in lymph viscosity, but lymph speed was found to be decreased in mice with collapsed PLN compared to WT and expanding PLN mice. LPP was measured indirectly by slowly releasing a pressurized cuff occluding ICG fluorescent dye; we found that mice with expanding PLN exhibit a higher LPP compared to WT and mice with collapsed PLN show an extremely low LPP. Direct measurement of LNP demonstrated a decrease in expanding PLN versus WT pressure, which dramatically increased in collapsed PLN. The decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of rheumatoid arthritis progression and possibly other chronic inflammatory conditions Abstract Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF‐Tg mice, a model of inflammatory–erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre‐arthritic ‘expanding’ phase, and then ‘collapses’ with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild‐type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP‐FRAP) of injected FITC‐BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P < 0.05). Direct measurement of intra‐lymph node pressure (LNP) demonstrated a decrease in expanding PLN versus WT pressure (3.41 ± 0.43 vs. 6.86 ± 0.56 cmH2O; P < 0.01), which dramatically increased to 9.92 ± 1.79 cmH2O in collapsed PLN. Lymphatic pumping pressure (LPP), measured indirectly by slowly releasing a pressurized cuff occluding indocyanine green (ICG), demonstrated an increase in vessels afferent to expanding PLN versus WT (18.76 ± 2.34 vs. 11.04 ± 1.47 cmH2O; P < 0.01), which dropped to 2.61 ± 0.72 cmH2O (P < 0.001) after PLN collapse. Herein, we document the first in vivo measurements of murine lymph viscosity and lymphatic pressure, and provide evidence to support the hypothesis that lymphangiogenesis and lymphatic transport are compensatory mechanisms to prevent synovitis via increased drainage of inflamed joints. Furthermore, the decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of RA progression and possibly other chronic inflammatory conditions.

  February 12, 2014   doi: 10.1113/jphysiol.2013.266700   open full text
• Resting pulmonary haemodynamics and shunting: a comparison of sea‐level inhabitants to high altitude Sherpas.
  Glen E. Foster, Philip N. Ainslie, Mike Stembridge, Trevor A. Day, Akke Bakker, Samuel J. E. Lucas, Nia C. S. Lewis, David B. MacLeod, Andrew T. Lovering.
  The Journal of Physiology. February 12, 2014
  Key points Evolutionary pressure to improve gas exchange and/or resting pulmonary haemodynamics in hypoxic environments may have led to differences in the amount of blood that flows through right‐to‐left shunt pathways between Sherpas and sea‐level inhabitants. We studied sea‐level inhabitants during rest at sea level and acute isocapnic hypoxia and during rest at high altitude following 3 weeks of acclimatization and compared their responses to those of Sherpas during rest at high altitude. Contrary to some previous literature, we found similar resting pulmonary pressure and total pulmonary resistance between acclimatized sea‐level inhabitants and Sherpas at high altitude. We also found a similar number of subjects from each group with intracardiac shunt and intrapulmonary shunt at high altitude. These results help us better understand resting cardiopulmonary adaptations to high altitude by comparing life‐long high altitude residents with sea‐level inhabitants acclimatized to high altitude. Abstract The incidence of blood flow through intracardiac shunt and intrapulmonary arteriovenous anastomoses (IPAVA) may differ between Sherpas permanently residing at high altitude (HA) and sea‐level (SL) inhabitants as a result of evolutionary pressure to improve gas exchange and/or resting pulmonary haemodynamics. To test this hypothesis we compared sea‐level inhabitants at SL (SL‐SL; n = 17), during acute isocapnic hypoxia (SL‐HX; n = 7) and following 3 weeks at 5050 m (SL‐HA; n = 8 non‐PFO subjects) to Sherpas at 5050 m (n = 14). S pO 2, heart rate, pulmonary artery systolic pressure (PASP) and cardiac index (Qi) were measured during 5 min of room air breathing at SL and HA, during 20 min of isocapnic hypoxia (SL‐HX; P ETO 2 = 47 mmHg) and during 5 min of hyperoxia (F IO 2 = 1.0; Sherpas only). Intracardiac shunt and IPAVA blood flow was evaluated by agitated saline contrast echocardiography. Although PASP was similar between groups at HA (Sherpas: 30.0 ± 6.0 mmHg; SL‐HA: 32.7 ± 4.2 mmHg; P = 0.27), it was greater than SL‐SL (19.4 ± 2.1 mmHg; P < 0.001). The proportion of subjects with intracardiac shunt was similar between groups (SL‐SL: 41%; Sherpas: 50%). In the remaining subjects, IPAVA blood flow was found in 100% of subjects during acute isocapnic hypoxia at SL, but in only 4 of 7 Sherpas and 1 of 8 SL‐HA subjects at rest. In conclusion, differences in resting pulmonary vascular regulation, intracardiac shunt and IPAVA blood flow do not appear to account for any adaptation to HA in Sherpas. Despite elevated pulmonary pressures and profound hypoxaemia, IPAVA blood flow in all subjects at HA was lower than expected compared to acute normobaric hypoxia.

  February 12, 2014   doi: 10.1113/jphysiol.2013.266593   open full text
• A novel computational model of mouse myocyte electrophysiology to assess the synergy between Na+ loading and CaMKII.
  S. Morotti, A. G. Edwards, A. D. McCulloch, D. M. Bers, E. Grandi.
  The Journal of Physiology. February 12, 2014
  Key points Intracellular [Na+] ([Na+]i) is elevated in heart failure (HF) and causes arrhythmogenic cellular [Ca2+]i loading. In HF, hyperactivity of Ca2+–calmodulin‐dependent protein kinase II (CaMKII), a key mediator of electrical and mechanical dysfunction in myocytes, causes elevated [Na+]i. We developed a computational model of mouse ventricular myocyte electrophysiology including Ca2+ and CaMKII signalling and quantitatively confirmed evidence suggesting that not only does CaMKII cause elevated [Na+]i, but this additional [Na+]i also promotes further CaMKII activation by increasing [Ca2+]i. We found that a 3–4 mm gain in [Na+]i (similar to that reported in HF) perturbs Ca2+ and membrane potential homeostasis in part via CaMKII activation. This disrupted Ca2+ homeostasis is exacerbated by CaMKII overexpression, and strongly relies upon CaMKII–Na+–Ca2+–CaMKII feedback. CaMKII inhibition in HF may be beneficial, in part by inhibiting [Na+]i loading, and thereby normalizing Ca2+ and membrane potential dynamics without disrupting systolic function. Abstract Ca2+–calmodulin‐dependent protein kinase II (CaMKII) hyperactivity in heart failure causes intracellular Na+ ([Na+]i) loading (at least in part by enhancing the late Na+ current). This [Na+]i gain promotes intracellular Ca2+ ([Ca2+]i) overload by altering the equilibrium of the Na+–Ca2+ exchanger to impair forward‐mode (Ca2+ extrusion), and favour reverse‐mode (Ca2+ influx) exchange. In turn, this Ca2+ overload would be expected to further activate CaMKII and thereby form a pathological positive feedback loop of ever‐increasing CaMKII activity, [Na+]i, and [Ca2+]i. We developed an ionic model of the mouse ventricular myocyte to interrogate this potentially arrhythmogenic positive feedback in both control conditions and when CaMKIIδC is overexpressed as in genetically engineered mice. In control conditions, simulation of increased [Na+]i causes the expected increases in [Ca2+]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca2+ handling and electrophysiology at high [Na+]i gain. Notably, clamping CaMKII activity to basal levels ameliorates but does not completely offset this outcome, suggesting that the increase in [Ca2+]i per se plays an important role. The effect of this CaMKII–Na+–Ca2+–CaMKII feedback is more striking in CaMKIIδC overexpression, where high [Na+]i causes delayed afterdepolarizations, which can be prevented by imposing low [Na+]i, or clamping CaMKII phosphorylation of L‐type Ca2+ channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na+ loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca2+ and membrane potential homeostasis. High [Na+]i is also required to produce instability when CaMKII is further activated by increased Ca2+ loading due to β‐adrenergic activation. Our results support recent experimental findings of a synergistic interaction between perturbed Na+ fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na+ loading can contribute to normalizing Ca2+ and membrane potential dynamics in heart failure.

  February 12, 2014   doi: 10.1113/jphysiol.2013.266676   open full text
• Exercise training decreases mitogen‐activated protein kinase phosphatase‐3 expression and suppresses hepatic gluconeogenesis in obese mice.
  Luciana Santos Souza Pauli, Eloize Cristina Chiarreotto Ropelle, Claudio Teodoro Souza, Dennys Esper Cintra, Adelino Sanchez Ramos Silva, Bárbara Almeida Rodrigues, Leandro Pereira Moura, Rodolfo Marinho, Vanessa Oliveira, Carlos Kiyoshi Katashima, José Rodrigo Pauli, Eduardo Rochete Ropelle.
  The Journal of Physiology. February 07, 2014
  Key points summary When the hepatic insulin signaling is compromised, there is an inadequate suppression of gluconeogenic pathways, leading the organism to high levels of glucose. Studies with animals with obesity induced by high fat diet or genetically modified showed increased MKP‐3 expression and MKP‐3/Foxo1 association in liver, with a consequent increase in blood glucose concentration, development of insulin resistance and DM2. As a non‐pharmacological strategy recognized and indicated for prevention and treatment of diabetes is the regular practice of physical exercise. In this study we demostrated that physical training is an important tool capable of reducing insulin resistance in the liver by reducing the inflammatory process, including the inhibition of MKP‐3 and, therefore, suppress gluconeogenic program in obesity rats. The understanding of these new mechanisms by which physical training regulates glucose homeostasis has critical importance to health professionals for the understanding and prevention of diabetes. Abstract Insulin plays an important role in the control of hepatic glucose production. Insulin resistant states are commonly associated with excessive hepatic glucose production, which contributes to both fasting hyperglycaemia and exaggerated postprandial hyperglycaemia. In this regard, increased activity of phosphatases may contribute to the dysregulation of gluconeogenesis. Mitogen‐activated protein kinase phosphatase‐3 (MKP‐3) is a key protein involved in the control of gluconeogenesis. MKP‐3‐mediated dephosphorylation activates FoxO1 (a member of the forkhead family of transcription factors) and subsequently promotes its nuclear translocation and binding to the promoters of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase). In this study, we investigated the effects of exercise training on the expression of MKP‐3 and its interaction with FoxO1 in the livers of obese animals. We found that exercised obese mice had a lower expression of MKP‐3 and FoxO1/MKP‐3 association in the liver. Further, the exercise training decreased FoxO1 phosphorylation and protein levels of Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) and gluconeogenic enzymes (PEPCK and G6Pase). These molecular results were accompanied by physiological changes, including increased insulin sensitivity and reduced hyperglycaemia, which were not caused by reductions in total body mass. Similar results were also observed with oligonucleotide antisense (ASO) treatment. However, our results showed that only exercise training could reduce an obesity‐induced increase in HNF‐4α protein levels while ASO treatment alone had no effect. These findings could explain, at least in part, why additive effects of exercise training treatment and ASO treatment were not observed. Finally, the suppressive effects of exercise training on MKP‐3 protein levels appear to be related, at least in part, to the reduced phosphorylation of Extracellular signal‐regulated kinases (ERK) in the livers of obese mice.

  February 07, 2014   doi: 10.1113/jphysiol.2013.264002   open full text
• Glycinergic feedback enhances synaptic gain in the distal retina.
  Zheng Jiang, Jinnan Yang, Lauren A. Purpura, Yufei Liu, Harris Ripps, Wen Shen.
  The Journal of Physiology. February 07, 2014
  Key points This study provides experimental evidence that glycinergic interplexiform cells create a centrifugal feedback loop in the vertebrate retina that regulates the transmission of glutamatergic signals between photoreceptors and second‐order neurons. This mechanism serves to reduce glutamate uptake and enhance glutamate release. Glycine receptors containing the GlyRα3 subunit are expressed on bipolar cell dendrites, and their activation leads to a depolarizing response in a group of rod‐dominated ON bipolar cells, and hyperpolarizing responses in OFF bipolar cells. Using strychnine to block endogenous glycine feedback reduces the amplitudes of light‐evoked responses in both ON and OFF bipolar cells, indicating that glycine feedback regulates signal propagation in the distal retina. Glycinergic feedback provides a neural mechanism that enhances synaptic gain and improves visual sensitivity. Abstract Glycine input originates with interplexiform cells, a group of neurons situated within the inner retina that transmit signals centrifugally to the distal retina. The effect on visual function of this novel mechanism is largely unknown. Using gramicidin‐perforated patch whole cell recordings, intracellular recordings and specific antibody labelling techniques, we examined the effects of the synaptic connections between glycinergic interplexiform cells, photoreceptors and bipolar cells. To confirm that interplexiform cells make centrifugal feedback on bipolar cell dendrites, we recorded the postsynaptic glycine currents from axon‐detached bipolar cells while stimulating presynaptic interplexiform cells. The results show that glycinergic interplexiform cells activate bipolar cell dendrites that express the α3 subunit of the glycine receptor, as well as a subclass of unidentified receptors on photoreceptors. By virtue of their synaptic contacts, glycine centrifugal feedback increases glutamate release from photoreceptors and suppresses the uptake of glutamate by the type 2A excitatory amino acid transporter on photoreceptors. The net effect is a significant increase in synaptic gain between photoreceptors and their second‐order neurons.

  February 07, 2014   doi: 10.1113/jphysiol.2013.265785   open full text
• Remodeling at the calyx of Held‐MNTB synapse in mice developing with unilateral conductive hearing loss.
  Giovanbattista Grande, Jaina Negandhi, Robert V. Harrison, Lu‐Yang Wang.
  The Journal of Physiology. February 06, 2014
  Structure and function of central synapses are profoundly influenced by experience during developmental sensitive periods. Sensory synapses, which are the indispensable interface for the developing brain to interact with its environment, are particularly plastic. In the auditory system, moderate forms of unilateral hearing loss during development are prevalent but the pre‐ and postsynaptic modifications that occur when hearing symmetry is perturbed are not well understood. We investigated this issue by performing experiments at the large calyx of Held synapse. Principal neurons of the medial nucleus of the trapezoid body (MNTB) are innervated by calyx of Held terminals that originate from the axons of globular bushy cells located in the contralateral ventral cochlear nucleus. We compared populations of synapses in the same animal that were either sound‐deprived (SD) or sound‐experienced (SE) after unilateral conductive hearing loss (CHL). Middle ear ossicles were removed one week prior to hearing onset (∼P12) and morphological and electrophysiological approaches were applied to auditory brainstem slices taken from these mice at P17–19. Calyces in the SD and SE MNTB acquired their mature digitated morphology but these were structurally more complex than those in normal hearing mice. This was accompanied by bilateral decreases in initial EPSC amplitude and synaptic conductance despite the CHL being unilateral. During high‐frequency stimulation, some SD synapses displayed short‐term depression whereas others displayed short‐term facilitation followed by slow depression similar to the heterogeneities observed in normal hearing mice. However SE synapses predominantly displayed short‐term facilitation followed by slow depression which could be explained in part by the decrease in release probability. Furthermore, the excitability of principal cells in the SD MNTB had increased significantly. Despite these unilateral changes in short‐term plasticity and excitability, heterogeneities in the spiking fidelity among the population of both SD and SE synapses showed similar continuums to those in normal hearing mice. Our study suggests preservations in the heterogeneity in spiking fidelity via synaptic remodeling ensures symmetric functional stability which is likely important for retaining the capability to maximally code sound localization cues despite moderate asymmetries in hearing experience. This article is protected by copyright. All rights reserved

  February 06, 2014   doi: 10.1113/jphysiol.2013.268839   open full text
• Blunted sympathoinhibitory responses in obesity‐related hypertension are due to aberrant central but not peripheral signalling mechanisms.
  Jackie. M.Y. How, Suhail. A. Wardak, Shaik I. Ameer, Rachel A. Davey, Daniela M. Sartor.
  The Journal of Physiology. February 06, 2014
  The gut hormone cholecystokinin (CCK) acts at subdiaphragmatic vagal afferents to induce renal and splanchnic sympathoinhibition and vasodilation, via reflex inhibition of a subclass of cardiovascular‐controlling neurons in the rostroventrolateral medulla (RVLM). These sympathoinhibitory and vasodilator responses are blunted in obese hypertensive rats and our aim in the present study was to determine whether this is attributable to (i) altered sensitivity of presympathetic vasomotor RVLM neurons, and (ii) aberrant peripheral or central signalling mechanisms. Using a diet induced obesity model, male Sprague Dawley rats exhibited either an obesity‐prone (OP) or obesity‐resistant (OR) phenotype when placed on a medium high‐fat diet for 13–15 weeks; control animals were placed on a low fat diet. OP animals had elevated resting arterial pressure compared to OR/control animals (P<0.05). Barosensitivity of RVLM neurons was significantly attenuated in OP animals (P < 0.05), suggesting altered baroreflex gain. CCK induced inhibitory responses in RVLM neurons of OR/control animals but not OP animals. Subdiaphragmatic vagal nerve responsiveness to CCK and CCK1 receptor mRNA expression in nodose ganglia did not differ between the groups, however CCK‐induced significantly less Fos‐like immunoreactivity in both the nucleus of the solitary tract and the caudal ventrolateral medulla of OP animals compared to controls (P < 0.05). These results suggest that blunted sympathoinhibitory and vasodilator responses in obesity‐related hypertension are due to alterations in RVLM neuronal responses, resulting from aberrant central but not peripheral signalling mechanisms. In obesity, blunted sympathoinhibitory mechanisms may lead to increased regional vascular resistance and contribute to the development of hypertension. This article is protected by copyright. All rights reserved

  February 06, 2014   doi: 10.1113/jphysiol.2013.269670   open full text
• Extracellular signal‐regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms.
  Julien Dine, Vincent R. R. Ducourneau, Valérie S. Fénelon, Pascal Fossat, Aurélie Amadio, Matthias Eder, Jean‐Marc Israel, Stéphane H. R. Oliet, Daniel L. Voisin.
  The Journal of Physiology. February 05, 2014
  Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal‐Regulated protein Kinases (ERK) are mitogen‐activated protein kinases that transduce extracellular stimuli into intracellular post‐translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 minutes after intraperitoneal injections of hypertonic saline (3 M, 6 M) in rats, many phosphoERK‐immunopositive neurones were observed in osmosensitive forebrain regions including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose‐dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 prior to a hyperosmotic challenge reduced the number of both phosphoERK‐immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically‐induced depolarisation and increase in firing of supraoptic MNCs recorded in vitro. It finally reduced hypertonically‐induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of vasopressin release. This article is protected by copyright. All rights reserved

  February 05, 2014   doi: 10.1113/jphysiol.2013.261008   open full text
• Activity‐dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones.
  Angela R. Wild, Susan Jones, Alasdair J. Gibb.
  The Journal of Physiology. January 31, 2014
  Key points Calcium ion influx through N‐methyl‐d‐aspartate receptors (NMDARs) may contribute to substantia nigra pars compacta (SNc) dopaminergic neurone dysfunction in Parkinson's disease. Responses of NMDARs in dopaminergic neurones showed use‐dependent run‐down that was not readily reversible and was partly dependent on Ca2+ influx and partly dependent on clathrin‐mediated endocytosis. Thus, we report regulation of NMDARs in SNc dopaminergic neurones by intracellular Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity‐dependent changes in receptor trafficking. Abstract N‐Methyl‐d‐aspartate receptors (NMDARs) are Ca2+‐permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca2+‐dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca2+‐independent mechanisms. We have investigated the activity‐dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole‐cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run‐down) that was use dependent and not readily reversible. Run‐down was reduced by increasing intracellular Ca2+ buffering or by reducing Ca2+ influx but did not appear to be mediated by the same regulatory proteins that cause Ca2+‐dependent run‐down in hippocampal neurones. The NMDAR current run‐down may be mediated in part by a Ca2+‐independent mechanism, because intracellular dialysis with a dynamin‐inhibitory peptide reduced run‐down, suggesting a role for clathrin‐mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run‐down during repeated low‐frequency synaptic stimulation in a Ca2+‐dependent but dynamin‐independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity‐dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca2+ homeostasis in dopaminergic neurones by limiting Ca2+ influx through the NMDAR.

  January 31, 2014   doi: 10.1113/jphysiol.2013.267310   open full text
• Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue.
  Bin Wei, Yingru Lu, J.‐P. Jin.
  The Journal of Physiology. January 27, 2014
  The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu180 causes a lethal form of recessively inherited nemaline myopathy (Amish Nemaline Myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused approximately 60% decrease in ssTnT protein expression whereas cre‐loxP‐mediated deletion of exons 11–13 resulted in total loss of ssTnT as that seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre‐specific myofilament proteins whereas fast fibre‐specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT deficient soleus muscle also contains a significant numbers of small size central nuclei type I fibres, indicating active regeneration. The data provide strong supports for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved. This article is protected by copyright. All rights reserved

  January 27, 2014   doi: 10.1113/jphysiol.2013.268177   open full text
• Purinergic signalling contributes to chemoreception in the retrotrapezoid nucleus but not the nucleus of the solitary tract or medullary raphe.
  Cleyton R. Sobrinho, Ian C. Wenker, Erin M. Poss, Ana C. Takakura, Thiago S. Moreira, Daniel K. Mulkey.
  The Journal of Physiology. January 22, 2014
  Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H+‐mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2‐evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H+‐ responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H+‐sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2‐responsiveness in vitro and in vivo. In brain slices, cell‐attached recordings of membrane potential show that CO2/H+‐sensitive NTS neurons are activated by focal ATP application; however, P2‐receptor blockade did not affect their CO2/H+‐responsiveness. CO2/H+‐sensitive raphe neurons were unaffected by ATP or P2‐receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2‐receptor into this region no effect on baseline breathing or CO2/H+‐responsiveness. Injections of ATP or a P2‐receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2‐receptor‐dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception. This article is protected by copyright. All rights reserved

  January 22, 2014   doi: 10.1113/jphysiol.2013.268490   open full text
• Vasoactive agonists exert dynamic and coordinated effects on vascular smooth muscle cell elasticity, cytoskeletal remodelling and adhesion.
  Zhongkui Hong, Zhe Sun, Min Li, Zhaohui Li, Filiz Bunyak, Ilker Ersoy, Jerome P. Trzeciakowski, Marius Catalin Staiculescu, Minshan Jin, Luis Martinez‐Lemus, Michael A. Hill, Kannappan Palaniappan, Gerald A. Meininger.
  The Journal of Physiology. January 22, 2014
  In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real‐time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen‐decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004, and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10−6 m) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10−4 m) reduced oscillation amplitude (‐30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the sub‐membranous actin cytoskeleton (100–300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased density of the stress fibres by 45%. AFM data were supported by Western Blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the ECM are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists likely invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMC. This article is protected by copyright. All rights reserved

  January 22, 2014   doi: 10.1113/jphysiol.2013.264929   open full text
• Peripherally driven low‐threshold inhibitory inputs to lamina I local‐circuit and projection neurones: a new circuit for gating pain responses.
  Liliana L. Luz, Peter Szucs, Boris V. Safronov.
  The Journal of Physiology. January 17, 2014
  Spinal lamina I is a key element of the pain processing system relaying primary afferent input to supraspinal areas. However, little is known about how the signal is modulated by its intrinsic network including local‐circuit neurones (LCNs) and much less numerous anterolateral‐tract projection neurones (PNs). Here, we used whole‐cell patch‐clamp recordings in an isolated spinal cord preparation to examine properties of identified LCNs (n = 85) and PNs (n = 73) in their functionally preserved local networks. Forty LCNs showed spontaneous rhythmic firing (2–7 Hz) at zero current injection, which persisted in the presence of blockers of fast synaptic transmission. In remaining cases, most LCNs and PNs fired tonically in response to depolarizing current injections. We identified LCNs and PNs receiving low‐threshold primary afferent driven inhibitory inputs, which in many cases were disynaptic and temporally preceded classical high‐threshold excitatory inputs. This direct inhibitory link between the low‐threshold afferents and PNs can function as a postsynaptic gate controlling the nociceptive information flow in the spinal cord. The LCNs were found to be integrated into the superficial dorsal horn network by receiving monosynaptic and disynaptic inputs from other lamina I and II neurones. One‐third of LCNs and two‐thirds of PNs tested responded to substance P application. Thus, substance P released by a noxious afferent stimulation could excite PNs in two ways, directly and via activation of their presynaptic LCN circuitries. In conclusion, we have described important properties of identified lamina I neurones and their role in a new circuit for gating pain responses. This article is protected by copyright. All rights reserved

  January 17, 2014   doi: 10.1113/jphysiol.2013.269472   open full text
• Synaptic noise is an information bottleneck in the inner retina during dynamic visual stimulation.
  Michael A. Freed, Zhiyin Liang.
  The Journal of Physiology. January 09, 2014
  Key points At chemical synapses, vesicles fuse with the presynaptic membrane at random, generating noise in postsynaptic currents. To determine how much noise synapses generate, we recorded excitatory postsynaptic currents from ganglion cells in an in vitro preparation of the mammalian retina during flickering visual stimulation. Postsynaptic currents received noise from three sources: substantial noise from bipolar cell synapses, somewhat more from the presynaptic retinal circuitry, but little from sources intrinsic to the ganglion cell. Presynaptic circuit elements but not bipolar cell synapses were significant sources of noise shared by pairs of ganglion cells. Signal‐to‐noise ratio was substantially reduced from the presynaptic bipolar cell array to the postsynaptic ganglion cell, indicating that synaptic noise can reduce the amount of information transmitted to a neuron. Abstract In daylight, noise generated by cones determines the fidelity with which visual signals are initially encoded. Subsequent stages of visual processing require synapses from bipolar cells to ganglion cells, but whether these synapses generate a significant amount of noise was unknown. To characterize noise generated by these synapses, we recorded excitatory postsynaptic currents from mammalian retinal ganglion cells and subjected them to a computational noise analysis. The release of transmitter quanta at bipolar cell synapses contributed substantially to the noise variance found in the ganglion cell, causing a significant loss of fidelity from bipolar cell array to postsynaptic ganglion cell. Virtually all the remaining noise variance originated in the presynaptic circuit. Circuit noise had a frequency content similar to noise shared by ganglion cells but a very different frequency content from noise from bipolar cell synapses, indicating that these synapses constitute a source of independent noise not shared by ganglion cells. These findings contribute a picture of daylight retinal circuits where noise from cones and noise generated by synaptic transmission of cone signals significantly limit visual fidelity.

  January 09, 2014   doi: 10.1113/jphysiol.2013.265744   open full text
• Properties of cholinergic and non‐cholinergic submucosal neurons along the mouse colon.
  Jaime Pei Pei Foong, Iain R. Tough, Helen M. Cox, Joel C. Bornstein.
  The Journal of Physiology. January 08, 2014
  Key points Submucosal neurons are crucial regulators of gut secretion. Despite significant interest in using mouse models for enteric neuropathies, much is still unknown about their submucous innervation. We examined properties of submucosal neurons in the mouse distal colon using immunohistochemical and intracellular recording techniques, and investigated colonic regional differences in neurochemistry and neurally mediated ion transport responses. Two main neurochemical but not electrophysiological classes of neurons were identified: cholinergic (containing choline acetyltransferase) and non‐cholinergic. Non‐cholinergic neurons had one or two axons; the cholinergic neurons examined were uniaxonal. Neurons exhibited predominantly nicotinic fast excitatory postsynaptic potentials and somatic action potentials mediated by tetrodotoxin‐resistant voltage‐gated channels. The distal colon had smaller ganglia, a higher proportion of cholinergic neurons (they remain a minority) and a larger nicotinic secretory component than the proximal colon. Properties of submucosal neurons in the mouse distal colon differ from other colonic regions, and from submucosal neurons in other species. Abstract Submucosal neurons are vital regulators of water and electrolyte secretion and local blood flow in the gut. Due to the availability of transgenic models for enteric neuropathies, the mouse has emerged as the research model of choice, but much is still unknown about the murine submucosal plexus. The progeny of choline acetyltransferase (ChAT)‐Cre × ROSA26YFP reporter mice, ChAT‐Cre;R26R‐yellow fluorescent protein (YFP) mice, express YFP in every neuron that has ever expressed ChAT. With the aid of the robust YFP staining in these mice, we correlated the neurochemistry, morphology and electrophysiology of submucosal neurons in distal colon. We also examined whether there are differences in neurochemistry along the colon and in neurally mediated vectorial ion transport between the proximal and distal colon. All YFP+ submucosal neurons also contained ChAT. Two main neurochemical but not electrophysiological groups of neurons were identified: cholinergic (containing ChAT) or non‐cholinergic. The vast majority of neurons in the middle and distal colon were non‐cholinergic but contained vasoactive intestinal peptide. In the distal colon, non‐cholinergic neurons had one or two axons, whereas the cholinergic neurons examined had only one axon. All submucosal neurons exhibited S‐type electrophysiology, shown by the lack of long after‐hyperpolarizing potentials following their action potentials and fast excitatory postsynaptic potentials (EPSPs). Fast EPSPs were predominantly nicotinic, and somatic action potentials were mediated by tetrodotoxin‐resistant voltage‐gated channels. The size of submucosal ganglia decreased but the proportion of cholinergic neurons increased distally along the colon. The distal colon had a significantly larger nicotinic ion transport response than the proximal colon. This work shows that the properties of murine submucosal neurons and their control of epithelial ion transport differ between colonic regions. There are several key differences between the murine submucous plexus and that of other animals, including a lack of conventional intrinsic sensory neurons, which suggests there is an incomplete neuronal circuitry within the murine submucous plexus.

  January 08, 2014   doi: 10.1113/jphysiol.2013.265686   open full text
• Morphological, biophysical and synaptic properties of glutamatergic neurons of the mouse spinal dorsal horn.
  Pradeep Punnakkal, Carolin Schoultz, Karen Haenraets, Hendrik Wildner, Hanns Ulrich Zeilhofer.
  The Journal of Physiology. January 08, 2014
  Key points Excitatory and inhibitory interneurons of the spinal dorsal horn are critically involved in normal sensory processing and in the generation of pathological pain, but their physiological properties, especially those of excitatory interneurons, are only incompletely characterised. Here, we identified a vGluT2::eGFP BAC transgenic mouse line in which enhanced green fluorescent protein (eGFP) is specifically expressed in a subset of neurons that are likely to be representative of the whole population of excitatory dorsal horn neurons. We compared the physiological properties of vGluT2::eGFP neurons with those of inhibitory neurons in Gad67::eGFP and GlyT2::eGFP transgenic mice: vGluT2::eGFP neurons required stronger depolarising currents than inhibitory neurons to fire action potentials and fired fewer action potentials during prolonged depolarisations. Both excitatory or inhibitory dorsal horn neurons received synaptic input from capsaicin‐sensitive fibres and primary afferent fibre‐evoked (polysynaptic) inhibitory input. These findings should contribute to a better mechanistic understanding of normal and pathological sensory processing in the spinal dorsal horn. Abstract Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one‐third of all excitatory dorsal horn neurons and, as demonstrated by the co‐expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell‐type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after‐hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin‐induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent‐evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory dorsal horn neurons thus differ from their inhibitory counterparts in several biophysical properties and possibly also in their integration into the local neuronal circuitry.

  January 08, 2014   doi: 10.1113/jphysiol.2013.264937   open full text
• Consequences of Peripheral Chemoreflex Inhibition with Low‐Dose Dopamine in Humans.
  Piotr Niewinski, Stanislaw Tubek, Waldemar Banasiak, Julian F.R. Paton, Piotr Ponikowski.
  The Journal of Physiology. January 07, 2014
  Low‐dose dopamine inhibits peripheral chemoreceptors and attenuates the hypoxic ventilatory response (HVR) in humans. However, it is unknown whether it also modulates: (1) the hemodynamic reactions to acute hypoxia, (2) cardiac baroreflex sensitivity (BRS) and (3) if there is any effect of dopamine withdrawal. We performed a double‐blinded, placebo controlled study on 11 healthy male volunteers. At sea level over two days every subject was administered low‐dose dopamine (2 mcg/kg/min) or saline infusion, during which we assessed both ventilatory and hemodynamic responses to acute hypoxia. Separately, we evaluated effects of initiation and withdrawal of each infusion and BRS. The initiation of dopamine infusion did not affect minute ventilation (MV) and mean blood pressure (MAP), but increased both heart rate (HR) and cardiac output. Concomitantly it decreased systemic vascular resistance. Dopamine blunted the ventilatory, MAP and HR reactions (hypertension, tachycardia) to acute hypoxia. Dopamine attenuated cardiac BRS to falling blood pressure. Dopamine withdrawal evoked an increase in MV. The magnitude of the increment in MV due to dopamine withdrawal correlated with the size of the HVR and depended on the duration of dopamine administration. The ventilatory reaction to dopamine withdrawal constitutes a novel index of peripheral chemoreceptor function. This article is protected by copyright. All rights reserved

  January 07, 2014   doi: 10.1113/jphysiol.2012.266858   open full text
• Bi‐directional modulation of somatosensory mismatch negativity with transcranial direct current stimulation: an event related potential study.
  Jui‐Cheng Chen, Dorothea Hämmerer, Kevin D'Ostilio, Elias P. Casula, Louise Marshall, Chon‐Haw Tsai, John C. Rothwell, Mark J. Edwards.
  The Journal of Physiology. January 03, 2014
  Key points Sensory mismatch negativity is impaired in patients with cerebellar lesions, suggesting that the cerebellum may play an important role in this form of sensory processing. Anodal transcranial direct current stimulation over the right cerebellar hemisphere increased the amplitude of sensory mismatch negativity to stimuli delivered to the right hand while cathodal transcranial direct current stimulation reduced it. The cerebellum appears to be an important node in the network mediating sensory mismatch negativity, and tDCS is a useful method with which to manipulate sensory mismatch negativity for experimental studies. Abstract Appropriate orientation towards potentially salient novel environmental stimuli requires a system capable of detecting change in the sensorium. Mismatch negativity (MMN), an evoked potential calculated by subtracting the response to a standard repeated stimulus and a rare ‘oddball’ stimulus, is proposed as such a change detection mechanism. It is most widely studied in the auditory domain, but here we chose to explore the mechanism of somatosensory MMN, and specifically its dependence on the cerebellum. We recorded event‐related potentials (ERPs) evoked in response to auditory and sensory stimuli from 10 healthy subjects before and after anodal, cathodal and sham transcranial direct current stimulation (tDCS) of the right cerebellar hemisphere. There was a significant increase in peak amplitude of somatosensory MMN after anodal tDCS (F(1,9) = 8.98, P < 0.02, mean difference anodal pre–post: −1.02 μV) and a significant reduction in peak amplitude of somatosensory MMN after cathodal tDCS (F(1,9) = 7.15, P < 0.03, mean difference cathodal pre–post: 0.65 μV). The amplitude of auditory MMN was unchanged by tDCS. These results reveal the capability of tDCS to cause bidirectional modulation of somatosensory MMN and the dependence of somatosensory MMN on the cerebellum.

  January 03, 2014   doi: 10.1113/jphysiol.2013.260331   open full text
• Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.
  Helga Vitzthum, Anika Seniuk, Laura Helene Schulte, Maxie Luise Müller, Hannah Hetz, Heimo Ehmke.
  The Journal of Physiology. January 03, 2014
  A network of kinases including WNKs, SPAK, and Sgk1 is critical for the independent regulation of K+ and Na+ transport in the distal nephron. Angiotensin II is thought to act as key hormone to orchestrate these kinases to switch from K+ secretion during hyperkalaemia to Na+ reabsorption during intravascular volume depletion, thus keeping disturbances in electrolyte and blood pressure homeostasis at a minimum. It remains unclear, however, how K+ and Na+ transport are regulated during a high Na+ intake, which is associated with suppressed angiotensin II levels and a high distal tubular Na+ load. We therefore investigated the integrated blood pressure, renal, hormonal, and gene and protein expression responses to large changes of K+ intake in Na+ replete mice. Both low and high K+ intake increased blood pressure and caused Na+ retention. Low K+ intake was accompanied by an upregulation of the sodium‐chloride‐cotransporter (NCC) and its activating kinase SPAK, and inhibition of NCC normalized blood pressure. The renal responses were unaffected by angiotensin AT1 receptor antagonism, indicating that low K+ intake activates the distal nephron by an angiotensin‐independent mode of action. High K+ intake was associated with elevated plasma aldosterone concentrations and an upregulation of the epithelial sodium channel (ENaC) and its activating kinase Sgk1. Surprisingly, high K+ intake increased blood pressure even during ENaC or mineralocorticoid receptor antagonism, suggesting the contribution of aldosterone‐independent mechanisms. These findings show that in a Na+ replete state changes in K+ intake induce specific molecular and functional changes in the distal nephron that cause a functional coupling of renal K+ and Na+ handling, resulting in Na+ retention and high blood pressure when K+ intake is either restricted or excessively increased. This article is protected by copyright. All rights reserved

  January 03, 2014   doi: 10.1113/jphysiol.2014.266924   open full text
• Neuronal detection thresholds during vestibular compensation: contributions of response variability and sensory substitution.
  Mohsen Jamali, Diana E. Mitchell, Alexis Dale, Jerome Carriot, Soroush G. Sadeghi, Kathleen E. Cullen.
  The Journal of Physiology. January 03, 2014
  The vestibular system is responsible for processing self‐motion, allowing normal subjects to discriminate the direction of rotational movements as slow as 1–2 deg s−1. After unilateral vestibular injury patients’ direction‐discrimination thresholds worsen to ∼20deg s−1, and despite some improvement thresholds remain substantially elevated following compensation. To date, however, the underlying neural mechanisms of this recovery have not been addressed. Here, we recorded from first‐order central neurons in the macaque monkey that provide vestibular information to higher brain areas for self‐motion perception. Immediately following unilateral labyrinthectomy, neuronal detection thresholds increased by more than two‐fold (from 14 to 30deg s−1). While thresholds showed slight improvement by week 3 (25deg s−1), they never recovered to control values – a trend mirroring the time course of perceptual thresholds in patients. We further discovered that changes in neuronal response variability paralleled changes in sensitivity for vestibular stimulation during compensation, thereby causing detection thresholds to remain elevated over time. However, we found that in a subset of neurons, the emergence of neck proprioceptive responses combined with residual vestibular modulation during head‐on‐body motion led to better neuronal detection thresholds. Taken together, our results emphasize that increases in response variability to vestibular inputs ultimately constrain neural thresholds and provide evidence that sensory substitution with extravestibular (i.e. proprioceptive) inputs at the first central stage of vestibular processing provides a neural substrate for improvements in self‐motion perception following vestibular loss. Thus, our results provide a neural correlate for the patient benefits provided by rehabilitative strategies that take advantage of the convergence of these multisensory cues. This article is protected by copyright. All rights reserved

  January 03, 2014   doi: 10.1113/jphysiol.2014.267534   open full text
• Short‐term hypoxic vasodilation in vivo is mediated by bioactive nitric oxide metabolites, rather than free nitric oxide derived from haemoglobin‐mediated nitrite reduction.
  Michele Umbrello, Alex Dyson, Bernardo Bollen Pinto, Bernadette O. Fernandez, Verena Simon, Martin Feelisch, Mervyn Singer.
  The Journal of Physiology. January 03, 2014
  Local increases in blood flow – ‘hypoxic vasodilation’ – confer cellular protection in the face of reduced oxygen delivery. The physiological relevance of this response is well established, yet ongoing controversy surrounds its underlying mechanisms. We sought to confirm that early hypoxic vasodilation is a nitric oxide (NO)‐mediated phenomenon and to study putative pathways for increased levels of NO, namely production from NO synthases, intravascular nitrite reduction, release from pre‐formed stores, and reduced deactivation by cytochrome c oxidase. Experiments were performed on spontaneously breathing, anaesthetized, male Wistar rats undergoing short‐term systemic hypoxaemia, who received pharmacological inhibitors and activators of the various NO pathways. Arterial blood pressure, cardiac output, tissue oxygen tension and the circulating pool of NO metabolites (oxidation, nitrosation and nitrosylation products) were measured in plasma and erythrocytes. Hypoxaemia caused a rapid and sustained vasodilation, which was only partially reversed by non‐selective NOS inhibition. This was associated with significantly lower plasma nitrite, and marginally elevated nitrate levels, suggestive of nitrite bioinactivation. Administration of sodium nitrite had little effect in normoxia, but produced significant vasodilation and increased nitrosylation during hypoxaemia that could not be reversed by NO scavenging. Methodological issues prevented assessment of the contribution, if any, of reduced deactivation of NO by cytochrome c oxidase. In conclusion, acute hypoxic vasodilation is an adaptive NO‐mediated response that is conferred through bioactive metabolites rather than free NO from haemoglobin‐mediated reduction of nitrite. This article is protected by copyright. All rights reserved

  January 03, 2014   doi: 10.1113/jphysiol.2014.255687   open full text
• A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short‐term plasticity and vesicle number.
  Brandon J. Walters, Jada J. Hallengren, Christopher S. Theile, Hidde L. Ploegh, Scott M. Wilson, Lynn E. Dobrunz.
  The Journal of Physiology. January 02, 2014
  Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short‐term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short‐term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short‐term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome‐bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short‐term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination‐independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over‐expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination‐independent function for USP14 in influencing synaptic architecture and plasticity.

  January 02, 2014   doi: 10.1113/jphysiol.2013.266015   open full text
• Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility.
  Veit‐Simon Eckle, Aleksandr Shcheglovitov, Iuliia Vitko, Deblina Dey, Chan Choo Yap, Bettina Winckler, Edward Perez‐Reyes.
  The Journal of Physiology. January 02, 2014
  Key points Mutations in the Cav3.2 T‐type Ca2+ channel were found in patients with idiopathic generalized epilepsies, yet the mechanisms by which these mutations increase neuronal excitability and susceptibility to seizures remain to be determined. Using electrophysiological and transfection methods, we validate in cultured hippocampal neurons the hypothesis that an epilepsy mutation increases neuronal excitability. Mutations in the I–II loop of the channel increase trafficking to the plasma membrane without altering trafficking into dendrites. Mutations also enhance dendritic arborization. Additionally, we provide the first evidence that Cav3.2 can signal to Ca2+‐regulated transcription factors, which are known to play important roles in neuronal development and gene expression. Abstract T‐type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T‐type Ca2+ channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I–II loop (the region in which most childhood absence epilepsy‐associated mutations are found) substantially increase the surface expression of T‐channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T‐channel blocker, TTA‐P2. As Ca2+‐regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain‐of‐function mutations in Cav3.2 T‐type Ca2+ channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.

  January 02, 2014   doi: 10.1113/jphysiol.2013.264176   open full text
• Cholesterol and F‐actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane.
  Jeffrey S. Dason, Alex J. Smith, Leo Marin, Milton P. Charlton.
  The Journal of Physiology. January 02, 2014
  Key points Extraction of cholesterol from synaptic vesicles trapped on the presynaptic plasma membrane causes synaptic vesicle proteins to disperse after exocytosis. Vesicular cholesterol regulates both presynaptic phosphatidylinositol (4,5)‐bisphosphate levels and actin distribution during synaptic vesicle recycling. Inhibition of actin polymerization results in the dispersal of proteins from trapped synaptic vesicles and impairs synaptic vesicle recycling. Vesicular cholesterol and actin together confine synaptic vesicle proteins on the presynaptic plasma membrane during synaptic vesicle recycling. Alteration of membrane or synaptic vesicle lipids might therefore affect the ability of synapses to undergo sustained exocytosis and endocytosis by compromising the recycling of synaptic vesicle proteins. Abstract Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature‐sensitive dynamin mutant shibire‐ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl‐β‐cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)‐bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

  January 02, 2014   doi: 10.1113/jphysiol.2013.265447   open full text
• Transition between fast and slow gamma modes in rat hippocampus area CA1 in vitro is modulated by slow CA3 gamma oscillations.
  Alexander N. J. Pietersen, Peter D. Ward, Nicholas Hagger‐Vaughan, James Wiggins, John G. R. Jefferys, Martin Vreugdenhil.
  The Journal of Physiology. January 02, 2014
  Key points The synchronisation of neuronal activity at gamma frequencies (30–100 Hz) could determine the effectiveness of neuronal communication. Gamma oscillations in the CA1 region of the hippocampus in vitro was thought to be dependent on gamma oscillations generated in area CA3, but in vivo CA1 can generate gamma oscillations independently. In this study we found that activating acetylcholine receptors induced stable gamma oscillations in the CA1 local network isolated in slices in vitro that were faster than those in CA3, but relied on similar neuronal circuitry involving feedback inhibition. Gamma frequency inputs from CA3 (spontaneous in intact hippocampal slices or stimulated in isolated CA1) can suppress the local fast gamma oscillation in CA1 and force it to adopt the slower CA3 oscillation through feed‐forward inhibition. This modulation could allow CA1 to alternate between effective communication with the entorhinal cortex and CA3, which may regulate memory encoding and memory recall phases. Abstract Hippocampal gamma oscillations have been associated with cognitive functions including navigation and memory encoding/retrieval. Gamma oscillations in area CA1 are thought to depend on the oscillatory drive from CA3 (slow gamma) or the entorhinal cortex (fast gamma). Here we show that the local CA1 network can generate its own fast gamma that can be suppressed by slow gamma‐paced inputs from CA3. Moderate acetylcholine receptor activation induces fast (45 ± 1 Hz) gamma in rat CA1 minislices and slow (33 ± 1 Hz) gamma in CA3 minislices in vitro. Using pharmacological tools, current‐source density analysis and intracellular recordings from pyramidal cells and fast‐spiking stratum pyramidale interneurons, we demonstrate that fast gamma in CA1 is of the pyramidal–interneuron network gamma (PING) type, with the firing of principal cells paced by recurrent perisomal IPSCs. The oscillation frequency was only weakly dependent on IPSC amplitude, and decreased to that of CA3 slow gamma by reducing IPSC decay rate or reducing interneuron activation through tonic inhibition of interneurons. Fast gamma in CA1 was replaced by slow CA3‐driven gamma in unlesioned slices, which could be mimicked in CA1 minislices by sub‐threshold 35 Hz Schaffer collateral stimulation that activated fast‐spiking interneurons but hyperpolarised pyramidal cells, suggesting that slow gamma frequency CA3 outputs can suppress the CA1 fast gamma‐generating network by feed‐forward inhibition and replaces it with a slower gamma oscillation driven by feed‐forward inhibition. The transition between the two gamma oscillation modes in CA1 might allow it to alternate between effective communication with the medial entorhinal cortex and CA3, which have different roles in encoding and recall of memory.

  January 02, 2014   doi: 10.1113/jphysiol.2013.263889   open full text
• Daily variation in the electrophysiological activity of mouse medial habenula neurones.
  Kanwal Sakhi, Mino D. C. Belle, Nicole Gossan, Philippe Delagrange, Hugh D. Piggins.
  The Journal of Physiology. December 16, 2013
  Key points Neurones of the suprachiasmatic nucleus (SCN) contain a molecular clock that drives these cells to exhibit daily rhythms in electrical activity. The molecular clock may also be present in another brain structure, the medial habenula, and here we tested whether medial habenula neurones show daily changes in their electrical activity. Using a brain slice preparation in which the medial habenula is isolated from inputs from the SCN, we made recordings from mouse medial habenula neurones and determined that they exhibit daily variation in their electrical properties. By contrast, in mice lacking functional molecular clocks, medial habenula neurones did not show overt daily change in their electrical activity. These studies indicate for the first time that medial habenula neurones exhibit daily changes in electrical activity that require a functional molecular clock, but do not depend on signals from the SCN. Abstract Intrinsic daily or circadian rhythms arise through the outputs of the master circadian clock in the brain's suprachiasmatic nuclei (SCN) as well as circadian oscillators in other brain sites and peripheral tissues. SCN neurones contain an intracellular molecular clock that drives these neurones to exhibit pronounced day–night differences in their electrical properties. The epithalamic medial habenula (MHb) expresses clock genes, but little is known about the bioelectric properties of mouse MHb neurones and their potential circadian characteristics. Therefore, in this study we used a brain slice preparation containing the MHb to determine the basic electrical properties of mouse MHb neurones with whole‐cell patch clamp electrophysiology, and investigated whether these vary across the day–night cycle. MHb neurones (n = 230) showed heterogeneity in electrophysiological state, ranging from highly depolarised cells (∼ −25 to −30 mV) that are silent with no membrane activity or display depolarised low‐amplitude membrane oscillations, to neurones that were moderately hyperpolarised (∼40 mV) and spontaneously discharging action potentials. These electrical states were largely intrinsically regulated and were influenced by the activation of small‐conductance calcium‐activated potassium channels. When considered as one population, MHb neurones showed significant circadian variation in their spontaneous firing rate and resting membrane potential. However, in recordings of MHb neurones from mice lacking the core molecular circadian clock, these temporal differences in MHb activity were absent, indicating that circadian clock signals actively regulate the timing of MHb neuronal states. These observations add to the extracellularly recorded rhythms seen in other brain areas and establish that circadian mechanisms can influence the membrane properties of neurones in extra‐SCN sites. Collectively, the results of this study indicate that the MHb may function as an intrinsic secondary circadian oscillator in the brain, which can shape daily information flow in key brain processes, such as reward and addiction.

  December 16, 2013   doi: 10.1113/jphysiol.2013.263319   open full text
• Transcranial direct current stimulation reverses neurophysiological and behavioural effects of focal inhibition of human pharyngeal motor cortex on swallowing.
  Dipesh H. Vasant, Satish Mistry, Emilia Michou, Samantha Jefferson, John C. Rothwell, Shaheen Hamdy.
  The Journal of Physiology. December 13, 2013
  Key points Cortical control of swallowing exhibits functional asymmetry with brain lesions involving the strongest projection being implicated in the pathophysiology of dysphagia after unilateral stroke. Swallowing recovery is associated with neuroplastic adaptation in the unlesioned hemisphere, a process which can be facilitated by excitatory neurostimulation techniques including transcranial direct current stimulation (tDCS). Unilateral suppression of the strongest pharyngeal motor projection using 1 Hz repetitive transcranial magnetic stimulation (rTMS) can disrupt swallowing neurophysiology and behaviour making it a useful model for trialling novel neurostimulation interventions in healthy subjects. In this healthy participant study we examined the effects of tDCS after unilateral pre‐conditioning with 1 Hz rTMS to determine its ability to restore swallowing neurophysiology and behaviour. We show that application of optimised parameters of tDCS (anodal stimulation, 1.5 mA, 10 min) over the unconditioned hemisphere reverses the brain and behavioural consequences of inhibitory pre‐conditioning, supporting the use of tDCS in clinical trials. Abstract The human cortical swallowing system exhibits bilateral but functionally asymmetric representation in health and disease as evidenced by both focal cortical inhibition (pre‐conditioning with 1 Hz repetitive transcranial magnetic stimulation; rTMS) and unilateral stroke, where disruption of the stronger (dominant) pharyngeal projection alters swallowing neurophysiology and behaviour. Moreover, excitatory neurostimulation protocols capable of reversing the disruptive effects of focal cortical inhibition have demonstrated therapeutic promise in post‐stroke dysphagia when applied contralaterally. In healthy participants (n = 15, 8 males, mean age (±SEM) 35 ± 9 years), optimal parameters of transcranial direct current stimulation (tDCS) (anodal, 1.5 mA, 10 min) were applied contralaterally after 1 Hz rTMS pre‐conditioning to the strongest pharyngeal projection. Swallowing neurophysiology was assessed in both hemispheres by intraluminal recordings of pharyngeal motor‐evoked responses (PMEPs) to single‐pulse TMS as a measure of cortical excitability. Swallowing behaviour was examined using a pressure‐based reaction time protocol. Measurements were made before and for up to 60 min post intervention. Subjects were randomised to active or sham tDCS after 1 Hz rTMS on separate days and data were compared using repeated measures ANOVA. Active tDCS increased PMEPs bilaterally (F1,14 = 7.4, P = 0.017) reversing the inhibitory effects of 1 Hz rTMS in the pre‐conditioned hemisphere (F1,14 = 10.1, P = 0.007). Active tDCS also enhanced swallowing behaviour, increasing the number of correctly timed challenge swallows compared to sham (F1,14 = 6.3, P = 0.025). Thus, tDCS to the contralateral pharyngeal motor cortex reverses the neurophysiological and behavioural effects of focal cortical inhibition on swallowing in healthy individuals and has therapeutic potential for dysphagia rehabilitation.

  December 13, 2013   doi: 10.1113/jphysiol.2013.263475   open full text
• PET imaging detects greater blood flow and less blood flow heterogeneity in the exercising skeletal muscles of old compared with young men during fatiguing contractions.
  Thorsten Rudroff, Jessica A. Weissman, Marco Bucci, Marko Seppänen, Kimmo Kaskinoro, Ilkka Heinonen, Kari K. Kalliokoski.
  The Journal of Physiology. November 19, 2013
  Abstract  The purpose of this study was to investigate blood flow and its heterogeneity within and among knee muscles of five young (26 ± 6 years) and five old (77 ± 6 years) healthy men with similar physical activity levels when they performed two types of submaximal fatiguing isometric contractions that required either force or position control. Positron emission tomography (PET) and [15O]‐H2O were used to determine blood flow two minutes (beginning) and 12 min (end) after the start of the tasks. Young and old men had similar maximal forces and endurance times for the fatiguing tasks. Although muscle volumes were lower in the old subjects, total muscle blood flow was similar between the groups (young: 25.8 ± 12.6; old: 25.1 ± 15.4 ml min−1; age main effect, P = 0.77) since blood flow per unit mass of muscle in the exercising knee extensors was greater for the old men (12.5 ± 6.2 ml min−1 100 g−1) than for the young men (8.6 ± 3.6 ml min−1 100 g−1; age main effect, P = 0.001). Further, blood flow heterogeneity in the exercising knee extensors was significantly lower in the old (56 ± 27%) compared to the young (67 ± 34%) men. Taken together, despite smaller skeletal muscles, the intact neural drive to the muscle and the greater, less heterogeneous blood flow per gram of muscle indicates that old fit muscle achieves adequate exercise hyperaemia.

  November 19, 2013   doi: 10.1113/jphysiol.2013.264614   open full text
• Phase‐shifting response to light in older adults.
  Seong Jae Kim, Susan Benloucif, Kathryn Jean Reid, Sandra Weintraub, Nancy Kennedy, Lisa F. Wolfe, Phyllis C. Zee.
  The Journal of Physiology. November 18, 2013
  •  Ageing is characterized by changes in circadian rhythms. •  Reduced light exposure or reduced responsiveness to light in older adults may contribute to age‐related circadian changes. •  We hypothesized that the aged circadian clock would exhibit a decreased response to light at a lower intensity (2000 lux) but not to light at a higher intensity (8000 lux). Here, we assessed phase‐shifting responses to 2 h of broad‐spectrum white light at two different intensities in 29 healthy younger and 16 healthy older subjects. •  Older subjects had a significantly earlier phase and lower amplitude of melatonin rhythm compared with younger subjects. •  There was no evidence of age‐related changes in the magnitude or direction of phase shifts of melatonin mid‐point in response to 2 h of broad‐spectrum white light at either 2000 lux or 8000 lux; this indicates that the acute phase‐shifting response to light is not significantly affected by age. Abstract  Age‐related changes in circadian rhythms may contribute to the sleep disruption observed in older adults. A reduction in responsiveness to photic stimuli in the circadian timing system has been hypothesized as a possible reason for the advanced circadian phase in older adults. This project compared phase‐shifting responses to 2 h of broad‐spectrum white light at moderate and high intensities in younger and older adults. Subjects included 29 healthy young (25.1 ± 4.1 years; male to female ratio: 8 : 21) and 16 healthy older (66.5 ± 6.0 years; male to female ratio: 5 : 11) subjects, who participated in two 4‐night and 3‐day laboratory stays, separated by at least 3 weeks. Subjects were randomly assigned to one of three different time‐points, 8 h before (−8), 3 h before (−3) or 3 h after (+3) the core body temperature minimum (CBTmin) measured on the baseline night. For each condition, subjects were exposed in a randomized order to 2 h light pulses of two intensities (2000 lux and 8000 lux) during the two different laboratory stays. Phase shifts were analysed according to the time of melatonin mid‐point on the nights before and after light exposure. Older subjects in this study showed an earlier baseline phase and lower amplitude of melatonin rhythm compared to younger subjects, but there was no evidence of age‐related changes in the magnitude or direction of phase shifts of melatonin mid‐point in response to 2 h of light at either 2000 lux or 8000 lux. These results indicate that the acute phase‐shifting response to moderate‐ or high‐intensity broad spectrum light is not significantly affected by age.

  November 18, 2013   doi: 10.1113/jphysiol.2013.262899   open full text
• Muscle nuclei remember to cheat death.
  Lawrence M. Schwartz.
  The Journal of Physiology. November 18, 2013
  Abstract  Getting buff is hard work and invariably involves lots of repetitive and exhausting resistance exercise. Unfortunately, stop the workouts and those hard fought gains can be lost easily. The only heartening part is that getting back into shape is far easier the second time around, a phenomenon known as “muscle memory” (Staron et al. 1991).

  November 18, 2013   doi: 10.1113/jphysiol.2013.268243   open full text
• Branching patterns emerge in a mathematical model of the dynamics of lung development (Revised2).
  Yina Guo, Ting‐Hsuan Chen, Xingjuan Zeng, David Warburton, Kristina I. Boström, Chih‐Ming Ho, Xin Zhao, Alan Garfinkel.
  The Journal of Physiology. November 18, 2013
  Abstract  Recent experimental work has described an elegant pattern of branching in the development of the lung. Multiple forms of branching have been identified, including side branching and tip bifurcation. A particularly interesting feature is the phenomenon of ‘orthogonal rotation of the branching plane’. The lung must fill 3D space with the essentially 2D phenomenon of branching. It accomplishes this by rotating the branching plane by 90 degrees with each generation. The mechanisms underlying this rotation are not understood. In general, the programs that underlie branching have been hypothetically attributed to genetic ‘subroutines’ under the control of a ‘global master routine’ to invoke particular subroutines at the proper time and location, but the mechanisms of these routines are not known. Here, we demonstrate that fundamental mechanisms, the reaction and diffusion of biochemical morphogens, can create these patterns. We used a Partial Differential Equation model that postulates 3 morphogens, which we identify with specific molecules in lung development. We found that cascades of branching events, including side branching, tip splitting and orthogonal rotation of the branching plane, all emerge immediately from the model, without further assumptions. In addition, we found that one branching mode can be easily switched to another, by increasing or decreasing the values of key parameters. This shows how a ‘global master routine’ could work by the alteration of a single parameter. Being able to simulate cascades of branching events is necessary to understand the critical features of branching, such as orthogonal rotation of the branching plane between successive generations, and branching mode switch during lung development. Thus, our model provides a paradigm for how genes could possibly act to produce spatial structures. Our low‐dimensional model gives a qualitative understanding of how generic physiological mechanisms can produce branching phenomena, and how the system can switch from one branching pattern to another using low‐dimensional ‘control knobs’. The model provides a number of testable predictions, some of which have already been observed (though not explained) in experimental work.

  November 18, 2013   doi: 10.1113/jphysiol.2013.261099   open full text
• Diversity of vestibular nuclei neurons targeted by cerebellar nodulus inhibition.
  Hui Meng, Pablo M. Blázquez, J. David Dickman, Dora E. Angelaki.
  The Journal of Physiology. November 13, 2013
  •  Electrical stimulation of the cerebellar nodulus and ventral uvula decreases the time constant of the horizontal vestibulo‐ocular reflex during yaw rotation. •  Unlike the flocculus and ventral paraflocculus which target a particular cell group, nodulus/ventral uvula inhibition targets a large diversity of cell types in the vestibular nuclei. •  Twenty per cent of nodulus/ventral uvula‐target neurons were sensitive to both vestibular stimuli and eye movements, whereas the majority was only sensitive to vestibular stimuli. •  Most nodulus/ventral uvula‐target cells responded to both rotation and translation and only approximately half discriminated translational and gravitational accelerations. •  Projections of the nodulus/ventral uvula to both eye movement‐and non‐eye movement‐sensitive vestibular nuclei neurons suggest a role in both eye movement generation and vestibulo‐spinal or thalamo‐cortical systems. Abstract  A functional role of the cerebellar nodulus and ventral uvula (lobules X and IXc,d of the vermis) for vestibular processing has been strongly suggested by direct reciprocal connections with the vestibular nuclei, as well as direct vestibular afferent inputs as mossy fibres. Here we have explored the types of neurons in the macaque vestibular nuclei targeted by nodulus/ventral uvula inhibition using orthodromic identification from the caudal vermis. We found that all nodulus‐target neurons are tuned to vestibular stimuli, and most are insensitive to eye movements. Such non‐eye‐movement neurons are thought to project to vestibulo‐spinal and/or thalamo‐cortical pathways. Less than 20% of nodulus‐target neurons were sensitive to eye movements, suggesting that the caudal vermis can also directly influence vestibulo‐ocular pathways. In general, response properties of nodulus‐target neurons were diverse, spanning the whole continuum previously described in the vestibular nuclei. Most nodulus‐target cells responded to both rotation and translation stimuli and only a few were selectively tuned to translation motion only. Other neurons were sensitive to net linear acceleration, similar to otolith afferents. These results demonstrate that, unlike the flocculus and ventral paraflocculus which target a particular cell group, nodulus/ventral uvula inhibition targets a large diversity of cell types in the vestibular nuclei, consistent with a broad functional significance contributing to vestibulo‐ocular, vestibulo‐thalamic and vestibulo‐spinal pathways.

  November 13, 2013   doi: 10.1113/jphysiol.2013.259614   open full text
• Hydrogen sulphide inhibits Ca2+ release through InsP3 receptors and relaxes airway smooth muscle.
  Isabel Castro‐Piedras, Jose F. Perez‐Zoghbi.
  The Journal of Physiology. November 12, 2013
  •  The novel signalling molecule hydrogen sulphide (H2S) regulates diverse cell physiological processes in several organs and systems including airway smooth muscle contractility. •  We explored the mechanisms of H2S‐induced smooth muscle relaxation in small intrapulmonary airways using lung slices and imaging approaches. •  We found that exogenous and endogenous H2S inhibited intracellular Ca2+ release specifically through the inositol‐1,4,5‐trisphosphate (InsP3) receptor in smooth muscle cells and reversibly inhibited acetylcholine‐induced intracellular Ca2+ oscillations, thus leading to airway dilatation. •  The effects of H2S on InsP3‐induced Ca2+ release and airway contraction were mimicked by the reducing agent dithiothreitol and inhibited by the oxidizing agent diamide, suggesting that H2S acts as a thiol‐reducing agent to reduce Ca2+ release through InsP3 receptors and to evoke relaxation. •  Our results suggest that endogenously produced H2S is a novel modulator of InsP3‐mediated Ca2+ signalling in airway smooth muscle and thus promotes bronchodilatation. Abstract  Hydrogen sulphide (H2S) is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including vascular and airway smooth muscle cell (SMC) contraction. Decreases in endogenous H2S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2S in small intrapulmonary airways using mouse lung slices and confocal and phase‐contrast video microscopy. Exogenous H2S donor Na2S (100 μm) reversibly inhibited Ca2+ release and airway contraction evoked by inositol‐1,4,5‐trisphosphate (InsP3) uncaging in airway SMCs. Similarly, InsP3‐evoked Ca2+ release and contraction was inhibited by endogenous H2S precursor l‐cysteine (10 mm) but not by l‐serine (10 mm) or either amino acid in the presence of dl‐propargylglycine (PPG). Consistent with the inhibition of Ca2+ release through InsP3 receptors (InsP3Rs), Na2S reversibly inhibited acetylcholine (ACh)‐induced Ca2+ oscillations in airway SMCs. In addition, Na2S, the H2S donor GYY‐4137, and l‐cysteine caused relaxation of airways pre‐contracted with either ACh or 5‐hydroxytryptamine (5‐HT). Na2S‐induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp‐8‐pCPT‐cGMPS). The effects of H2S on InsP3‐evoked Ca2+ release and contraction as well as on the relaxation of agonist‐contracted airways were mimicked by the thiol‐reducing agent dithiothreitol (DTT, 10 mm) and inhibited by the oxidizing agent diamide (30 μm). These studies indicate that H2S causes airway SMC relaxation by inhibiting Ca2+ release through InsP3Rs and consequent reduction of agonist‐induced Ca2+ oscillations in SMCs. The results suggest a novel role for endogenously produced H2S that involves the modulation of InsP3‐evoked Ca2+ release – a cell‐signalling system of critical importance for many physiological and pathophysiological processes.

  November 12, 2013   doi: 10.1113/jphysiol.2013.257790   open full text
• Velocity storage mechanism in zebrafish larvae.
  Chien‐Cheng Chen, Christopher J. Bockisch, Giovanni Bertolini, Itsaso Olasagasti, Stephan C. F. Neuhauss, Konrad P. Weber, Dominik Straumann, Melody Ying‐Yu Huang.
  The Journal of Physiology. November 12, 2013
  Abstract  The optokinetic reflex (OKR) and the angular vestibulo‐ocular reflex (aVOR) complement each other to stabilize images on the retina despite self‐ or world motion, a joint mechanism that is critical for effective vision. It is currently hypothesized that signals from both systems integrate, in a mathematical sense, in a network of neurons operating as a velocity storage mechanism (VSM). When exposed to a rotating visual surround, subjects display the OKR, slow following eye movements frequently interrupted by fast resetting eye movements. Subsequent to light‐off during optokinetic stimulation, eye movements do not stop abruptly, but decay slowly, a phenomenon referred to as the optokinetic after response (OKAR). The OKAR is most likely generated by the VSM. In this study, we observed the OKAR in developing larval zebrafish before the horizontal aVOR emerged. Our results suggest that the VSM develops prior to and without the need for a functional aVOR. It may be critical to ocular motor control in early development as it increases the efficiency of the OKR. This article is protected by copyright. All rights reserved.

  November 12, 2013   doi: 10.1113/jphysiol.2013.258640   open full text
• Image based assessment of microvascular function and structure in Collagen XV and XVIII deficient mice.
  C.B. Rygh, G. Løkka, R. Heljasvaara, T. Taxt, T. Pavlin, R. Sormunen, T. Pihlajaniemi, F.R. Curry, O. Tenstad, R.K. Reed.
  The Journal of Physiology. November 12, 2013
  Abstract  Aims. Collagen XV and XVIII are ubiquitous constituents of basement membranes. We aimed to study the physiological roles of these two components of the permeability barrier non‐invasively in striated muscle in mice deficient of collagen XV or XVIII by dynamic‐contrast‐enhanced magnetic resonance imaging (DCE‐MRI). Structural information was obtained with transmission electron tomography (TEM). MR data was analysed by two different analysis methods to quantify tissue perfusion and microcirculatory exchange parameters to rule out data analysis method‐dependent results. Methods and Results. Control mice (C57BL/6J Ola/Hsd strain) or mice lacking either collagen XV (Col15a1−/−) or XVIII (Col18a1−/−) were included in the study. MR images were acquired using a preclinical system using gadodiamide (Gd‐DTPA‐BMA, molecular weight 0.58 kDa) as a tracer. Exchange capacity (permeability (P)‐surface area (S) product relative to blood flow, (FB)), was increased in test mice compared to controls, but the contributions from P, S, and FB were different in these two phenotypes. FB was significantly increased in Col18a1−/−, but slightly decreased in Col15a1−/−. PS was significantly increased only in Col18a1−/− even though P was increased in both phenotypes suggesting S might also be reduced in Col15a1−/− mice. Immunohistochemistry and electron microscopy demonstrated alterations in capillary density and morphology in both knockout mouse strains in comparison to the control mice. Conclusion. Both collagen XV and XVIII are important for maintaining normal capillary permeability in the striated muscle. DCE‐MRI and the perfusion analyses successfully determined microvascular hemodynamic parameters of genetically modified mice and gave results consistent with more invasive methods. This article is protected by copyright. All rights reserved.

  November 12, 2013   doi: 10.1113/jphysiol.2013.263574   open full text
• Functional properties of extrasynaptic AMPA and NMDA receptors during postnatal hippocampal neurogenesis.
  Charlotte Schmidt‐Salzmann, Liyi Li, Josef Bischofberger.
  The Journal of Physiology. November 12, 2013
  Abstract  In the mammalian hippocampus, new granule cells are continuously generated throughout life. Although it is well known that they rapidly form several thousand new glutamatergic synapses, the underlying mechanisms are not well understood. As extrasynaptic NMDA receptors are believed to support the generation of new spines, we have studied the functional properties of extrasynaptic ionotropic glutamate receptors in newborn granule cells in juvenile rats during and after synaptic integration. Using fast application of glutamate to outside‐out membrane patches, we show that all immature granule cells express functional AMPA and NMDA receptors. The density of AMPA receptors was small in cells starting to receive excitatory synaptic input (∼30 pS μm−2) but substantially increased during synaptic integration to finally reach ∼ 120 pS μm−2 in fully mature cells. Interestingly, AMPA receptors showed a biphasic change in desensitisation time constant which was slowest during synaptic integration and substantially faster before and afterwards. This was paralleled by a change in the non‐desensitising current component which was maximal during synaptic integration and about 50% smaller afterwards. Surprisingly, the NMDA‐receptor kinetics and density in young cells was already comparable to mature cells (∼10 pS μm−2), leading to an enhanced NMDA‐/AMPA‐receptor density ratio. Similar to somatic outside‐out patches, iontophoretic application of glutamate onto dendrites also revealed an enhanced dendritic NMDA/AMPA ratio in young cells. These data indicate that prolonged AMPAR currents in newly generated young granule cells might support the effective activation of extrasynaptic NMDA receptors and therefore constitute a competitive advantage over mature cells for new synapse formation. This article is protected by copyright. All rights reserved.

  November 12, 2013   doi: 10.1113/jphysiol.2013.267203   open full text
• Identification of unique release kinetics of serotonin from guinea‐pig and human enterochromaffin cells.
  Ravinarayan Raghupathi, Michael D. Duffield, Leah Zelkas, Adrian Meedeniya, Simon J. H. Brookes, Tiong Cheng Sia, David A. Wattchow, Nick J. Spencer, Damien J. Keating.
  The Journal of Physiology. November 12, 2013
  •  Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the body's total serotonin (5‐HT). •  Although 5‐HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level. •  This study provides the first functional characterisation of single primary guinea‐pig and human EC cells. •  EC cells release 5‐HT from large dense core vesicles in a calcium‐dependent manner with kinetics surprisingly resembling release from synaptic vesicles. •  3D modelling indicates that the quantity of 5‐HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5‐HT receptors. •  These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control. Abstract  The major source of serotonin (5‐HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5‐HT, and that this 5‐HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5‐HT release from single primary EC cells. We have developed a rapid primary culture of guinea‐pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca2+ imaging, immunocytochemistry and 3D modelling. Ca2+ enters EC cells upon stimulation and triggers quantal 5‐HT release via L‐type Ca2+ channels. Real time amperometric techniques reveal that EC cells release 5‐HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5‐HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5‐HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three‐dimensional (3D) simulations of 5‐HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5‐HT release in the whole‐tissue milieu. These models indicate that local 5‐HT levels are likely to be maintained around the activation threshold for mucosal 5‐HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5‐HT release in primary EC cells. The mode of 5‐HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.

  November 12, 2013   doi: 10.1113/jphysiol.2013.259796   open full text
• Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1.
  Dante J. Heredia, Michael D. Gershon, Sang Don Koh, Robert D. Corrigan, Takanubu Okamoto, Terence K. Smith.
  The Journal of Physiology. November 11, 2013
  •  Previous studies have indicated that neither neuronal nor mucosal 5‐hydroxytryptamine (5‐HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5‐HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5‐HT in enterochromaffin cells in the mucosa. •  Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5‐HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. •  TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. •  In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. •  Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. •  This suggests that 5‐HT release from the mucosa and stretch reflexes are important for normal colonic propulsion. Abstract  Although there is general agreement that mucosal 5‐hydroxytryptamine (5‐HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5‐HT is critical. We therefore tested the hypothesis that the secretion of 5‐HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1−/− mice), which is the rate‐limiting enzyme in the biosynthesis of mucosal but not neuronal 5‐HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1+/+) mice, Tph1−/− animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1+/+ colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1+/+); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1−/− colon; and (9) the 5‐HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1+/+ mice, failed to alter CMMCs in Tph1−/− animals. Our observations suggest that mucosal 5‐HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.

  November 11, 2013   doi: 10.1113/jphysiol.2013.256230   open full text
• Optogenetic identification of an intrinsic cholinergically‐driven inhibitory oscillator sensitive to cannabinoids and opioids in hippocampal CA1.
  Daniel A. Nagode, Ai‐Hui Tang, Kun Yang, Bradley E. Alger.
  The Journal of Physiology. November 08, 2013
  Abstract  Neuronal electrical oscillations in the theta (4–14 Hz) and gamma (30–80 Hz) ranges are necessary for performance of certain animal behaviors and cognitive processes. Perisomatic GABAergic inhibition is prominently involved in cortical oscillations driven by acetylcholine (ACh) release from septal cholinergic afferents. In neocortex and hippocampal CA3 regions, parvalbumin–positive (PV) basket cells, activated by ACh and glutamatergic agonists, largely mediate oscillations. However, in CA1 hippocampus in vitro, cholinergic agonists or optogenetic release of endogenous ACh from septal afferents induce rhythmic, theta‐frequency inhibitory post‐synaptic currents (IPSCs) in pyramidal cells, even with glutamatergic transmission blocked. The IPSCs are regulated by exogenous and endogenous cannabinoids, suggesting that they arise from type 1 cannabinoid‐receptor expressing (CB1R+) interneurons – mainly cholecystokinin (CCK) cells. Nevertheless, an occult contribution of PV interneurons to these rhythms remained conceivable. Here we directly test this hypothesis by selectively silencing CA1 PV cells optogenetically with halorhodopsin or archaerhodopsin. However, this had no effect on theta‐frequency IPSC rhythms induced by carbachol (CCh). In contrast, silencing glutamic‐acid‐decarboxylase 2+ interneurons, which include the CCK basket cells, strongly suppressed inhibitory oscillations; PV interneurons appear to play no role. The low‐frequency IPSC oscillations induced by CCh or optogenetically‐stimulated ACh release were also inhibited by a μ‐opioid receptor (MOR) agonist, which was unexpected because MORs in CA1 are not usually associated with CCK cells. Our results reveal novel properties of an inhibitory oscillator circuit within CA1 that is activated by muscarinic agonists. The oscillations could contribute to behaviourally‐relevant, atropine‐sensitive, theta‐rhythms and link cannabinoid and opioid actions functionally.

  November 08, 2013   doi: 10.1113/jphysiol.2013.257428   open full text
• Maximal heart rate does not limit cardiovascular capacity in healthy humans: insight from right atrial pacing during maximal exercise.
  G.D.W. Munch, J.H. Svendsen, R. Damsgaard, N.H. Secher, J. González‐Alonso, S.P. Mortensen.
  The Journal of Physiology. November 08, 2013
  Abstract  In humans, maximal aerobic power (VO2max) is associated with a plateau in cardiac output (Q), but the mechanisms regulating the interplay between maximal heart rate (HRmax) and stroke volume (SV) are unclear. To evaluate the effect of tachycardia and elevations in HRmax on cardiovascular function and capacity during maximal exercise in healthy humans, twelve young male cyclists performed incremental cycling and one‐legged knee‐extensor exercise (KEE) to exhaustion with and without right atrial pacing to increase HR. During control cycling, Q and leg blood flow increased up to 85% of maximal workload (WLmax) and remained unchanged until exhaustion. SV initially increased, plateaued and then decreased before exhaustion (P < 0.05) despite an increase in right atrial pressure (RAP) and a tendency (P = 0.056) for a reduction in left ventricular transmural filling pressure (LVFP). Atrial pacing increased HRmax from 184 ± 2 to 206 ± 3 beats min−1 (P < 0.05), but Q remained similar to the control condition at all intensities because of a lower SV and LVTP (P < 0.05). No differences in arterial pressure, peripheral haemodynamics, catecholamines or VO2 were observed, but pacing increased the rate pressure product and RAP (P < 0.05). Atrial pacing had a similar effect on haemodynamics during KEE, except that pacing decreased RAP. In conclusion, the human heart can be paced to a higher HR than observed during maximal exercise, suggesting that HRmax and myocardial work capacity do not limit VO2max in healthy individuals. A limited left ventricular filling and possibly altered contractility reduce SV during atrial pacing, whereas a plateau in LVFP appears to restrict Q close to VO2max.

  November 08, 2013   doi: 10.1113/jphysiol.2013.262246   open full text
• The contribution of motor commands to position sense differs between elbow and wrist.
  Lee D. Walsh, Uwe Proske, Trevor J. Allen, Simon C. Gandevia.
  The Journal of Physiology. November 08, 2013
  •  Knowing the position of our limbs is critical for accurate movement. Central motor command signals generated by the brain contribute to position sense at the human wrist, but this could not be demonstrated at the elbow. •  We tested whether this represents a fundamental difference between the two joints or whether it reflects the two different methods used to measure position sense. •  For both measurement methods, contraction of wrist muscles led to illusions that the wrist is displaced. No such illusions were detected at the elbow during muscle contraction. •  Thus, the contribution of centrally generated command signals to position sense differs between joints. Any contribution at the elbow joint is small and new methods will be needed to reveal it. Abstract  Recent studies have suggested that centrally generated motor commands contribute to the perception of position and movement at the wrist, but not at the elbow. Because the wrist and elbow experiments used different methods, this study was designed to resolve the discrepancy. Two methods were used to test both the elbow and wrist (20 subjects each). For the wrist, subjects sat with their right arm strapped to a device that restricted movement to the wrist. Before each test, voluntary contraction of wrist flexor or extensor muscles controlled for muscle spindle thixotropy. After relaxation, the wrist was moved to a test angle. Position was indicated either with a pointer, or by matching with the contralateral wrist, under two conditions: when the reference wrist was relaxed or when its muscles were contracted isometrically (30% maximum). The elbow experiment used the same design to measure position sense in the passive elbow and with elbow muscles contracting (30% maximum). At the wrist when using a pointer, muscle contraction altered significantly the perceived wrist angle in the direction of contraction by 7 deg [3 deg, 12 deg] (mean [95% confidence interval]) with a flexor contraction and 8 deg [4 deg, 12 deg] with an extensor contraction. Similarly, in the wrist matching task, there was a change of 13 deg [9 deg, 16 deg] with a flexor contraction and 4 deg [1 deg, 8 deg] with an extensor contraction. In contrast, contraction of elbow flexors or extensors did not alter significantly the perceived position of the elbow, compared with rest. The contribution of central commands to position sense differs between the elbow and the wrist.

  November 08, 2013   doi: 10.1113/jphysiol.2013.259127   open full text
• Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres.
  C. R. Lamboley, R. M. Murphy, M. J. McKenna, G. D. Lamb.
  The Journal of Physiology. November 08, 2013
  •  Ca2+ release from the sarcoplasmic reticulum (SR) controls contraction in vertebrate skeletal muscle. Calsequestrin (CSQ) is thought to be the principal Ca2+ binding protein in the SR but little is known about SR Ca2+ content and loading characteristics, or CSQ isoform distribution, in human skeletal muscle fibres. •  Type I (slow‐twitch) and type II (fast‐twitch) skeletal muscle fibres in young healthy adults show highly‐stereotyped patterns of isoform expression of CSQ and SR Ca2+ pumps, in tight correspondence with isoform expression of the contractile proteins, which probably facilitates optimal contractile function in the individual fibre types. •  Endogenous Ca2+ content of the SR is slightly larger in type II fibres than in type I fibres, but its maximal capacity is substantially greater, probably due to the larger amount of the CSQ1 isoform present. SR Ca2+ content and capacity in type I fibres is probably determined by their content of both CSQ1 and CSQ2. Abstract  The relationship between sarcoplasmic reticulum (SR) Ca2+ content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre‐lysing assay was used to quantify the endogenous Ca2+ content and maximal Ca2+ capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca2+ content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca2+ per litre of fibre, respectively), with virtually all of this Ca2+ evidently being in the SR, as it could be rapidly released with a caffeine‐low [Mg2+] solution (only 0.08 ± 0.01 and <0.07 mmol l−1, respectively, remaining). The maximal Ca2+ content that could be reached with SR Ca2+ loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l−1 in type I and type II fibres, respectively (P < 0.05). In non‐lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine‐induced Ca2+ release and subsequent Ca2+ reloading similarly indicated that (i) maximal SR Ca2+ content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca2+ content represented a greater percentage of maximal content in type I fibres compared to type II fibres (∼59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ∼3–fold more CSQ1 and ∼5–fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca2+ content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.

  November 08, 2013   doi: 10.1113/jphysiol.2013.265900   open full text
• Development of heart failure is independent of K+ channel‐interacting protein 2 expression.
  Tobias Speerschneider, Søren Grubb, Artina Metoska, Søren‐Peter Olesen, Kirstine Calloe, Morten B. Thomsen.
  The Journal of Physiology. November 07, 2013
  •  Previous studies have suggested that the K+ channel auxiliary subunit K+ channel‐interacting protein 2 (KChIP2) serves as a regulator of cardiac remodelling leading to heart failure and increased risk of arrhythmias. •  The results presented here show that the progression of cardiac remodelling and heart failure induced by transverse aortic constriction follows a similar time course in wild‐type and KChIP2−/− mice. •  Protein expression analysis shows that ventricular KChIP2 is significantly downregulated in heart failure in wild‐type mice. •  The electrophysiological analysis reveals enlarged J and T wave amplitudes and lower vulnerability to pacing‐induced ventricular arrhythmias in KChIP2−/− control mice compared to wild‐type control mice. Heart failure in wild‐type and KChIP2−/− mice prompted comparable prolongation of QT intervals and ventricular effective refractory periods. •  Collectively, these results demonstrate that KChIP2 does not influence the structural and functional development of heart failure. Moreover, in contrast to previously reported data, downregulation of KChIP2 expression in heart failure may reduce the risk of cardiac arrhythmia. Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K+ channel‐interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K+ current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild‐type (WT) and KChIP2−/− mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post‐surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC‐operated WT and KChIP2−/− mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2−/− control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham‐operated mice of either genotype. Pacing‐induced ventricular tachycardia (VT) was observed in 5/10 sham‐operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham‐operated KChIP2−/− mice were resistant to pacing‐induced VT resulting in only 1/10 inducible mice. KChIP2−/− with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K+ current.

  November 07, 2013   doi: 10.1113/jphysiol.2013.263483   open full text
• Exercise counteracts the effects of short‐term overfeeding and reduced physical activity independent of energy imbalance in healthy young men.
  Jean‐Philippe Walhin, Judith D. Richardson, James A. Betts, Dylan Thompson.
  The Journal of Physiology. November 06, 2013
  Abstract  Physical activity can affect many aspects of metabolism but it is unclear to what extent this relies on manipulation of energy balance. Twenty‐six active men (age 25 ± 7 years) were randomly‐assigned either to consume 50% more energy than normal by over‐consuming their habitual diet for 7 days whilst simultaneously restricting their physical activity below 4000 steps day−1 to induce an energy surplus (SUR group; n= 14) or to the same regimen but with 45 min of daily treadmill running at 70% of maximum oxygen uptake (SUR+EX group; n= 12). Critically, the SUR+EX group received additional dietary energy intake to account for the energy expended during exercise; thus maintaining a matched energy surplus. At baseline and follow‐up, fasted blood samples and abdominal subcutaneous adipose tissue biopsies were obtained and oral glucose tolerance tests conducted. Insulinaemic responses to a standard glucose load increased 2‐fold from baseline to follow‐up in the SUR group (Δ17 ± 16 nmol × 120 min l−1; P= 0.002) whereas there was no change in the SUR+EX group (Δ1 ± 6 nmol × 120 min l−1). Seven of 17 genes within adipose tissue were differentially‐expressed in the SUR group; expression of SREBP1c, FAS and GLUT4 was significantly up‐regulated and expression of PDK4, IRS2, HSL and VISFATIN was significantly down‐regulated (P≤ 0.05). The pAMPK/AMPK protein ratio in adipose was significantly down‐regulated in the SUR group (P= 0.005). Vigorous‐intensity exercise counteracted most of the effects from short‐term overfeeding and under‐activity at the whole‐body level and in adipose tissue, even in the face of a standardised energy surplus.

  November 06, 2013   doi: 10.1113/jphysiol.2013.262709   open full text
• Cellular properties and chemosensory responses of the human carotid body.
  Patricia Ortega‐Sáenz, Ricardo Pardal, Konstantin Levitsky, Javier Villadiego, Ana Belén Muñoz‐Manchado, Rocío Durán, Victoria Bonilla‐Henao, Ignacio Arias‐Mayenco, Verónica Sobrino, Antonio Ordóñez, María Oliver, Juan José Toledo‐Aral, José López‐Barneo.
  The Journal of Physiology. November 06, 2013
  Abstract  The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular. However, the functional characteristics of human CB cells remain practically unknown. Herein, we use tissue slices or enzymatically dispersed cells to determine characteristics of human CB cells. The adult human CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin‐positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line‐derived neurotrophic factor (GDNF). We found that GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. Moreover, glomus cells exhibited voltage‐dependent Na+, Ca2+ and K+ currents that were qualitatively similar to those reported in lower mammals. These cells responded to hypoxia with an external Ca2+‐dependent increase of cytosolic Ca2+ and quantal catecholamine secretion, as reported for other mammalian species. Interestingly, human glomus cells are also responsive to hypoglycaemia and together these two stimuli can potentiate each other's effects. The chemosensory responses of glomus cells are also preserved at an advance age. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.

  November 06, 2013   doi: 10.1113/jphysiol.2013.263657   open full text
• Down‐regulation of CaV1.2 channels during hypertension: How fewer CaV1.2 channels allow more Ca2+ into hypertensive arterial smooth muscle.
  Sendoa Tajada, Pilar Cidad, Olaia Colinas, L. Fernando Santana, José R. López‐López, M. Teresa Pérez‐García.
  The Journal of Physiology. November 06, 2013
  Abstract  Hypertension is a clinical syndrome characterized by increased arterial tone. Although the mechanisms are varied, the generally accepted view is that increased CaV1.2 channel function is a common feature of this pathological condition. Here, we investigated the mechanisms underlying vascular dysfunction in a mouse model of genetic hypertension. Contrary to expectation, we found that whole‐cell CaV1.2 currents (ICa) were lower in hypertensive (BPH) than normotensive (BPN) myocytes. However, local CaV1.2 sparklet activity was higher in BPH cells, suggesting that the relatively low ICa in these cells was produced by a few hyperactive CaV1.2 channels. Furthermore, our data suggest that while the lower expression of the pore‐forming α1c subunit of CaV1.2 s underlies the lower ICa in BPH myocytes, the increased sparklet activity was due to a different composition in the auxiliary subunits of the CaV1.2 complexes. ICa in BPN cells were produced by channels composed of α1c/α2δ/β3 subunits, while in BPH myocytes currents were likely generated by the opening of channels formed by α1c/α2δ/β2 subunits. In addition, Ca2+ sparks evoked BK currents of lower magnitude in BPH than in BPN myocytes, because BK channels were less sensitive to Ca2+. Our data are consistent with a model in which a decrease in the global number of CaV1.2 currents coexist with the existence of a subpopulation of channels highly active that dominate the resting Ca2+ influx. The decrease in BK channel activity turns ineffective the hyperpolarizing brake and leads BPH myocytes to a more contracted resting state.

  November 06, 2013   doi: 10.1113/jphysiol.2013.265751   open full text
• Structure and gating of tetrameric glutamate receptors.
  Alexander I. Sobolevsky.
  The Journal of Physiology. November 06, 2013
  Abstract  Ionotropic glutamate receptors (iGluRs) are ligand‐gated ion channels that open their ion‐conducting pores in response to the binding of agonist glutamate. In recent years, significant progress has been achieved in studies of iGluRs by obtaining numerous structures of isolated water soluble ligand binding and amino terminal domains as well as the first full length crystal structure of GluA2 in the closed, antagonist‐bound state. This structural data combined with electrophysiological and fluorescence recordings, biochemical experiments, mutagenesis and molecular dynamics simulations have greatly improved our understanding of iGluR assembly, activation and desensitization processes. This article reviews the recent structural and functional advances in iGluR field and summarizes them in a simplified model of full length iGluR gating.

  November 06, 2013   doi: 10.1113/jphysiol.2013.264911   open full text
• Myosin filaments in smooth muscle cells do not have a constant length.
  Jeffrey C.‐Y. Liu, Jörg Rottler, Lu Wang, Jenny Zhang, Chris D. Pascoe, Bo Lan, Brandon A. Norris, Ana M. Herrera, Peter D. Paré, Chun Y. Seow.
  The Journal of Physiology. October 31, 2013
  •  The length of myosin filaments was measured in three types of smooth muscle using serial electron microscopy. •  The frequency distribution of myosin filament length for all three types of smooth muscle followed an exponential decay pattern. •  The same frequency distribution pattern was observed in activated tracheal smooth muscle, although the average length was shorter compared with the filaments in relaxed smooth muscle. •  Analysis suggests that the distribution pattern reflects a dynamic equilibrium between competing processes of linear polymerization and de‐polymerization of myosin dimers. Abstract  Myosin molecules from smooth muscle and non‐muscle cells are known to self‐assemble into side‐polar filaments in vitro. However, the in situ mechanism of filament assembly is not clear and the question of whether there is a unique length for myosin filaments in smooth muscle is still under debate. In this study we measured the lengths of 16,587 myosin filaments in three types of smooth muscle cells using serial electron microscopy (EM). Sheep airway and pulmonary arterial smooth muscle as well as rabbit carotid arterial smooth muscle were fixed for EM and serial ultra‐thin (50–60 nm) sections were obtained. Myosin filaments were traced in consecutive sections to determine their lengths. The results indicate that there is not a single length for the myosin filaments; instead there is a wide variation in lengths. The plots of observation frequency versus myosin filament length follow an exponential decay pattern. Analysis suggests that in situ assembly of myosin filaments in smooth muscle is governed by random processes of linear polymerization and de‐polymerization, and that the dynamic equilibrium of these processes determines the observed length distribution.

  October 31, 2013   doi: 10.1113/jphysiol.2013.264168   open full text
• Endogenous zinc depresses GABAergic transmission via T‐type Ca2+ channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells.
  Antonia Grauert, Dominique Engel, Arnaud J. Ruiz.
  The Journal of Physiology. October 31, 2013
  •  Zinc inhibits ionotropic receptors commonly found at central synapses, as well as a wide variety of voltage‐activated ion channels that modulate neuronal excitability and neurotransmitter release. •  We found that zinc chelation facilitated GABAergic signalling in dentate granule cells and that blocking T‐type Ca2+ channel activity abolished this effect. Zinc chelation reduced spike threshold, increased spike width and shifted the input–output relationship in dentate interneurones, which is consistent with increased excitability. •  In granule cells, zinc chelation narrowed the window for the integration of glutamatergic inputs originating from perforant path synapses. •  These results demonstrate that zinc modulates dentate interneurone function and regulates spike routing to local and hippocampal targets. Abstract  Zinc actions on synaptic transmission span the modulation of neurotransmitter receptors, transporters, activation of intracellular cascades and alterations in gene expression. Whether and how zinc affects inhibitory synaptic signalling in the dentate gyrus remains largely unexplored. We found that mono‐ and di‐synaptic GABAergic inputs onto dentate granule cells were reversibly depressed by exogenous zinc application and enhanced by zinc chelation. Blocking T‐type Ca2+ channels prevented the effect of zinc chelation. When recording from dentate fast‐spiking interneurones, zinc chelation facilitated T‐type Ca2+ currents, increased action potential half‐width and decreased spike threshold. It also increased the offset of the input–output relation in a manner consistent with enhanced excitability. In granule cells, chelation of zinc reduced the time window for the integration of glutamatergic inputs originating from perforant path synapses, resulting in reduced spike transfer. Thus, zinc‐mediated modulation of dentate interneurone excitability and GABA release regulates information flow to local targets and hippocampal networks.

  October 31, 2013   doi: 10.1113/jphysiol.2013.261420   open full text
• A single point mutation reveals gating of the human ClC‐5 Cl−/H+ antiporter.
  Silvia De Stefano, Michael Pusch, Giovanni Zifarelli.
  The Journal of Physiology. October 31, 2013
  •  ClC‐5 is a 2Cl−/1H+ antiporter expressed in endosomes that is essential for proper endocytosis, but its molecular function is still not understood. •  In heterologous systems ClC‐5 elicits currents only at positive potentials. This rectifying behaviour conflicts with most of the models proposed to explain ClC‐5 function. The origin of this rectification is unknown. •  Here we identified a ClC‐5 mutation, D76H, that elicits inward tail currents at negative potentials. •  These currents reflect transmembrane transport that preserve the 2Cl−/1H+ stoichiometry. •  We conclude that a gating mechanism regulates ClC‐5 transport activity and is at least in part responsible for the strong rectification of ClC‐5 currents. Abstract  ClC‐5 is a 2Cl−/1H+ antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC‐5 cause Dent's disease, potentially leading to renal failure. However, the physiological role of ClC‐5 is still unclear. One of the main issues is whether the strong rectification of ClC‐5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and what is the origin of this rectification. In this work we identified a ClC‐5 mutation, D76H, which, besides the typical outward currents of the wild‐type (WT), shows inward tail currents at negative potentials that allow the estimation of the reversal of ClC‐5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl−] shows that they are generated by a coupled transport of Cl− and H+ with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are caused by a gating mechanism that regulates ClC‐5 transport activity and not by a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC‐5 is at least in part caused by a gating mechanism that activates the transporter at positive potentials. These results elucidate the biophysical properties of ClC‐5 and contribute to the understanding of its physiological role.

  October 31, 2013   doi: 10.1113/jphysiol.2013.260240   open full text
• Submaximal ADP‐stimulated respiration is impaired in ZDF rats and recovered by resveratrol.
  Brennan K. Smith, Christopher G. R. Perry, Eric A. F. Herbst, Ian R. Ritchie, Marie‐Soleil Beaudoin, Jeffrey C. Smith, P. Darrell Neufer, David C. Wright, Graham P. Holloway.
  The Journal of Physiology. October 30, 2013
  •  Disparity exists within the literature surrounding mitochondrial dysfunction and insulin resistance and previous reports have primarily examined mitochondrial function as a capacity measurement. •  We show that submaximal ADP‐stimulated respiration rates are lower in ZDF rats, which coincides with decreased adenine nucleotide translocase 2 (ANT2) protein content. •  Supplementation of ZDF rats with resveratrol improves skeletal muscle insulin sensitivity, increases submaximal ADP‐stimulated respiration rates and increases ANT2 protein content. •  Improvements in the ability of ADP to attenuate mitochondrial reactive oxygen species (ROS) emission and cellular redox balance were also observed following resveratrol supplementation. •  These data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for ROS formation. Abstract  Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP‐stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP‐stimulated respiration rates (250–2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP‐stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP‐stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

  October 30, 2013   doi: 10.1113/jphysiol.2013.259226   open full text
• Astroglial potassium clearance contributes to short‐term plasticity of synaptically evoked currents at the tripartite synapse.
  Jérémie Sibille, Ulrike Pannasch, Nathalie Rouach.
  The Journal of Physiology. October 30, 2013
  •  Astrocytes, active players in neurotransmission, display complex membrane ionic responses upon neuronal activity. •  However, the nature, plasticity and role of the activity‐dependent astroglial currents on synaptic plasticity remain unclear in the hippocampus. •  We here demonstrate, using simultaneous electrophysiological recordings of hippocampal neurons and astrocytes, that the complex astroglial current induced synaptically is dominated (80%) by potassium entry through Kir4.1 channels and also includes, in addition to the glutamate transporter current, a small residual current, partially mediated by GABA transporters and Kir4.1‐independent potassium channels. •  These synaptically evoked astroglial currents exhibit differential short‐term plasticity patterns, and astroglial potassium uptake mediated by Kir4.1 channels down‐regulates hippocampal short‐term plasticity. •  This study establishes astrocytes as integrators of excitatory and inhibitory synaptic activity, which may, through dynamic potassium handling, define the signal‐to‐noise ratio essential for specific strengthening of synaptic contacts and synchronization of neuronal ensembles, a prerequisite for learning and memory. Abstract  Astroglial processes enclose ∼60% of CA1 hippocampal synapses to form the tripartite synapse. Although astrocytes express ionic channels, neurotransmitter receptors and transporters to detect neuronal activity, the nature, plasticity and impact of the currents induced by neuronal activity on short‐term synaptic plasticity remain elusive in hippocampal astrocytes. Using simultaneous electrophysiological recordings of astrocytes and neurons, we found that single stimulation of Schaffer collaterals in hippocampal slices evokes in stratum radiatum astrocytes a complex prolonged inward current synchronized to synaptic and spiking activity in CA1 pyramidal cells. The astroglial current is composed of three components sensitive to neuronal activity, i.e. a long‐lasting potassium current mediated by Kir4.1 channels, a transient glutamate transporter current and a slow residual current, partially mediated by GABA transporters and Kir4.1‐independent potassium channels. We show that all astroglial membrane currents exhibit activity‐dependent short‐term plasticity. However, only the astroglial glutamate transporter current displays neuronal‐like dynamics and plasticity. As Kir4.1 channel‐mediated potassium uptake contributes to 80% of the synaptically evoked astroglial current, we investigated in turn its impact on short‐term synaptic plasticity. Using glial conditional Kir4.1 knockout mice, we found that astroglial potassium uptake reduces synaptic responses to repetitive stimulation and post‐tetanic potentiation. These results show that astrocytes integrate synaptic activity via multiple ionic channels and transporters and contribute to short‐term plasticity in part via potassium clearance mediated by Kir4.1 channels.

  October 30, 2013   doi: 10.1113/jphysiol.2013.261735   open full text
• Modulation of stimulus‐specific adaptation by GABAA receptor activation or blockade in the medial geniculate body of the anaesthetized rat.
  Daniel Duque, Manuel S. Malmierca, Donald M. Caspary.
  The Journal of Physiology. October 30, 2013
  Key points Neurons in the medial geniculate body (MGB), the auditory thalamus, give stronger responses to rare sounds than to repetitive sounds, a phenomenon referred to as stimulus‐specific adaptation (SSA). The present study sought to elucidate how the inhibitory thalamic circuitry acting at GABAA receptors affects the generation and/or modulation of SSA from recordings of single unit responses from MGB. Microiontophoretic application of GABAergic agonists selectively increased SSA indices, whereas application of antagonists selectively reduced SSA values. We found that GABAA‐mediated inhibition did not generate the SSA response but regulated the magnitude of SSA sensitivity in a gain control manner. These findings advance our understanding of the role of inhibition in coding deviance detection in the MGB. Abstract Stimulus‐specific adaptation (SSA), which describes adaptation to repeated sounds concurrent with the maintenance of responsiveness to uncommon ones, may be an important neuronal mechanism for the detection of and attendance to rare stimuli or for the detection of deviance. It is well known that GABAergic neurotransmission regulates several different response properties in central auditory system neurons and that GABA is the major inhibitory neurotransmitter acting in the medial geniculate body (MGB). The mechanisms underlying SSA are still poorly understood; therefore, the primary aim of the present study was to examine what role, if any, MGB GABAergic circuits play in the generation and/or modulation of SSA. Microiontophoretic activation of GABAA receptors (GABAARs) with GABA or with the selective GABAAR agonist gaboxadol significantly increased SSA (computed with the common SSA index, CSI) by decreasing responses to common stimuli while having a lesser effect on responses to novel stimuli. In contrast, GABAAR blockade using gabazine resulted in a significant decrease in SSA. In all cases, decreases in the CSI during gabazine application were accompanied by an increase in firing rate to the stimulus paradigm. The present findings, in conjunction with those of previous studies, suggest that GABAA‐mediated inhibition does not generate the SSA response, but can regulate the level of SSA sensitivity in a gain control manner. The existence of successive hierarchical levels of processing through the auditory system suggests that the GABAergic circuits act to enhance mechanisms to reduce redundant information.

  October 30, 2013   doi: 10.1113/jphysiol.2013.261941   open full text
• Quantification of human urinary exosomes by nanoparticle tracking analysis.
  Wilna Oosthuyzen, Nicole E. L. Sime, Jessica R. Ivy, Emma J. Turtle, Jonathan M. Street, John Pound, Louise E. Bath, David J. Webb, Christopher D. Gregory, Matthew A. Bailey, James W. Dear.
  The Journal of Physiology. October 29, 2013
  •  Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively. •  Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. •  In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins. •  Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus. •  With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non‐invasive tool for investigating human renal physiology. Abstract  Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24‐ and AQP2‐positive particles of characteristic exosomal size. Extensive pre‐NTA processing of urine was not necessary. However, the intra‐assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at −80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

  October 29, 2013   doi: 10.1113/jphysiol.2013.264069   open full text
• Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex.
  Sarah F. Hassan, Jennifer L. Cornish, Ann K. Goodchild.
  The Journal of Physiology. October 29, 2013
  •  The medial prefrontal cortex (mPFC) is often referred to as the ‘visceral cortex’, largely based on anatomical connections and cardiovascular influences. •  Although an extensive network of inhibitory interneurons regulates PFC function, their roles in modifying central respiratory, metabolic and cardiac functions have not been explored. •  This study provides the first integrative investigation describing central respiratory, metabolic and cardiac variables altered following chemical disinhibition of discrete subregions of the mPFC. •  Changes were evoked in a site‐dependent manner, with central respiratory function modified throughout the mPFC, and exclusively from dorsal regions. By contrast, central respiratory, metabolic and cardiac functions were simultaneously increased in the ventral mPFC, particularly the infralimbic cortex. •  These data provide reference material for future investigations into chronic changes in the activity of neurons within the mPFC, as seen in stress and mental health disorders, which are often accompanied by autonomic and respiratory dysfunction. Abstract  The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane‐anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA‐A receptor antagonist) into 90 sites in the mPFC at 0.72–4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site‐dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress‐related and mental health disorders, and suggests how basic physiological systems may be affected.

  October 29, 2013   doi: 10.1113/jphysiol.2013.262071   open full text
• Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio).
  K.S. Wilson, G. Matrone, D.E.W. Livingstone, E.A.S. Al‐Dujaili, J.J. Mullins, C.S. Tucker, P.W.F. Hadoke, C.J. Kenyon, M.A. Denvir.
  The Journal of Physiology. October 29, 2013
  Abstract  While glucocorticoids (GCs) are known to be present in the zebrafish embryo little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid‐related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immune sorbent assay (ELISA) and quantitative real‐time polymerase chain reaction (qRT‐PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress‐response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met) an inhibitor of 11β hydroxylase (encoded by cyp11b1) and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress‐induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research. This article is protected by copyright. All rights reserved

  October 29, 2013   doi: 10.1113/jphysiol.2013.256826   open full text
• A cellular memory mechanism aids overload hypertrophy in muscle long after an episodic exposure to anabolic steroids
Muscle memory induced by steroid use.
  Ingrid M. Egner, Jo C. Bruusgaard, Einar Eftestøl, Kristian Gundersen.
  The Journal of Physiology. October 27, 2013
  Abstract  Previous strength training with or without the use of anabolic steroids facilitates subsequent re‐acquisition of muscle mass even after long intervening periods of inactivity. Based on in vivo and ex vivo microscopy we here propose a cellular memory mechanism residing in the muscle cells. Female mice were treated with testosterone propionate for 14 days, inducing a 66% increase in the number of myonuclei and a 77% increase in fibre cross sectional area. Three weeks after removing the drug, fibre size was decreased to the same level as in sham treated animals, but the number of nuclei remained elevated for at least 3 months (>10% of the mouse lifespan). At this time, when the myonuclei‐rich muscles were exposed to overload‐exercise for 6 days, the fibre cross sectional area increased by 31% while control muscles did not grow significantly. We suggest that the lasting, elevated number of myonuclei constitutes a cellular memory facilitating subsequent muscle overload hypertrophy. Our findings might have consequences for the exclusion time of doping offenders. Since the ability to generate new myonuclei is impaired in the elderly our data also invites speculation that it might be beneficial to perform strength training when young in order to benefit in senescence. This article is protected by copyright. All rights reserved

  October 27, 2013   doi: 10.1113/jphysiol.2013.264457   open full text
• Motor unit recruitment by size does not provide functional advantages for motor performance.
  Jakob L. Dideriksen, Dario Farina.
  The Journal of Physiology. October 25, 2013
  Abstract  It is commonly assumed that the orderly recruitment of motor units by size provides a functional advantage for the performance of movements compared with a random recruitment order. On the other hand, the excitability of a motor neuron depends on its size and this is intrinsically linked to its innervation number. A range of innervation numbers among motor neurons corresponds to a range of sizes and thus to a range of excitabilities ordered by size. Therefore, if the excitation drive is similar among motor neurons, the recruitment by size is inevitably due to the intrinsic properties of motor neurons and may not have arisen to meet functional demands. In this view, we tested the assumption that orderly recruitment is necessarily beneficial by determining if this type of recruitment produces optimal motor output. Using evolutionary algorithms and without any a‐priori assumptions, the parameters of neuromuscular models were optimized with respect to several criteria for motor performance. Interestingly, the optimized model parameters matched well known neuromuscular properties, but none of the optimization criteria determined a consistent recruitment order by size unless this was imposed by an association between motor neuron size and excitability. Further, when the association between size and excitability was imposed, the resultant model of recruitment did not improve the motor performance with respect to the absence of orderly recruitment. A consistent observation was that optimal solutions for a variety of criteria of motor performance always required a broad range of innervation numbers in the population of motor neurons, skewed towards the small values. These results indicate that orderly recruitment of motor units in itself does not provide substantial functional advantages for motor control. Rather, the reason for its near‐universal presence in human movements is that motor functions are optimized by a broad range of innervation numbers. This article is protected by copyright. All rights reserved

  October 25, 2013   doi: 10.1113/jphysiol.2013.262477   open full text
• Modelling genetic reorganization in the mouse spinal cord affecting left–right coordination during locomotion.
  Ilya A. Rybak, Natalia A. Shevtsova, Ole Kiehn.
  The Journal of Physiology. October 25, 2013
  •  EphA4, Netrin‐1 and DCC are axon guidance molecules involved in mid‐line crossing of axons of commissural and other spinal interneurons. Absence of these molecules in mice lead to specific locomotor pattern transformations. •  We use a computational model of the bilateral spinal neural networks to simulate the genetically induced transformations in EphA4, Netrin‐1 and DCC mutant mice. •  The model closely reproduces the changes in the locomotor patterns in these mutants, including transformations from the normal left–right alternating pattern to a synchronized hopping pattern in EphA4 and Netrin‐1 mutants, uncoordinated left–right activity in the DCC mutant, and re‐establishment of the alternating patterns in EphA4 and DCC mutant cords by augmentation of inhibitory interactions. •  The model suggests mechanistic explanations for the locomotor transformations and provides insights into the organization of the locomotor network in the spinal cord. Abstract  The spinal neural circuit contains inhibitory (CINi) and excitatory (CINe) commissural interneurons with axons crossing the mid‐line. Direction of these axons to the other side of the cord is controlled by axon guidance molecules, such as Netrin‐1 and DCC. The cord also contains glutamatergic interneurons, whose axon guidance involves the EphA4 receptor. In EphA4 knockout (KO) and Netrin‐1 KO mice, the normal left–right alternating pattern is replaced with a synchronized hopping gait, and the cord of DCC KO mice exhibits uncoordinated left and right oscillations. To investigate the effects of these genetic transformations, we used a computational model of the spinal circuits containing left and right rhythm‐generating neuron populations (RGs), each with a subpopulation of EphA4‐positive neurons, and CINi and CINe populations mediating mutual inhibition and excitation between the left and right RGs. In the EphA4 KO circuits, half of the EphA4‐positive axons crossed the mid‐line and excited the contralateral RG neurons. In the Netrin‐1 KO model, the number of contralateral CINi projections was significantly reduced, while in the DCC KO model, the numbers of both CINi and CINe connections were reduced. In our simulations, the EphA4 and Netrin‐1 KO circuits switched from the left–right alternating pattern to a synchronized hopping pattern, and the DCC KO network exhibited uncoordinated left–right activity. The amplification of inhibitory interactions re‐established an alternating pattern in the EphA4 and DCC KO circuits, but not in the Netrin‐1 KO network. The model reproduces the genetic transformations and provides insights into the organization of the spinal locomotor network.

  October 25, 2013   doi: 10.1113/jphysiol.2013.261115   open full text
• Distribution and Ca2+ signaling of fibroblast‐like (PDGFRα+) cells in the murine gastric fundus.
  Salah A. Baker, Grant W. Hennig, Anna K. Salter, Masaki Kurahashi, Sean M. Ward, Kenton M. Sanders.
  The Journal of Physiology. October 22, 2013
  Abstract  Platelet Derived Growth Factor Receptor α positive (PDGFRα+) cells are suggested to mediate purinergic inputs in GI muscles, but responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα+ cells in murine gastric fundus, load cells with Ca2+ indicators, and follow their activity via digital imaging. Immuno‐labeling demonstrated a high density of PDGFRα+ cells in the fundus. Cells were isolated and purified by FACS using endogenous expression of eGFP driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of P2Y1 receptors and SK3 channels in PDGFRα+ cells. Ca2+ imaging was used to characterize spontaneous Ca2+ transients and responses to purines in PDGFRα+ cells in situ. ATP, ADP, UTP and β‐NAD elicited robust Ca2+ transients in PDGFRα+ cells. Ca2+ transients were also elicited by the P2Y1‐specific agonist N)‐methanocarba‐2MeSADP (MRS‐2365), and inhibited by MRS‐2500, a P2Y1‐specific antagonist. Responses to ADP, MRS‐2365 and β‐NAD were absent in PDGFRα+ cells from P2ry1(‐/‐) mice, but responses to ATP were retained. Purine evoked Ca2+ transients were mediated through Ca2+ release mechanisms. Inhibitors of PLC (U‐73122), IP3 and ryanodine receptors, SERCA pump (cyclopiazonic acid (CPA) and thapsigargin) abolished Ca2+ transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα+ cells. Activation of Ca2+ release is likely to be the signaling mechanism in PDGFRα+ cells responsible for transduction of purinergic enteric inhibitory input in gastric fundus muscles. This article is protected by copyright. All rights reserved

  October 22, 2013   doi: 10.1113/jphysiol.2013.264747   open full text
• Low voltage‐activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse.
  Gabriela M. Rozanski, Arup R. Nath, Michael E. Adams, Elise F. Stanley.
  The Journal of Physiology. October 17, 2013
  •  Sensory neurons in dorsal root ganglia (DRG) lack direct inter‐somatic synaptic contacts but a subpopulation can communicate with their immediate neighbours via transglial, neuron–glial cell–neuron ‘sandwich synapses’. •  We used gently dissociated chick DRG to explore the properties and identity of the voltage sensitive calcium channel responsible for gating transmitter (ATP) release at the neuron‐to‐glial cell synapse. •  A combined pharmacological and biophysical characterization identified the T type, CaV3.2 calcium channel. •  The low voltage‐activated and inactivation‐sensitive properties of CaV3.2 suggest that sandwich synapse transmission is gated not only by action potentials but also by sub‐threshold membrane depolarizations. •  CaV3.2 modulating agents are of interest as anaesthetics, raising the possibility that sandwich synapse transmission plays a role in the aetiology of DRG‐derived abnormal sensation and pain. Abstract  A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi‐, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca2+ entry via voltage‐gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (−50 mV) or low‐concentration Ni2+ (0.1 mm). Transmission persisted using a voltage pulse to −40 mV from a holding potential of −80 mV, confirming the involvement of a low voltage‐activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation‐ and Ni2+‐sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter‐somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub‐threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.

  October 17, 2013   doi: 10.1113/jphysiol.2013.260281   open full text
• Redox modification of ryanodine receptors by mitochondria‐derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts.
  Leroy L. Cooper, Weiyan Li, Yichun Lu, Jason Centracchio, Radmila Terentyeva, Gideon Koren, Dmitry Terentyev.
  The Journal of Physiology. October 17, 2013
  •  Ageing is associated with increased risk of sudden cardiac death due to malignant arrhythmias. •  Shortened refractoriness of Ca2+ release due to increased activity of Ca2+ release channels (RyRs) is recognized as an important contributor to cardiac‐triggered arrhythmias. However, molecular mechanisms of RyR dysfunction and its contribution to arrhythmias in ageing remain to be examined. •  Using ventricular myocytes isolated from old rabbit hearts we demonstrate that age‐associated increase in rate of production of reactive oxygen species (ROS) by mitochondria leads to the thiol‐oxidation of RyRs, which underlies the hyperactivity of the channels and thus shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. Mitochondria‐specific scavenging of ROS in old myocytes restored the redox status of RyRs, reducing SR Ca2+ leak and arrhythmogenic spontaneous Ca2+ waves. •  We conclude that increased ROS production by mitochondria contributes to age‐associated increased risk of stress‐induced arrhythmia and sudden cardiac death through thiol‐modifications of RyRs. Abstract  Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca2+) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca2+ handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5–9 months) and old (4–6 years) rabbit hearts to test the hypothesis that changes in Ca2+ homeostasis are caused by post‐translational modification of ryanodine receptors (RyRs) by mitochondria‐derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca2+ handling were determined by measuring cytosolic and intra‐sarcoplasmic reticulum (SR) Ca2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age‐related changes in ROS production and mitochondria membrane potential using a ROS‐sensitive dye and a mitochondrial voltage‐sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca2+ load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR‐mediated SR Ca2+ leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β‐adrenergic stimulation, Ca2+ transient amplitude, SR Ca2+ load, and latency of pro‐arrhythmic spontaneous Ca2+ waves (SCWs) were decreased while RyR‐mediated SR Ca2+ leak was increased in cardiomyocytes from old rabbits. Additionally, with β‐adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca2+ leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age‐associated increase of ROS production by mitochondria leads to the thiol‐oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population.

  October 17, 2013   doi: 10.1113/jphysiol.2013.260521   open full text
• Mechanisms contributing to cluster formation in the inferior olivary nucleus in brainstem slices from postnatal mice.
  Mathias Kølvraa, Felix C. Müller, Henrik Jahnsen, Jens C. Rekling.
  The Journal of Physiology. October 16, 2013
  •  One of the two main inputs to the cerebellum consists of climbing fibres from neurons in the inferior olivary nucleus (IO). •  The IO spontaneously forms clusters of co‐active neurons in transverse brainstem slices from 1‐ to 2‐week‐old animals, coinciding with a critical time period in cerebellar development. •  Here, we studied the cluster‐forming mechanisms, and find that randomly occurring spontaneous clusters overlap extensively, and contain 10 to hundreds of IO neurons with an average somatodendritic field size that is slightly smaller than the average IO cluster size. •  Cluster formation is dependent on sodium action potentials and electrical coupling between IO neurons, and may spread with decreasing velocity and may involve active dendritic properties. •  The results help us better understand the basic mechanism underlying cluster formation in the IO, which is an important feature in the generation of patterned input to the cerebellum. Abstract  The inferior olivary nucleus (IO) in in vitro slices from postnatal mice (P5.5–P15.5) spontaneously generates clusters of neurons with synchronous calcium transients, and intracellular recordings from IO neurons suggest that electrical coupling between neighbouring IO neurons may serve as a synchronizing mechanism. Here, we studied the cluster‐forming mechanism and find that clusters overlap extensively with an overlap distribution that resembles the distribution for a random overlap model. The average somatodendritic field size of single curly IO neurons was ∼6400 μm2, which is slightly smaller than the average IO cluster size. Eighty‐seven neurons with overlapping dendrites were estimated to be contained in the principal olive mean cluster size, and about six non‐overlapping curly IO neurons could be contained within the largest clusters. Clusters could also be induced by iontophoresis with glutamate. Induced clusters were inhibited by tetrodotoxin, carbenoxelone and 18β‐glycyrrhetinic acid, suggesting that sodium action potentials and electrical coupling are involved in glutamate‐induced cluster formation, which could also be induced by activation of N‐methyl‐d‐aspartate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors. Spikelets and a small transient depolarizing response were observed during glutamate‐induced cluster formation. Calcium transients spread with decreasing velocity during cluster formation, and somatic action potentials and cluster formation are accompanied by large dendritic calcium transients. In conclusion, cluster formation depends on gap junctions, sodium action potentials and spontaneous clusters occur randomly throughout the IO. The relative slow signal spread during cluster formation, combined with a strong dendritic influx of calcium, may signify that active dendritic properties contribute to cluster formation.

  October 16, 2013   doi: 10.1113/jphysiol.2013.260067   open full text
• Reciprocal regulation of inhibitory synaptic transmission by nicotinic and muscarinic receptors in rat nucleus accumbens shell.
  Kiyofumi Yamamoto, Katsuko Ebihara, Noriaki Koshikawa, Masayuki Kobayashi.
  The Journal of Physiology. October 11, 2013
  •  While cholinergic interneurones are few in number, their axons densely innervate and provide abundant acetylcholine‐containing terminals in the nucleus accumbens (NAc). •  Although cholinergic effects on inhibitory postsynaptic currents (IPSCs) via nicotinic and muscarinic receptors have been reported, the relationship between cholinergic modulation of IPSCs and presynaptic cell subtypes in the NAc has remained elusive. •  Here we show that muscarinic receptor activation suppresses IPSCs in medium spiny neurone (MSN)→MSN connections, whereas nicotinic receptor activation enhances IPSCs in fast‐spiking neurone→MSN connections. •  These reciprocal regulatory mechanisms for IPSCs help us to understand the role of cholinergic processes in physiological and pathophysiological functions of the NAc. Abstract  Medium spiny neurones (MSNs) in the nucleus accumbens (NAc) are the principal neurones whose activities are regulated by GABAergic inputs from MSNs and fast‐spiking interneurones (FSNs). Cholinergic interneurones play important roles in the regulation of activity in MSNs; however, how acetylcholine modulates inhibitory synaptic transmission from MSNs/FSNs to MSNs remains unknown. We performed paired whole‐cell patch‐clamp recordings from MSNs and FSNs in rat NAc shell slice preparations and examined cholinergic effects on unitary inhibitory postsynaptic currents (uIPSCs). Carbachol (1 μm) suppressed uIPSC amplitude by 58.3 ± 8.0% in MSN→MSN connections, accompanied by increases in paired‐pulse ratio and failure rate, suggesting that acetylcholine reduces the probability of GABA release from the synaptic terminals of MSNs. Carbachol‐induced uIPSC suppression was antagonised by 100 μm atropine, and was mimicked by pilocarpine (1 μm) and acetylcholine (1 μm) but not nicotine (1 μm). Application of AM251 slightly reduced carbachol‐induced uIPSC suppression (30.8 ± 8.9%), suggesting an involvement of endocannabinoid signalling in muscarinic suppression of uIPSCs. In contrast, FSN→MSN connections showed that pilocarpine had little effect on the uIPSC amplitude, whereas both nicotine and acetylcholine facilitated uIPSC amplitude, with decreases in failure rate and paired‐pulse ratio, suggesting that nicotine‐induced uIPSC facilitation is mediated by presynaptic mechanisms. Miniature IPSC recordings support these hypotheses of presynaptic cholinergic mechanisms. These results suggest a differential role for muscarinic and nicotinic receptors in GABA release, which depends on presynaptic neuronal subtypes in the NAc shell.

  October 11, 2013   doi: 10.1113/jphysiol.2013.258558   open full text
• Electrotonic suppression of early afterdepolarizations in the neonatal rat ventricular myocyte monolayer.
  Herman D. Himel, Alan Garny, Penelope J. Noble, Raj Wadgoankar, Joseph Savarese, Nian Liu, Gil Bub, Nabil El‐Sherif.
  The Journal of Physiology. October 11, 2013
  •  Early afterdepolarizations (EADs) are a known trigger for arrhythmias, but the effect of surrounding tissue on EADs is poorly understood. •  Neurotoxin anthopleurin‐A (AP‐A) increases action potential duration and gives rise to EADs in isolated myocytes. We investigate the effect of AP‐A on connected networks of cultured cardiac cells. •  We show that EADs are markedly suppressed in well‐coupled neonatal rat ventricular monolayers treated with AP‐A, but reappear when gap junction connectivity is blocked. •  The ability of cell coupling to electrotonically damp EADs is confirmed in a two‐cell simulation where connectivity is systematically varied. •  Taken together, these results suggest that cell–cell coupling can act to suppress EADs in normal cardiac tissue. Results also suggest that EADs may emerge and propagate in poorly coupled tissue. Abstract  Pathologies that result in early afterdepolarizations (EADs) are a known trigger for tachyarrhythmias, but the conditions that cause surrounding tissue to conduct or suppress EADs are poorly understood. Here we introduce a cell culture model of EAD propagation consisting of monolayers of cultured neonatal rat ventricular myocytes treated with anthopleurin‐A (AP‐A). AP‐A‐treated monolayers display a cycle length dependent prolongation of action potential duration (245 ms untreated, vs. 610 ms at 1 Hz and 1200 ms at 0.5 Hz for AP‐A‐treated monolayers). In contrast, isolated single cells treated with AP‐A develop prominent irregular oscillations with a frequency of 2.5 Hz, and a variable prolongation of the action potential duration of up to several seconds. To investigate whether electrotonic interactions between coupled cells modulates EAD formation, cell connectivity was reduced by RNA silencing gap junction Cx43. In contrast to well‐connected monolayers, gap junction silenced monolayers display bradycardia‐dependent plateau oscillations consistent with EADs. Further, simulations of a cell displaying EADs electrically connected to a cell with normal action potentials show a coupling strength‐dependent suppression of EADs consistent with the experimental results. These results suggest that electrotonic effects may play a critical role in EAD‐mediated arrhythmogenesis.

  October 11, 2013   doi: 10.1113/jphysiol.2013.262923   open full text
• Optical determination of intracellular water in apoptotic cells.
  Michael A. Model, Ethan Schonbrun.
  The Journal of Physiology. October 11, 2013
  Abstract  Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport‐of‐intensity equation. At the same time, cell thickness was determined by transmission through an externally applied dye (“transmission‐through‐dye” microscopy); switching between these two imaging modalities was accomplished by simply changing the illumination wavelength. The sets of data thus obtained allow computation of the refractive index and cell water content within individual cells. The method was illustrated using cells treated with apoptotic agents staurosporine and actinomycin D and with necrosis inducer ionomycin. Water imaging allows discrimination between apoptotic volume decrease due to dehydration from that due to detachment of apoptotic bodies and can be used on samples where cell volume determination alone would be difficult or insufficient. This article is protected by copyright. All rights reserved

  October 11, 2013   doi: 10.1113/jphysiol.2013.263228   open full text
• Important considerations for protein analyses using antibody based techniques: Down‐sizing western blotting up‐sizes outcomes.
  Robyn M. Murphy, Graham D. Lamb.
  The Journal of Physiology. October 11, 2013
  Abstract  Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Single cell (fibre) western blotting demonstrates the ability to detect proteins in small sample sizes, 5‐10 μg total mass (1‐3 μg total protein). That is an order of magnitude less than often used. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake western blotting in sample sizes equivalent to single fibre segments is demonstrated. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable. This article is protected by copyright. All rights reserved

  October 11, 2013   doi: 10.1113/jphysiol.2013.263251   open full text
• Glucose utilization rates regulate intake levels of artificial sweeteners.
  Luis A. Tellez, Xueying Ren, Wenfei Han, Sara Medina, Jozélia G. Ferreira, Catherine W. Yeckel, Ivan E. de Araujo.
  The Journal of Physiology. October 10, 2013
  •  Much remains to be determined regarding the physiological signals and brain systems that mediate the attribution of greater reward to sugars compared to artificial sweeteners. •  We show that disruption of glucose utilization in mice produces an enduring inhibitory effect on artificial sweetener intake. •  Consistently, hungry mice shifted their preferences away from artificial sweeteners and in favour of glucose after experiencing glucose in a hungry state. •  Disrupting glucose oxidation suppressed dorsal striatum dopamine efflux during sugar intake. •  Glucose oxidation controls intake levels of sweet tastants by modulating extracellular dopamine levels in dorsal striatum. Abstract  It is well established that animals including humans attribute greater reinforcing value to glucose‐containing sugars compared to their non‐caloric counterparts, generally termed ‘artificial sweeteners’. However, much remains to be determined regarding the physiological signals and brain systems mediating the attribution of greater reinforcing value to sweet solutions that contain glucose. Here we show that disruption of glucose utilization in mice produces an enduring inhibitory effect on artificial sweetener intake, an effect that did not depend on sweetness perception or aversion. Indeed, such an effect was not observed in mice presented with a less palatable, yet caloric, glucose solution. Consistently, hungry mice shifted their preferences away from artificial sweeteners and in favour of glucose after experiencing glucose in a hungry state. Glucose intake was found to produce significantly greater levels of dopamine efflux compared to artificial sweetener in dorsal striatum, whereas disrupting glucose oxidation suppressed dorsal striatum dopamine efflux. Conversely, inhibiting striatal dopamine receptor signalling during glucose intake in sweet‐naïve animals resulted in reduced, artificial sweetener‐like intake of glucose during subsequent gluco‐deprivation. Our results demonstrate that glucose oxidation controls intake levels of sweet tastants by modulating extracellular dopamine levels in dorsal striatum, and suggest that glucose utilization is one critical physiological signal involved in the control of goal‐directed sweetener intake.

  October 10, 2013   doi: 10.1113/jphysiol.2013.263103   open full text
• The emergence of subcellular pacemaker sites for calcium waves and oscillations.
  Michael Nivala, Christopher Y. Ko, Melissa Nivala, James N. Weiss, Zhilin Qu.
  The Journal of Physiology. October 10, 2013
  •  Calcium (Ca2+) is fundamental to biological cell function, and Ca2+ waves generating oscillatory Ca2+ signals are widely observed in many cell types. •  Some experimental studies have shown that Ca2+ waves initiate from random locations within the cell, while other studies have shown that waves occur repetitively from preferred locations (pacemaker sites). •  In both ventricular myocyte experiments and computer simulations of a heterogeneous model of coupled Ca2+ release units (CRUs), we show that Ca2+ waves occur randomly in space and time when the Ca2+ level is low, but as the Ca2+ level increases, waves occur repetitively from the same sites. •  Ca2+ waves are self‐organized dynamics of the CRU network, and the wave frequency strongly depends on CRU coupling. •  Using these results, we develop a theory for the entrainment of random oscillators, which provides a unified explanation for the experimental and computational observations. Abstract  Calcium (Ca2+) waves generating oscillatory Ca2+ signals are widely observed in biological cells. Experimental studies have shown that under certain conditions, initiation of Ca2+ waves is random in space and time, while under other conditions, waves occur repetitively from preferred locations (pacemaker sites) from which they entrain the whole cell. In this study, we use computer simulations to investigate the self‐organization of Ca2+ sparks into pacemaker sites generating Ca2+ oscillations. In both ventricular myocyte experiments and computer simulations of a heterogeneous Ca2+ release unit (CRU) network model, we show that Ca2+ waves occur randomly in space and time when the Ca2+ level is low, but as the Ca2+ level increases, waves occur repetitively from the same sites. Our analysis indicates that this transition to entrainment can be attributed to the fact that random Ca2+ sparks self‐organize into Ca2+ oscillations differently at low and high Ca2+ levels. At low Ca2+, the whole cell Ca2+ oscillation frequency of the coupled CRU system is much slower than that of an isolated single CRU. Compared to a single CRU, the distribution of interspike intervals (ISIs) of the coupled CRU network exhibits a greater variation, and its ISI distribution is asymmetric with respect to the peak, exhibiting a fat tail. At high Ca2+, however, the coupled CRU network has a faster frequency and lesser ISI variation compared to an individual CRU. The ISI distribution of the coupled network no longer exhibits a fat tail and is well‐approximated by a Gaussian distribution. This same Ca2+ oscillation behaviour can also be achieved by varying the number of ryanodine receptors per CRU or the distance between CRUs. Using these results, we develop a theory for the entrainment of random oscillators which provides a unified explanation for the experimental observations underlying the emergence of pacemaker sites and Ca2+ oscillations.

  October 10, 2013   doi: 10.1113/jphysiol.2013.259960   open full text
• Stomatin‐domain protein interactions with acid‐sensing ion channels modulate nociceptor mechanosensitivity.
  Rabih A. Moshourab, Christiane Wetzel, Carlos Martinez‐Salgado, Gary R. Lewin.
  The Journal of Physiology. October 09, 2013
  •  Gene deletion studies revealed that membrane proteins stomatin and STOML3, as well as the acid‐sensing ion channels ASIC2 and ASIC3, regulate mechanosensory transduction. •  Both stomatin and STOML3 interact with ASIC proteins and we asked if deletion of two interacting proteins has a more than additive effect on the mechanosensitivity of cutaneous sensory afferents. •  A detailed electrophysiological comparison of sensory afferent phenotypes observed in asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− mutant mice compared to their respective single gene mutants revealed especially strong effects on the mechanosensitivity of thinly myelinated mechanonociceptors in double mutants. •  Deletion of the asic3 gene or pharmacological blockade of this channel decreased adaptation rates specifically in rapidly adapting mechanoreceptors, an effect not exacerbated by deletion of stomatin‐domain genes. •  This study reveals that loss of stomatin–ASIC interactions can have profound effects on mechanosensitivity in specific subsets of skin afferents; interfering with such interactions could have potential for treating mechanical pain. Abstract  Acid‐sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ‐ and C‐fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ‐nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:stomatin−/− mutant mice many Aδ‐nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or stomatin−/− mice. Examination of stomatin−/−:stoml3−/− mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ‐nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.

  October 09, 2013   doi: 10.1113/jphysiol.2013.261180   open full text
• Correlation of cellular expression with function of NO‐sensitive guanylyl cyclase in the murine lower urinary tract.
  Barbara Lies, Dieter Groneberg, Andreas Friebe.
  The Journal of Physiology. October 09, 2013
  •  Diseases of the lower urinary tract are associated with dysfunctions of cellular mechanisms that regulate smooth muscle tone. Nitric oxide (NO) mediates relaxation of most smooth muscle‐containing tissues via NO‐sensitive guanylyl cyclase (NO‐GC). Correlation of cellular localization with function of NO‐GC in the murine lower urinary tract has not been previously performed. •  Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed exclusively in smooth muscle cells of the urethral sphincter and mediates NO‐induced relaxation. •  In bladder detrusor, NO‐GC is not detected in smooth muscle cells but rather in platelet‐derived growth factor receptor α‐positive interstitial cells. NO‐GC in these cells does not contribute to NO‐induced relaxation; therefore, bladder detrusor smooth muscle appears to be unique as it is not relaxed by NO. •  The correlation of NO‐GC localization and function regarding smooth muscle relaxation allows the clinical use of compounds acting within NO/cGMP signalling to be assessed. Abstract  The action of nitric oxide (NO) to stimulate NO‐sensitive guanylyl cyclase (NO‐GC), followed by production of cGMP, and eventually to cause smooth muscle relaxation is well known. In the lower urinary tract (LUT), in contrast to the cardiovascular system and the gastrointestinal tract, specific localization in combination with function of NO‐GC has not been investigated to date. Consequently, little is known about the mechanisms regulating relaxation of the lower urinary tract in general and the role of NO‐GC‐expressing cells in particular. To study the distribution and function of NO‐GC in the murine lower urinary tract, we used internal urethral sphincter and bladder detrusor from global (GCKO) and smooth muscle cell‐specific (SM‐GCKO) NO‐GC knock‐out mice for immunohistochemical analyses and organ bath experiments. In urethral sphincter, NO‐GC‐positive immunofluorescence was confined to smooth muscle cells (SMCs). Deletion of NO‐GC in SMCs abolished NO‐induced relaxation. In bladder detrusor, exposure to NO did not cause relaxation although immunohistochemistry uncovered the existence of NO‐GC in the tissue. In contrast to the urethral sphincter, expression of NO‐GC in bladder detrusor was limited to platelet‐derived growth factor receptor α (PDGFRα)‐positive interstitial cells. In conclusion, NO‐GC found in SMCs of the urethral sphincter mediates NO‐induced relaxation; bladder detrusor is unique as NO‐GC is not expressed in SMCs and, thus, NO does not induce relaxation. Nevertheless, NO‐GC expression was found in PDGFRα‐positive interstitial cells of the murine bladder with an as yet unknown function. Further investigation is needed to clarify the role of NO‐GC in the detrusor.

  October 09, 2013   doi: 10.1113/jphysiol.2013.262410   open full text
• Neural circuits in movement control.
  Henrik Jörntell.
  The Journal of Physiology. October 08, 2013
  Abstract  This symposium took place at Trolleholm Castle near Lund on May 27–28, 2011. The meeting was a celebration of the lifetime achievements of Carl‐Fredrik Ekerot, who is now retired. The meeting drew together participants from many different disciplines of motor systems neuroscience, but with a focus on the cerebellum and the spinocerebellar systems, which was the main field of interest of Carl‐Fredrik Ekerot. Ekerot pioneered the field of climbing fibre microzones in the forelimb area of the C3 zone (Ekerot & Larson, 1980; Ekerot et al., 1991) and the role of the microzones as a functional unit in the cerebellar cortex and in the cerebellar nuclei (Garwicz & Ekerot, 1994; Ekerot et al., 1995), provided early evidence of the congruence between mossy fibre and climbing fibre inputs in the cerebellar cortex (Ekerot & Larson, 1973, 1980; Garwicz et al., 1998), made important contributions to the early days of cerebellar plasticity research centred on the classical climbing‐fibre dependent LTD of parallel fibre synapses on Purkinje cells (Ekerot & Kano, 1985, 1989), later discovered of several new forms of plasticity between the parallel fibres and the interneurons and Purkinje cells (Ekerot & Jorntell, 2001; Jorntell & Ekerot, 2002) and made a unique and detailed series of studies on the spinal and motor information processed by the cells of the lateral reticular nucleus (Clendenin et al., 1974a; Clendenin et al., 1974c, b; Ekerot, 1990c, b, a), an important source of mossy fibres to the cerebellum. This article is protected by copyright. All rights reserved

  October 08, 2013   doi: 10.1113/jphysiol.2013.265603   open full text
• Is this my finger? Proprioceptive illusions of body ownership and representation.
  Martin E. Héroux, Lee D. Walsh, Annie A. Butler, Simon C. Gandevia.
  The Journal of Physiology. October 04, 2013
  •  The brain keeps a representation of which things are part of our body. This sense of ownership is easily manipulated using brushing of the skin or movement of a limb to create an illusion of ownership over an inanimate object, such as a rubber hand. •  We induced a sense of ownership of an artificial finger using movement of the index finger without vision of the hands. As cutaneous receptors had been anaesthetised, this illusion depended on proprioceptive signals from muscle receptors. •  In addition, we found a new grasp illusion in which perceived distance between the index fingers decreases when subjects hold an artificial finger. •  These results increase understanding of how the brain generates our body representation and may help in understanding diseases in which the sense of ownership is disrupted. Abstract  Body ‘ownership’ defines which things belong to us and can be manipulated by signals from cutaneous or muscle receptors. Whether signals from muscle proprioceptors on their own influence perceived ownership is unknown. We used finger‐joint movement to induce illusory ownership of an artificial finger without vision. We coupled the subject's index finger to an artificial finger 12 cm above it. The experimenter held the subject's other index finger and thumb on the artificial finger and passively moved them congruently or incongruently for 3 min with the index finger and the grasping index finger and thumb intact or anaesthetised. When intact, congruent movement (19 subjects) reduced perceived vertical distance between index fingers to 1.0 (0.0, 2.0) cm [median (IQR)] from 3.0 (3.0, 4.0) cm with incongruent movement (P < 0.01). Simply grasping the artificial finger reduced perceived spacing between the grasping and test index fingers from 6.0 (5.0, 9.0) cm to 3.0 (3.0, 6.0) cm (P < 0.01), a new grasp illusion. Digital anaesthesia eliminated this grasp effect, after which congruent movement still reduced the perceived spacing between the index fingers to 1.0 (0.0, 2.75) cm compared to 4.0 (3.25, 6.0) cm with incongruent movement (P < 0.001). Subjects more strongly agreed that they were holding their own finger after congruent but not incongruent movement (P < 0.01). We propose that the brain generates possible scenarios and tests them against available sensory information. This process can function without vision or motor commands, and with only one channel of somatic information.

  October 04, 2013   doi: 10.1113/jphysiol.2013.261461   open full text
• Texture‐dependent motion signals in primate middle temporal area.
  Saba Gharaei, Chris Tailby, Selina S. Solomon, Samuel G. Solomon.
  The Journal of Physiology. October 04, 2013
  •  The receptive fields of neurons in the middle temporal (MT) area of primate visual cortex are an important stage in motion analysis. Some neurons in MT (pattern cells) can signal motion independent of contour orientation, but others (component cells) cannot; there is no systematic account of how responses in area MT depend on the spatial structure of images. •  We measured the extracellular response of neurons in area MT of anaesthetised marmoset monkeys to synthetic textures and natural images. •  Direction tuning of pattern cells was broad and largely stable against variation in spatial texture. Direction tuning of component cells was narrower than that of pattern cells when spatial textures contained few orientations, but tuning was not stable against variation in spatial texture. •  Response variability in all neurons was lower for rich spatial texture. •  Pattern and component cells may provide parallel analyses for motion vision. Abstract  Neurons in the middle temporal (MT) area of primate cortex provide an important stage in the analysis of visual motion. For simple stimuli such as bars and plaids some neurons in area MT – pattern cells – seem to signal motion independent of contour orientation, but many neurons – component cells – do not. Why area MT supports both types of receptive field is unclear. To address this we made extracellular recordings from single units in area MT of anaesthetised marmoset monkeys and examined responses to two‐dimensional images with a large range of orientations and spatial frequencies. Component and pattern cell response remained distinct during presentation of these complex spatial textures. Direction tuning curves were sharpest in component cells when a texture contained a narrow range of orientations, but were similar across all neurons for textures containing all orientations. Response magnitude of pattern cells, but not component cells, increased with the spatial bandwidth of the texture. In addition, response variability in all neurons was reduced when the stimulus was rich in spatial texture. Fisher information analysis showed that component cells provide more informative responses than pattern cells when a texture contains a narrow range of orientations, but pattern cells had more informative responses for broadband textures. Component cells and pattern cells may therefore coexist because they provide complementary and parallel motion signals.

  October 04, 2013   doi: 10.1113/jphysiol.2013.257568   open full text
• Blood pressure and the contractility of a human leg muscle.
  Billy L. Luu, Richard C. Fitzpatrick.
  The Journal of Physiology. October 04, 2013
  •  At identical activation, muscle force production in a small, non‐postural hand muscle that normally operates near heart level is sensitive to physiological changes in perfusion pressure. •  Here we investigate how perfusion pressure affects muscle contractility in a large leg muscle that is subject to high hydrostatic pressures in its normal role. •  We show that this large postural muscle and the small hand muscle have similar perfusion–contractility relationships, and that changes in local perfusion and systemic blood pressure produce proportionately the same effects on muscle contractility. •  The rapid and reversible modulation of contractility by perfusion pressure may be explained by interstitial [K+] that depends on the balance between energy‐dependent processes in the myocyte and passive vascular removal. •  These results help explain how blood pressure is controlled through the pressor response during exercise and how muscle force production and fatigue varies in different postures. Abstract  These studies investigate the relationships between perfusion pressure, force output and pressor responses for the contracting human tibialis anterior muscle. Eight healthy adults were studied. Changing the height of tibialis anterior relative to the heart was used to control local perfusion pressure. Electrically stimulated tetanic force output was highly sensitive to physiological variations in perfusion pressure showing a proportionate change in force output of 6.5% per 10 mmHg. This perfusion‐dependent change in contractility begins within seconds and is reversible with a 53 s time constant, demonstrating a steady‐state equilibrium between contractility and perfusion pressure. These stimulated contractions did not produce significant cardiovascular responses, indicating that the muscle pressor response does not play a major role in cardiovascular regulation at these workloads. Voluntary contractions at forces that would require constant motor drive if perfusion pressure had remained constant generated a central pressor response when perfusion pressure was lowered. This is consistent with a larger cortical drive being required to compensate for the lost contractility with lower perfusion pressure. The relationship between contractility and perfusion for this large postural muscle was not different from that of a small hand muscle (adductor pollicis) and it responded similarly to passive peripheral and active central changes in arterial pressure, but extended over a wider operating range of pressures. If we consider that, in a goal‐oriented motor task, muscle contractility determines central motor output and the central pressor response, these results indicate that muscle would fatigue twice as fast without a pressor response. From its extent, timing and reversibility we propose a testable hypothesis that this change in contractility arises through contraction‐ and perfusion‐dependent changes in interstitial K+ concentration.

  October 04, 2013   doi: 10.1113/jphysiol.2013.261107   open full text
• Increases in intracellular pH facilitate endocytosis and decrease availability of voltage‐gated proton channels in osteoclasts and microglia.
  Hiromu Sakai, Guangshuai Li, Yoshiko Hino, Yoshie Moriura, Junko Kawawaki, Makoto Sawada, Miyuki Kuno.
  The Journal of Physiology. October 03, 2013
  Abstract  Voltage‐gated proton channels (H+ channels) are highly proton‐selective transmembrane pathways. Although the primary determinants for activation are the pH‐ and voltage‐gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole‐cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H+ channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H+ channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage‐dependence for activation, were unchanged, the decreases in the H+ channel conductance were likely due to the decreases in H+ channels available at the plasma membrane. The decreases in the H+ channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker, FM1–43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V‐ATPases and H+ channels, pHi increases by these H+‐transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H+ channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin‐dependent endocytosis. This article is protected by copyright. All rights reserved

  October 03, 2013   doi: 10.1113/jphysiol.2013.263558   open full text
• Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non‐fast‐spiking GABAergic hippocampal interneurons using two‐photon laser microscopy and high‐ and low‐affinity dyes.
  Máté Kisfali, Tibor Lőrincz, E. Sylvester Vizi.
  The Journal of Physiology. September 30, 2013
  •  Previously, the use of two‐photon scanning microscopy to characterize Ca2+ transients at the individual bouton level has been primarily limited to glutamatergic terminals. •  No previous study has attempted to record Ca2+ dynamics in individual boutons in response to somatic stimulation or to compare them with the Ca2+ dynamics recorded in the dendrites of the same GABAergic interneurons located in the CA1 area of the rat hippocampus. In addition, the endogenous buffer capacity that affects the decay of transients was also estimated. •  This study was limited to interneurons that responded to somatic current stimulation at frequencies <60 Hz and that had cell bodies located in the stratum radiatum. •  The amplitudes of the Ca2+ transients and changes in [Ca2+]i that occurred in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP). These data were calculated as unperturbed values, which excluded the modulatory effect of the dye's buffer capacity and the possible failure to reach equilibrium in dye concentrations in dendrites and boutons. Therefore, the higher density of Ca2+ channels expressed in boutons might account for the nine‐fold difference in Δ[Ca2+]i observed in boutons and dendrites. •  Our results indicate that care should be taken when using high‐ and low‐affinity dyes to measure [Ca2+]i in boutons to avoid saturation and the erroneous calculation of [Ca2+]i. Abstract  Using two‐photon laser microscopy, high‐ and low‐affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation‐evoked Ca2+ transients simultaneously in the dendrites and axonal boutons of the same non‐fast‐spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca2+ transients and changes in [Ca2+]i in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP), leading to the conclusion that the much greater influx of Ca2+ observed in terminals might be due to a higher density of N‐type voltage‐sensitive Ca2+ channels compared to the L‐type channels present in dendrites. Whereas the decay of Ca2+ transients recorded in dendrites was primarily mono‐exponential, the decay in boutons was bi‐exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca2+ was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca2+]i, we assessed the limits of [Ca2+] measurements (which ranged between 6 and 82% of the applied dye saturation) when high‐ and low‐affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low‐affinity indicators OGB‐6F (KD= 3.0 μm) and OGB‐5N (KD= 20 μm) were able to accurately reflect the changes in ΔF/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (κE), which can shape Ca2+ signals, calculated in dendrites (κE= 354) or boutons (κE= 458).

  September 30, 2013   doi: 10.1113/jphysiol.2013.258863   open full text
• Inner retinal inhibition shapes the receptive field of retinal ganglion cells in primate.
  D.A. Protti, S. Di Marco, J.Y. Huang, C.R. Vonhoff, V. Nguyen, S.G. Solomon.
  The Journal of Physiology. September 30, 2013
  Abstract  The centre–surround organization of receptive fields is a feature of most retinal ganglion cells (RGCs) that is critical for spatial discrimination and contrast detection. Although lateral inhibitory processes are known to be important in generating the receptive field surround, the contribution of each of the two synaptic layers in the primate retina remains unclear. Here we studied the spatial organisation of excitatory and inhibitory synaptic inputs onto ON and OFF ganglion cells in the primate retina. All RGCs showed increase in excitation in response to stimulus of preferred polarity. Inhibition onto RGCs comprised two types of responses to preferred polarity: some RGCs showed increase in inhibition whilst others showed removal of tonic inhibition. Excitatory inputs were strongly spatially tuned but inhibitory inputs showed more variable organisation: in some neurons they were as strongly tuned as excitation, and in others inhibitory inputs showed no spatial tuning. We targeted one source of inner retinal inhibition by functionally ablating spiking amacrine cells with bath application of tetrodotoxin (TTX). TTX significantly reduced the spatial tuning of excitatory inputs. In addition, TTX reduced inhibition onto those RGCs where a stimulus of preferred polarity increased inhibition. Reconstruction of the spatial tuning properties by somatic injection of excitatory and inhibitory synaptic conductances verified that TTX‐mediated inhibition onto bipolar cells increases the strength of the surround in RGC spiking output. These results indicate that in the primate retina inhibitory mechanisms in the inner plexiform layer sharpen the spatial tuning of ganglion cells. This article is protected by copyright. All rights reserved

  September 30, 2013   doi: 10.1113/jphysiol.2013.257352   open full text
• The spinal reflex cannot be perceptually separated from voluntary movements.
  Arko Ghosh, Patrick Haggard.
  The Journal of Physiology. September 30, 2013
  Abstract  Both voluntary and involuntary movements activate sensors in the muscles, skin, tendon and joints. Since limb movement can result from a mixture of spinal reflexes and voluntary motor commands, the cortical centres underlying conscious proprioception might either aggregate or separate the sensory inputs generated by voluntary movements from those generated by involuntary movements such as spinal reflexes. We addressed whether healthy volunteers could perceive the contribution of a spinal reflex during movements that combined both reflexive and voluntary contributions. Volunteers reported the reflexive contribution in leg movements that were partly driven by the knee jerk reflex induced by a patellar tendon tap and partly by voluntary motor control. In one condition, participants were instructed to kick back in response to a tendon tap. The results were compared to reflexes in a resting baseline condition without voluntary movement. In a further condition, particpants were instructed to kick forwards after a tap. Volunteers reported the perceived reflex contribution by repositioning the leg to the perceived maximum displacement to which the reflex moved the leg after each tendon tap. In the resting baseline condition, the reflex was accurately perceived. We found a near‐unity slope of linear regressions of perceived on actual reflexive displacement. Both the slope value, and the quality of regression fit in individual volunteers, was significantly reduced when volunteers were instructed to generate voluntary backward kicks as soon as they detected the tap. In the kick forward condition, kinematic analysis showed continuity of reflex and voluntary movements, but the reflex contribution could be estimated from EMG amplitude on each trial. Again, participants’ judgements of reflexes showed a poor relation to reflex EMG, in contrast to the baseline condition. In sum, we show that reflexes can be accurately perceived from afferent information. However, the presence of voluntary movement significantly impairs reflex perception. We suggest that perceptual separation between voluntary and reflex movement is poor at best. Our results imply that the brain has no clear marker for perceptually separating voluntary and involuntary movement. Attribution of body movement to voluntary or involuntary motor commands is surprisingly poor when both are present. This article is protected by copyright. All rights reserved

  September 30, 2013   doi: 10.1113/jphysiol.2013.260588   open full text
• Chronic renin inhibition lowers blood pressure and reduces upright muscle sympathetic nerve activity in hypertensive seniors.
  Yoshiyuki Okada, Sara S. Jarvis, Stuart A. Best, Tiffany B. Bivens, Beverley Adams‐Huet, Benjamin D. Levine, Qi Fu.
  The Journal of Physiology. September 30, 2013
  Abstract  Cardiovascular risk remains high in patients with hypertension even with adequate blood pressure (BP) control. One possible mechanism may be sympathetic activation via the baroreflex. We tested the hypothesis that chronic inhibition of renin reduces BP without sympathetic activation, but diuresis augments sympathetic activity in elderly hypertensives. Fourteen patients with stage‐I hypertension [66±5 (SD) years] were treated with a direct renin inhibitor, aliskiren, (n = 7) or a diuretic, hydrochlorothiazide, (n = 7) for 6 months. Muscle sympathetic nerve activity (MSNA), BP, direct renin and aldosterone were measured during supine and a graded head‐up tilt (HUT; 5‐min 30° and 20‐min 60°), before and after treatment. Sympathetic baroreflex sensitivity (BRS) was assessed. Both groups had similar BP reductions after treatment (all P<0.01), while MSNA responses were different between hydrochlorothiazide and aliskiren (P = 0.006 pre/post×drug). Both supine and upright MSNA became greater after hydrochlorothiazide treatment (supine, 72±18 post vs. 64±15 pre; 60° HUT, 83±10 vs. 78±13 bursts·100beats−1; P = 0.002). After aliskiren treatment, supine MSNA remained unchanged (69±13 vs. 64±8 bursts·100beats−1), but upright MSNA was lower (74±15 vs. 85±10 bursts·100beats−1; P = 0.012 for pre/post×posture). Direct renin was greater after both treatments (both P<0.05), while upright aldosterone was greater after hydrochlorothiazide only (P = 0.002). The change in upright MSNA by the treatment was correlated with the change of aldosterone (r = 0.74, P = 0.002). Upright sympathetic BRS remained unchanged after either treatment. Thus, chronic renin inhibition may reduce upright MSNA through suppressed renin activity, while diuresis may evoke sympathetic activation via the upregulated renin‐angiotensin‐aldosterone system, without changing intrinsic sympathetic baroreflex function in elderly hypertensive patients. This article is protected by copyright. All rights reserved

  September 30, 2013   doi: 10.1113/jphysiol.2013.261362   open full text
• HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition.
  Arianna Rinaldi, Cagla Defterali, Antoine Mialot, Derek L. F. Garden, Mathieu Beraneck, Matthew F. Nolan.
  The Journal of Physiology. September 30, 2013
  •  Purkinje cells in the cerebellum are important for motor learning and have electrical signalling properties determined by several different types of ion channel. •  Using a restricted genetic deletion, we investigate the roles of HCN1 ion channels expressed by cerebellar Purkinje cells. •  This deletion causes specific learning impairments in a subset of behaviours to which Purkinje cells contribute. •  At a cellular level this specificity of function is mirrored by increases in the duration of responses to inhibitory synaptic input, without changes in responses to excitatory synaptic input activated in the absence of inhibition. •  The results help us to understand how behaviours are influenced by ion channels important for aspects of computation in a defined neuronal cell type. Abstract  Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type‐specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo‐ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour.

  September 30, 2013   doi: 10.1113/jphysiol.2013.259499   open full text
• Sign‐preserving and sign‐inverting synaptic interactions between rod and cone photoreceptors in the dark‐adapted retina.
  Fan Gao, Ji‐Jie Pang, Samuel M. Wu.
  The Journal of Physiology. September 30, 2013
  •  Five types of rods and cones in the dark‐adapted salamander retina are electrically coupled with linear and symmetrical junctional conductances Gj of different average values. •  The average Gj values of the five types of rod–cone pairs recorded at day and night times suggest that the the circadian‐dependent changes in rod–cone coupling observed in the fish and rodent retinas are not present in the tiger salamander. •  In addition to rod–cone coupling, there is a sign‐inverting, unidirectional rod→cone current IRC, and the current–voltage (IRC–VCone) relations are linear, with a reversal potential near the chloride reversal potential ECl. •  I RC can be observed in rods and cones separated by at least 260 μm, and its waveform resembles that of the rod‐elicited horizontal cell (HC) response IHC; a glutamate transporter‐associated chloride channel blocker TBOA suppresses IRC without affecting IHC. •  These results suggest that IRC is largely mediated by HCs via a sign‐inverting feedback chemical synapse associated with a chloride channel in cones. Abstract  We show that various types of rods and cones in the dark‐adapted salamander retina are electrically coupled with linear and symmetrical junctional conductances Gj (40–223 pS) and a rank order: RodC–large single cone, rod–large single cone, rod–small single cone, rod–accessory double cone and rod–principal double cone. By systematically comparing the transjunctional current–voltage (Ij–Vj) relations and average Gj values of the five types of rod–cone pairs recorded at day and night times, our results suggest that the differences in Gj values among various types of rod–cone pairs are not caused by circadian differences, and the circadian‐dependent changes in rod–cone coupling observed in the fish and rodent retinas are not present in the tiger salamander. In addition to rod–cone coupling, there is a sign‐inverting, unidirectional rod→cone current IRC, and the IRC–VCone relations are linear, with a reversal potential near the chloride reversal potential ECl. IRC can be observed in rods and cones separated by at least 260 μm, and its waveform resembles that of the rod‐elicited horizontal cell (HC) response IHC. A glutamate transporter‐associated chloride channel blocker TBOA suppresses IRC but not IHC. These results suggest that IRC is largely mediated by HCs via a sign‐inverting feedback chemical synapse associated with a chloride channel. IRC significantly reduced rod→cone coupling in the frequency range below 15 Hz, allowing better separation of rod and cone signals in the dark‐adapted retina.

  September 30, 2013   doi: 10.1113/jphysiol.2013.260984   open full text
• Long‐lasting hyperpolarization underlies seizure reduction by low frequency deep brain electrical stimulation.
  Sheela Toprani, Dominique M. Durand.
  The Journal of Physiology. September 25, 2013
  •  Deep brain electrical stimulation (DBS) is a promising treatment for mesial temporal lobe epilepsy (MTLE). However, treatment optimization and clinical application are limited by the fact that the mechanisms of seizure reduction by electrical stimulation remain unknown. •  We have shown that low frequency electrical stimulation (LFS) of a white matter target connecting the hippocampi effectively reduces chemically induced epileptic activity in bilateral hippocampi. •  LFS induces long‐lasting hyperpolarization (1–2 s) in the inter‐stimulus interval that protects cells from seizure activity. •  This long‐lasting hyperpolarization is mediated by (1) GABAB IPSPs and (2) the slow afterhyperpolarization (sAHP). Its magnitude, as measured by amplitude and area, is correlated with LFS efficacy of seizure reduction. •  Understanding the mechanisms of LFS could have therapeutic applications for seizure reduction in patients with MTLE. Abstract  Mesial temporal lobe epilepsy (MTLE) is a common medically refractory neurological disease. Deep brain electrical stimulation (DBS) of grey matter has been used for MTLE with limited success. However, stimulation of a white matter tract connecting the hippocampi, the ventral hippocampal commissure (VHC), with low frequencies that simulate interictal discharges has shown promising results, with seizure reduction greater than 98% in bilateral hippocampi during stimulation and greater than 50% seizure reduction in bilateral hippocampi after treatment. A major hurdle to the implementation and optimization of this treatment is that the mechanisms of seizure reduction by low frequency electrical stimulation (LFS) are not known. The goal of this study is to understand how commissural fibre tract stimulation reduces bilateral hippocampal epileptic activity in an in vitro slice preparation containing bilateral hippocampi connected by the VHC. It is our hypothesis that electrical stimuli induce hyperpolarization lasting hundreds of milliseconds following each pulse which reduces spontaneous epileptic activity during each inter‐stimulus interval (ISI). Stimulus‐induced long‐lasting‐hyperpolarization (LLH) can be mediated by GABAB inhibitory post‐synaptic potentials (IPSPs) or slow after‐hyperpolarization (sAHP). To test the role of LLH in effective bilateral seizure reduction by fibre tract stimulation, we measured stimulus‐induced hyperpolarization during LFS of the VHC using electrophysiology techniques. Antagonism of the GABAB IPSP and/or sAHP diminished stimulus‐induced hyperpolarization concurrently with LFS efficacy (greater than 50% reduction). Blocking both the GABAB IPSP and sAHP simultaneously eliminated the effect of electrical stimulation on seizure reduction entirely. These data show that LFS of the VHC is an effective protocol for bilateral hippocampal seizure reduction and that its efficacy relies on the induction of long‐lasting hyperpolarization mediated through GABAB IPSPs and sAHP. Based on this study, optimization of the timing of LFS and LFS‐induced‐LLH may lead to improved outcomes from DBS treatments for human epilepsy.

  September 25, 2013   doi: 10.1113/jphysiol.2013.253757   open full text
• Influence of spiking activity on cortical local field potentials.
  Stephan Waldert, Roger N. Lemon, Alexander Kraskov.
  The Journal of Physiology. September 25, 2013
  •  The intra‐cortical local field potential (LFP) reflects a variety of electrophysiological processes and is a fundamental signal used to enhance knowledge about neuroscience. •  For most investigations, spike‐free LFPs are mandatory for valid conclusions, but spikes can contaminate LFPs and falsify findings despite low‐pass filtering or other attempts to remove spiking activity from LFPs. The extent of this fundamental problem remains unclear. •  Using spikes recorded in the awake monkey, we revealed how spike amplitude, spike duration, firing rate and noise statistic influence the extent to which spikes contaminate LFPs. •  Contamination varies with these parameters and can affect LFPs down to around 10 Hz; below this it is theoretically possible but unlikely. LFP frequencies up to the (high‐) gamma band can remain unaffected, but signals above must always be carefully analysed. •  We propose a method to reveal modulations in spectrograms, which also allows the detection of spike contamination, and provide a systematic guide to assess spike contamination of intra‐cortical LFPs. Abstract  The intra‐cortical local field potential (LFP) reflects a variety of electrophysiological processes including synaptic inputs to neurons and their spiking activity. It is still a common assumption that removing high frequencies, often above 300 Hz, is sufficient to exclude spiking activity from LFP activity prior to analysis. Conclusions based on such supposedly spike‐free LFPs can result in false interpretations of neurophysiological processes and erroneous correlations between LFPs and behaviour or spiking activity. Such findings might simply arise from spike contamination rather than from genuine changes in synaptic input activity. Although the subject of recent studies, the extent of LFP contamination by spikes is unclear, and the fundamental problem remains. Using spikes recorded in the motor cortex of the awake monkey, we investigated how different factors, including spike amplitude, duration and firing rate, together with the noise statistic, can determine the extent to which spikes contaminate intra‐cortical LFPs. We demonstrate that such contamination is realistic for LFPs with a frequency down to ∼10 Hz. For LFP activity below ∼10 Hz, such as movement‐related potential, contamination is theoretically possible but unlikely in real situations. Importantly, LFP frequencies up to the (high‐) gamma band can remain unaffected. This study shows that spike–LFP crosstalk in intra‐cortical recordings should be assessed for each individual dataset to ensure that conclusions based on LFP analysis are valid. To this end, we introduce a method to detect and to visualise spike contamination, and provide a systematic guide to assess spike contamination of intra‐cortical LFPs.

  September 25, 2013   doi: 10.1113/jphysiol.2013.258228   open full text
• Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+‐sensitive K+ channels in rat mesenteric artery.
  Owain Llŷr Roberts, Tomoko Kamishima, Richard Barrett‐Jolley, John M. Quayle, Caroline Dart.
  The Journal of Physiology. September 25, 2013
  •  Relaxation of vascular smooth muscle, which increases blood vessel diameter, is often mediated through vasodilator‐induced elevations of intracellular 3′‐5′‐cyclic adenosine monophosphate (cAMP), although the mechanisms are incompletely understood. •  In this study we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. •  We show that Epac mediates vasorelaxation in mesenteric arteries by facilitating the opening of several subtypes of Ca2+‐sensitive K+ channel within the endothelium and on vascular smooth muscle. •  Epac‐mediated hyperpolarization of the smooth muscle membrane brought about by opening of these channels acts to limit Ca2+ entry via voltage‐gated Ca2+ channels leading to vasorelaxation. •  This represents a potentially important, previously uncharacterised mechanism through which vasodilator‐induced elevation of cAMP can regulate vascular tone and thus blood flow. Abstract  Vasodilator‐induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac‐selective cAMP analogue 8‐pCPT‐2′‐O‐Me‐cAMP‐AM (5 μm, subsequently referred to as 8‐pCPT‐AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine‐contracted arteries over a 5 min period (mean ± SEM; n= 6). 8‐pCPT‐AM induced only a 16.7 ± 2.4% relaxation in arteries pre‐contracted with high extracellular K+ over the same time period (n= 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca2+‐sensitive, large‐conductance K+ (BKCa) channel opening as iberiotoxin (100 nm) significantly reduced the ability of 8‐pCPT‐AM to reverse phenylephrine‐induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8‐pCPT‐AM, n= 5; P < 0.05). 8‐pCPT‐AM increased Ca2+ spark frequency in Fluo‐4‐AM‐loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s‐1μm‐1 (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s−1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n= 7; P < 0.05). 8‐pCPT‐AM‐activated STOCs were sensitive to iberiotoxin (100 nm) and to ryanodine (30 μm). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n= 10) hyperpolarization in response to 8‐pCPT‐AM that was sensitive to iberiotoxin (n= 5). Endothelial disruption suppressed 8‐pCPT‐AM‐mediated relaxation in phenylephrine‐contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n= 5; P < 0.05), as did apamin and TRAM‐34, blockers of Ca2+‐sensitive, small‐ and intermediate‐conductance K+ (SKCa and IKCa) channels, respectively, and NG‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). In Fluo‐4‐AM‐loaded mesenteric endothelial cells, 8‐pCPT‐AM induced a sustained increase in global Ca2+. Our data suggest that Epac hyperpolarizes smooth muscle by (1) increasing localized Ca2+ release from ryanodine receptors (Ca2+ sparks) to activate BKCa channels, and (2) endothelial‐dependent mechanisms involving the activation of SKCa/IKCa channels and NOS. Epac‐mediated smooth muscle hyperpolarization will limit Ca2+ entry via voltage‐sensitive Ca2+ channels and represents a novel mechanism of arterial relaxation.

  September 25, 2013   doi: 10.1113/jphysiol.2013.262006   open full text
• Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1.
  Samer Al‐Samir, Symeon Papadopoulos, Renate J. Scheibe, Joachim D. Meißner, Jean‐Pierre Cartron, William S. Sly, Seth L. Alper, Gerolf Gros, Volker Endeward.
  The Journal of Physiology. September 24, 2013
  •  Controversial results have been reported on the hypothesis that the cytosolic carbonic anhydrase II (CAII) of the red cell is largely bound to the cell's Cl−/HCO3− exchanger AE1, forming a ‘metabolon complex’ that greatly enhances transport activity of AE1. •  In examining so far untested aspects of this hypothesis, we report that fluorophore‐labelled AE1 and CAII proteins, expressed in tsA201 cells, neither colocalize at the cell membrane nor show close proximity by Förster resonance emission spectroscopy. •  Antibody against Flag‐tagged AE1 expressed in tsA201 cells co‐immunoprecipitates coexpressed ankyrin but not CAII. •  CAII‐deficient human red blood cells with substantial CAI activity exhibit HCO3− permeabilities identical to those of normal red cells. •  A mathematical model of CO2/HCO3− transport of red cells indicates that this process occurs more rapidly when the CA of the cell is distributed homogeneously across the cytoplasm rather than being bound to the membrane interior. Abstract  We have investigated the previously published ‘metabolon hypothesis’ postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII‐CyPet and YPet‐AE1. In these doubly transfected tsA201 cells, YPet‐AE1 is clearly associated with the cell membrane, whereas CAII‐CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet‐AE1 and CAII‐CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag‐antibody against Flag‐tagged AE1 expressed in tsA201 cells, which does not co‐precipitate native CAII but co‐precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3− permeability () sensitive to inhibition by 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid. Expression of the non‐catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N‐terminally truncated CAII lacking the proposed site of interaction with the C‐terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild‐type CAII. These variously co‐transfected tsA201 cells exhibit a positive correlation between cellular and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII–AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII–AE1 metabolon to erythroid AE1‐mediated HCO3− transport was further tested in normal red cells and red cells from CAII‐deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1‐binding sequence. Erythroid was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3− and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H+ makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.

  September 24, 2013   doi: 10.1113/jphysiol.2013.251181   open full text
• Activity‐dependent downregulation of D‐type K+ channel subunit Kv1.2 in rat hippocampal CA3 pyramidal neurons.
  Jung Ho Hyun, Kisang Eom, Kyu‐Hee Lee, Won‐Kyung Ho, Suk‐Ho Lee.
  The Journal of Physiology. September 23, 2013
  •  The intrinsic excitability of a hippocampal CA3 pyramidal cell (CA3‐PC), but not CA1‐PC, is enhanced by repetitive somatic firing at a physiologically relevant frequency (10 Hz for 2 s). •  Such an excitability change is mediated by the Ca2+‐ and Src family kinase‐dependent endocytosis of D‐type K+ channel subunit Kv1.2. •  We provide evidence that the surface expression of D‐type K+ channels is higher in the distal apical dendrites than in the proximal apical dendrites in CA3‐PCs. •  These results help us understand neuronal computational mechanisms underlying the cognitive functions of the hippocampal CA3 area. Abstract  The intrinsic excitability of neurons plays a critical role in the encoding of memory at Hebbian synapses and in the coupling of synaptic inputs to spike generation. It has not been studied whether somatic firing at a physiologically relevant frequency can induce intrinsic plasticity in hippocampal CA3 pyramidal cells (CA3‐PCs). Here, we show that a conditioning train of 20 action potentials (APs) at 10 Hz causes a persistent reduction in the input conductance and an acceleration of the AP onset time in CA3‐PCs, but not in CA1‐PCs. Induction of such long‐term potentiation of intrinsic excitability (LTP‐IE) was accompanied by a reduction in the D‐type K+ current, and was abolished by the inhibition of endocytosis or protein tyrosine kinase (PTK). Consistently, the CA3‐PCs from Kv1.2 knock‐out mice displayed no LTP‐IE with the same conditioning. Furthermore, the induction of LTP‐IE depended on the back‐propagating APs (bAPs) and intact distal apical dendrites. These results indicate that LTP‐IE is mediated by the internalization of Kv1.2 channels from the distal regions of apical dendrites, which is triggered by bAP‐induced dendritic Ca2+ signalling and the consequent activation of PTK.

  September 23, 2013   doi: 10.1113/jphysiol.2013.259002   open full text
• Three‐dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III–IV of the spinal dorsal horn.
  Go Kato, Masafumi Kosugi, Masaharu Mizuno, Andrew M. Strassman.
  The Journal of Physiology. September 23, 2013
  •  Axons of sensory neurons that detect painful and non‐painful stimulation of body tissues project centrally to the dorsal horn of the spinal cord, where they are partially segregated in the superficial and deep laminae, respectively. •  Interneuronal connections between superficial and deep laminae could potentially modulate sensory transmission and contribute to alterations that occur under conditions of pain hypersensitivity. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of local interneuronal synaptic connections to laminae III–IV neurons, combined with intracellular staining for morphological analysis, in an in vitro‘slice’ preparation of the rat lumbar spinal cord. •  Synaptic input from superficial laminae (I–II) was received by laminae III–IV neurons with long dorsal dendrites, supporting the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and, more generally, that local connectivity is governed by rules that are specific to the laminar position and morphology of the postsynaptic neuron. Abstract  Laser scanning photostimulation was used to map the distribution of the synaptic input zones (sites that give local synaptic inputs) for dorsal horn laminae III–IV neurons, in parasagittal and transverse slices of the rat lumbar spinal cord, and examine how these inputs differed for neurons of different morphologies. All neurons received local excitatory and inhibitory synaptic inputs from within laminae III–IV, while a subset of neurons also received excitatory input from the superficial laminae, especially lamina IIi, as well as the II/III border region. Two anatomical properties were found to be predictive of the dorsoventral position of a neuron's input zone relative to its soma: (1) both excitatory and inhibitory input zones were more dorsal for neurons with longer dorsal dendrites, and (2) excitatory, but not inhibitory, input zones were more dorsal (relative to the soma) for more ventral neurons, with the transition between the dorsal input zones of laminae III–IV neurons and the ventral input zones of lamina II neurons occurring at the II/III border. The observed morphophysiological correlations support the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and that, more generally, local connectivity within the dorsal horn is governed by rules relating the position of a neuron's soma and dendrites to the position of the local presynaptic neurons from which it receives inputs, which are specific to the axis and direction (dorsal vs. ventral), whether the input is excitatory or inhibitory, and the laminar position of the postsynaptic neuron.

  September 23, 2013   doi: 10.1113/jphysiol.2013.256016   open full text
• Intravital Förster resonance energy transfer imaging reveals elevated [Ca2+]i and enhanced sympathetic tone in femoral arteries of angiotensin II‐infused hypertensive biosensor mice.
  Youhua Wang, Ling Chen, W. Gil Wier, Jin Zhang.
  The Journal of Physiology. September 23, 2013
  •  It is desirable to study altered artery function in hypertension in living animals, where factors influencing artery function are intact. •  We infused ‘biosensor’ mice chronically with angiotensin II to produce hypertension, and used intravital Förster resonance energy transfer microscopy to measure, simultaneously, [Ca2+]i and artery diameter in vivo. •  Femoral arteries in hypertensive mice had increased basal (resting) [Ca2+]i and were more constricted than femoral arteries in saline‐infused mice. These differences were abolished by blocking (1) all peripheral sympathetic nerve activity (SNA), or (2) vascular α1‐adrenoceptors, but not by blocking vascular angiotensin II or arginine vasopressin receptors. •  Neither contractility nor structural changes were found in femoral arteries of angiotensin II‐infused mice. •  The results support previous suggestions that angiotensin II infusion raises blood pressure by acting on the CNS to increase SNA, which increases smooth muscle [Ca2+]i. Infused angiotensin II does not act directly on arterial angiotensin II receptors. Abstract  Artery narrowing in hypertension can only result from structural remodelling of the artery, or increased smooth muscle contraction. The latter may occur with, or without, increases in [Ca2+]i. Here, we sought to measure, in living hypertensive mice, possible changes in artery dimensions and/or [Ca2+]i, and to determine some of the mechanisms involved. Ca2+/calmodulin biosensor (Förster resonance energy transfer‐based) mice were made hypertensive by s.c. infusion of angiotensin II (Ang II, 400 ng kg−1 min−1, 2–3 weeks). Intravital fluorescence microscopy was used to determine [Ca2+]i and outer diameter of surgically exposed, intact femoral artery (FA) of anaesthetized mice. Active contractile FA ‘tone’ was calculated from the basal‐state diameter and the passive (i.e. Ca2+‐free) diameter (PD). Compared to saline control, FAs of Ang II‐infused mice had (1) ∼21% higher active tone and (2) ∼78 nm higher smooth muscle [Ca2+]i, but (3) the same PDs. The local Ang II receptor (AT1R) blocker losartan had negligible effect on tone or [Ca2+]i in control FAs, but reduced the basal tone by ∼9% in Ang II FAs. Both i.v. hexamethonium and locally applied prazosin abolished the difference in FA tone and [Ca2+]i, suggesting a dominant role of sympathetic nerve activity (SNA). Changes in diameter and [Ca2+]i in response to locally applied phenylephrine, Ang II, arginine vasopressin, elevated [K+]o and acetylcholine were not altered. In summary, FAs of living Ang II hypertensive mice have higher [Ca2+]i, and are more constricted, due, primarily, to elevated SNA and some increased arterial AT1R activation. Evidence of altered artery reactivity or remodeling was not found.

  September 23, 2013   doi: 10.1113/jphysiol.2013.257808   open full text
• Osmotic water transport in aquaporins: evidence for a stochastic mechanism.
  Thomas Zeuthen, Magnus Alsterfjord, Eric Beitz, Nanna MacAulay.
  The Journal of Physiology. September 23, 2013
  •  We test a novel model of osmosis in aquaporins. •  A solute molecule present at the pore mouth can be reflected or permeate the pore; we propose that only reflected molecules induce osmotic water transport, while permeating molecules give rise to no water transport. •  We tested a range of channel geometries using aquaporins AQP1 and AQP9 and mutants thereof; the aquaporins were expressed in Xenopus oocytes. Osmotic gradients were generated by solutes of molecular weights in the range 45–595 Daltons. The reflected fraction of a given solute was estimated optically and compared to the permeability obtained from uptakes of radio‐labelled solutes. •  In accordance with our model there was a linear relationship between solute permeability and reflection coefficient: solutes with high permeability had low reflection coefficients and vice versa. •  We found no evidence for significant coupling between water and solute fluxes inside the pore. Abstract  We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, PS, is proportional to 1 –σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mm of a range of different‐sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short‐term uptake of radio‐labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward‐facing end of the pore.

  September 23, 2013   doi: 10.1113/jphysiol.2013.261321   open full text
• Orexin neurons are indispensable for prostaglandin E2‐induced fever and defence against environmental cooling in mice.
  Yoshiko Takahashi, Wei Zhang, Kohei Sameshima, Chiharu Kuroki, Ami Matsumoto, Jinko Sunanaga, Yu Kono, Takeshi Sakurai, Yuichi Kanmura, Tomoyuki Kuwaki.
  The Journal of Physiology. September 19, 2013
  •  We recently showed that orexin neurons in the hypothalamus are indispensable for stress‐induced thermogenesis. •  In this study we examined whether the orexin neurons are also important for other forms of thermogenic processes, including brain prostaglandin E2 (PGE2) injection that mimics inflammatory fever and environmental cold exposure. •  As was the case with stress‐induced thermogenesis, orexin neuron‐ablated (ORX‐AB) mice exhibited a blunted PGE2‐induced fever and intolerance to cold (5°C) exposure. •  Injection of retrograde tracer into the medullary raphe nucleus, where sympathetic premotor neurons regulating thermogenesis by the brown adipose tissue are located, revealed direct and indirect projection from the orexin neurons, of which the latter seemed to be preserved in the ORX‐AB mice. •  These results suggest that orexin neurons are important in general thermogenic processes, and their importance is not restricted to stress‐induced thermogenesis. Abstract  We recently showed using prepro‐orexin knockout (ORX‐KO) mice and orexin neuron‐ablated (ORX‐AB) mice that orexin neurons in the hypothalamus, but not orexin peptides per se, are indispensable for stress‐induced thermogenesis. To examine whether orexin neurons are more generally involved in central thermoregulatory mechanisms, we applied other forms of thermogenic perturbations, including brain prostaglandin E2 (PGE2) injections which mimic inflammatory fever and environmental cold exposure, to ORX‐KO mice, ORX‐AB mice and their wild‐type (WT) litter mates. ORX‐AB mice, but not ORX‐KO mice, exhibited a blunted PGE2‐induced fever and intolerance to cold (5°C) exposure, and these findings were similar to the results previously obtained with stress‐induced thermogenesis. PGE2‐induced shivering was also attenuated in ORX‐AB mice. Both mutants responded similarly to environmental heating (39°C). In WT and ORX‐KO mice, the administration of PGE2 and cold exposure activated orexin neurons, as revealed by increased levels of expression of c‐fos. Injection of retrograde tracer into the medullary raphe nucleus revealed direct and indirect projection from the orexin neurons, of which the latter seemed to be preserved in the ORX‐AB mice. In addition, we found that glutamate receptor antagonists (d‐(–)‐2‐amino‐5‐phosphonopentanoic acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione) but not orexin receptor antagonists (SB334867 and OX2 29) successfully inhibited PGE2‐induced fever in WT mice. These results suggest that orexin neurons are important in general thermogenic processes, and their importance is not restricted to stress‐induced thermogenesis. In addition, these results indicate the possible involvement of glutamate in orexin neurons implicated in PGE2‐induced fever.

  September 19, 2013   doi: 10.1113/jphysiol.2013.261271   open full text
• The role of myogenic mechanisms in human cerebrovascular regulation.
  Can Ozan Tan, J. W. Hamner, J. Andrew Taylor.
  The Journal of Physiology. September 19, 2013
  •  The autoregulatory capacity of the cerebral vasculature allows for maintenance of relatively stable blood flow in the face of fluctuating arterial pressure to protect neural tissue from wide swings in oxygen and nutrient delivery. •  We recently found that neurogenic control plays an active role in autoregulation. Although myogenic pathways have also been hypothesized to play a role, previous data have not provided an unequivocal answer. •  We examined cerebral blood flow responses to augmented arterial pressure oscillations with and without calcium channel blockade, and characterized autoregulation via a robust non‐linear method. •  Blockade significantly altered the non‐linearity between pressure and flow, particularly at the slowest fluctuations, and the same rate of change in pressure elicited a larger change in flow than at baseline. •  These results show that myogenic mechanisms also play a significant role in cerebrovascular regulation, and help us better understand physiological mechanisms that underlie cerebral autoregulation in humans. Abstract  Although myogenic mechanisms have been hypothesized to play a role in cerebrovascular regulation, previous data from both animals and humans have not provided an unequivocal answer. However, cerebral autoregulation is explicitly non‐linear and most prior work relied on simple linear approaches for assessment, potentially missing important changes in autoregulatory characteristics. Therefore, we examined cerebral blood flow responses to augmented arterial pressure oscillations with and without calcium channel blockade (nicardipine) during blood pressure fluctuations (oscillatory lower body negative pressure, OLBNP) across a range of frequencies in 16 healthy subjects. Autoregulation was characterized via a robust non‐linear method (projection pursuit regression, PPR). Blockade resulted in significant tachycardia, a modest but significant elevation in mean arterial pressure, and reductions in mean cerebral blood flow and end‐tidal CO2 during OLBNP. The reductions in flow were directly related to the reductions in CO2 (r= 0.57). While linear cross‐spectral analysis showed that the relationship between pressure–flow fluctuations was preserved after blockade, PPR showed that blockade significantly altered the non‐linearity between pressure and flow, particularly at the slowest fluctuations. At 0.03 Hz, blockade reduced the range of pressure fluctuations that can be buffered (7.5 ± 1.0 vs. 3.7 ± 0.8 mmHg) while increasing the autoregulatory slope (0.10 ± 0.05 vs. 0.24 ± 0.08 cm s−1 mmHg−1). Furthermore, the same rate of change in pressure elicited a change in flow more than twice as large as at baseline. Thus, our results show that myogenic mechanisms play a significant role in cerebrovascular regulation but this may not be appreciated without adequately characterizing the non‐linearities inherent in cerebrovascular regulation.

  September 19, 2013   doi: 10.1113/jphysiol.2013.259747   open full text
• Modulation of the autonomic nervous system and behavior by acute glial cell Gq‐GPCR activation in vivo.
  Cendra Agulhon, Kristen M. Boyt, Alison Xiaoqiao Xie, Francois Friocourt, Bryan L. Roth, Ken D. McCarthy.
  The Journal of Physiology. September 19, 2013
  Abstract  Glial fibrillary acidic protein (GFAP)‐expressing cells (GFAP+ glial cells) are the predominant cell type in the central and peripheral nervous system (CNS and PNS). Our understanding of the role of GFAP+ glial cells and their signaling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell‐type specific and non‐invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP‐hM3Dq mice) that expresses an engineered Gq protein‐coupled receptor (Gq‐GPCR) known as hM3Dq DREADD (Designer Receptor Exclusively Activated by Designer Drug) selectively in GFAP+ glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine‐N‐oxide; CNO), while being non‐responsive to endogenous GPCR ligands. In GFAP‐hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature; parameters controlled by the autonomic nervous system (ANS). Additionally, changes in activity‐related behavior and motor coordination were observed following CNO administration. Genetically blocking IP3‐dependent Ca2+ increases in astrocytes failed to interfere with CNO‐mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP+ glial cells in modulating complex physiology and behavior in vivo and suggest that these effects are not dependent on IP3‐dependent increases in astrocytic Ca2+. This article is protected by copyright. All rights reserved

  September 19, 2013   doi: 10.1113/jphysiol.2013.261289   open full text
• The Lateral Reticular Nucleus: a precerebellar centre providing the Cerebellum with overview and integration of motor functions at systems level. A new hypothesis.
  Bror Alstermark, Carl‐Fredrik Ekerot.
  The Journal of Physiology. September 19, 2013
  Abstract  The lateral reticular nucleus (LRN) is a major precerebellar centre of mossy fibre information to the cerebellum from the spinal cord that is distinct from the direct spinocerebellar paths. The LRN has traditionally been considered to provide the cerebellum with segregated information from several spinal systems controlling posture, reaching, grasping, locomotion, scratching and respiration. However, results are presented that show extensive convergence on a majority of LRN neurons from spinal systems. We propose a new hypothesis suggesting that the LRN may use extensive convergence from the different input systems to provide overview and integration of linked motor components to the cerebellum. This integrated information is sent in parallel with the segregated information from the individual systems to the Cerebellum that finally may compare the activity and make necessary adjustments of various motor behaviour. This article is protected by copyright. All rights reserved

  September 19, 2013   doi: 10.1113/jphysiol.2013.256669   open full text
• Altered skeletal muscle mitochondrial biogenesis but improved endurance capacity in trained OPA1‐deficient mice.
  F. Caffin, A. Prola, J. Piquereau, M. Novotova, D.J. David, A. Garnier, D. Fortin, M. Alavi, V. Veksler, R. Ventura‐Clapier, F. Joubert.
  The Journal of Physiology. September 19, 2013
  Abstract  The role of OPA1, a GTPase dynamin protein mainly involved in the fusion of inner mitochondrial membranes, has been studied in many cell types, but only a few studies have been conducted on adult differentiated tissues such as cardiac or skeletal muscle cells. Yet, OPA1 is highly expressed in these cells, and could play different roles, especially in response to an environmental stress like exercise. Endurance exercise increases energy demand in skeletal muscle and repeated activity induces mitochondrial biogenesis and activation of fusion/fission cycles for the synthesis of new mitochondria. But currently, no study has clearly shown a link between mitochondrial dynamics and biogenesis. Using a mouse model of haploinsufficiency for the Opa1 gene (Opa1+/−), we therefore studied the impact of OPA1 deficiency on the adaptation ability of fast skeletal muscles to endurance exercise training. Our results show that surprisingly, Opa1+/− mice were able to perform the same physical activity as control mice. However, the adaptation strategies of both strains after training differed: while in control mice mitochondrial biogenesis was increased as expected, in Opa1+/− mice this process was blunted. Instead, training in Opa1+/− mice led to an increase in endurance capacity, and a specific adaptive response involving a metabolic remodeling towards enhanced fatty acid utilization. In conclusion, OPA1 appears necessary for the normal adaptive response and mitochondrial biogenesis of skeletal muscle to training. This work opens new perspectives on the role of mitochondrial dynamics in skeletal muscle cells and during adaptation to stress. This article is protected by copyright. All rights reserved

  September 19, 2013   doi: 10.1113/jphysiol.2013.263079   open full text
• Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium–bicarbonate cotransporter NBCn1 (SLC4A7).
  Ying Liu, Xue Qin, Deng‐Ke Wang, Yi‐Min Guo, Harindarpal S. Gill, Nathan Morris, Mark D. Parker, Li‐Ming Chen, Walter F. Boron.
  The Journal of Physiology. September 17, 2013
  •  The human SLC4A7 gene and the mouse Slc4a7 gene each have alternative promoters that can yield two groups of NBCn1 variants, one in which the extreme N terminus begins with MEAD (representing the first four residues of the N‐terminal domain (Nt)) and the other in which it begins with MERF. •  The mouse Slc4a7 gene contains, and the human SLC4A7 gene is predicted to contain, a novel exon that encodes an alternatively spliced cassette IV of 20 aa in the cytoplasmic Nt domain of NBCn1. This new cassette IV is in a position homologous to that of a previously described cassette in the Nt of NBCn2. •  From combinations of known optional structural elements (OSEs), SLC4A7 is theoretically able to produce 32 major variants, of which 16 have now been identified, 10 for the first time in the present study. •  With heterologous expression in Xenopus oocytes, the OSEs have strong effects on surface abundance and intrinsic HCO3− transport activity. Cassettes II, III and the novel cassette IV have stimulatory effects on the intrinsic HCO3− transport activity of NBCn1. Abstract  The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per‐molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD‐ and MERF‐NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full‐length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N‐terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3− transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.

  September 17, 2013   doi: 10.1113/jphysiol.2013.258673   open full text
• Phosphatidylethanolamine binding protein 1 in vacular endothelial cell autophagy and atherosclerosis.
  Li Wang, HaiYing Li, JinFeng Zhang, Wei Lu, Jing Zhao, Le Su, BaoXiang Zhao, Yun Zhang, ShangLi Zhang, JunYing Miao.
  The Journal of Physiology. September 17, 2013
  •  The enzyme phosphatidylcholine‐specific phospholipase C (PC‐PLC) participates in atherosclerosis development and may negatively regulate autophagy. •  Phosphatidylethanolamine binding protein 1 (PEBP1) represents a novel effector of signal transduction pathways that control cellular growth, motility, apoptosis, genomic integrity, and therapeutic resistance. •  The disruption of PEBP1 was reported to associate with a wide range of diseases, such as cancer, pancreatitis, and Alzheimer's disease, making it a potential target for disease therapy. However, the roles of PEBP1 in vascular endothelial cell (VEC) autophagy and arteriosclerosis are not clear. •  Here we report that PEBP1 interacts with PC‐PLC and positively regulates PC‐PLC activity, while both PEBP1 and PC‐PLC negatively regulate VEC autophagy. •  The PEBP1 level is elevated during the development of atherosclerosis, and the PC‐PLC inhibitor D609 significantly decreases the upregulated PEBP1 level in apolipoprotein E−/− mice, suggesting that PEBP1 may be a potential diagnostic indicator for atherosclerosis. Abstract  We previously found that phosphatidylcholine‐specific phospholipase C (PC‐PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC‐PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC‐PLC by using mass spectrometry (MS, MALDI‐TOF/TOF). We found that PEBP1 positively regulated PC‐PLC activity in HUVECs, and inhibition of PC‐PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC‐PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE−/− mice.

  September 17, 2013   doi: 10.1113/jphysiol.2013.262667   open full text
• Molecular transport machinery involved in orchestrating luminal acid‐induced duodenal bicarbonate secretion in vivo.
  Anurag Kumar Singh, Yongjian Liu, Brigitte Riederer, Regina Engelhardt, Basant Kumar Thakur, Manoocher Soleimani, Ursula Seidler.
  The Journal of Physiology. September 17, 2013
  Abstract  The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT‐1), the Na+/H+ exchanger isoform 3 (NHE3), as well as the anion channels CFTR and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9, or Slc9a3 (Na+/H+ exchanger isoform 3, NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal HCO3− secretory response (DBS‐R) to luminal acid; and to compare it to their role in DBS‐R elicited by the adenylyl cyclase agonist forskolin (FSK). While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS‐R to FSK was not altered. In contrast, the DBS‐R to a 5 min exposure to luminal acid, pH 2.5, was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)‐172] reduced the first phase of the acid‐induced DBS‐R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS‐R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis in the later stage. We conclude that the long‐lasting DBS‐R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3. This article is protected by copyright. All rights reserved

  September 17, 2013   doi: 10.1113/jphysiol.2013.254854   open full text
• Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischemia and involve resistin.
  Elie R. Chemaly, Soojeong Kang, Shihong Zhang, LaTronya McCollum, Jiqiu Chen, Ludovic Bénard, K‐Raman Purushothaman, Roger J. Hajjar, Djamel Lebeche.
  The Journal of Physiology. September 17, 2013
  Abstract  Pathological left ventricle (LV) hypertrophy (LVH) results in reactive and replacement fibrosis. Volume overload LVH (VOH) is less profibrotic than pressure overload LVH (POH). Studies attribute subendocardial fibrosis in POH to ischemia, and reduced fibrosis in VOH to collagen degradation favoring dilatation. However, the mechanical origin of the relative lack of fibrosis in VOH is incompletely understood. We hypothesized that reduced ischemia propensity in VOH compared to POH accounted for the reduced replacement fibrosis, along with reduced reactive fibrosis. Rats with POH (ascending aortic banding) evolved into either compensated‐concentric POH (POH‐CLVH) or dilated cardiomyopathy (POH‐DCM); they were compared to VOH (aorta‐caval fistula). We quantified LV fibrosis, structural and hemodynamic factors of ischemia propensity, and the activation of profibrotic pathways. Fibrosis in POH‐DCM was severe, subendocardial and subepicardial, in contrast with subendocardial fibrosis in POH‐CLVH and nearly no fibrosis in VOH. Propensity for ischemia was more important in POH versus VOH, explaining different patterns of replacement fibrosis. LV collagen synthesis and maturation, and matrix metalloproteinase‐2 expression, were more important in POH. The angiotensin‐II‐transforming growth‐factor ß axis was enhanced in POH, and connective tissue growth factor (CTGF) was overexpressed in all types of LVH. LV Resistin expression was markedly elevated in POH, mildly elevated in VOH and independently reflected chronic ischemic injury after myocardial infarction. In vitro, resistin is induced by angiotensin‐II and induces CTGF in cardiomyocytes. Based on these findings, we conclude that reduced ischemia propensity and attenuated upstream reactive fibrotic pathways account for the attenuated fibrosis in VOH versus POH. This article is protected by copyright. All rights reserved

  September 17, 2013   doi: 10.1113/jphysiol.2013.258731   open full text
• Specific amino acids inhibit food intake via the area postrema or vagal afferents.
  Josua Jordi, Brigitte Herzog, Simone M. R. Camargo, Christina N. Boyle, Thomas A. Lutz, François Verrey.
  The Journal of Physiology. September 16, 2013
  •  Proteins are more satiating than fats or lipids. Proteins are built by the 20 proteogenic amino acids. •  Here, we identified l‐arginine, l‐lysine and l‐glutamic acid as the most potent anorectic amino acids in rats. •  l‐Arginine and l‐glutamic acid require intact neurons in the area postrema to inhibit food intake, whereas l‐lysine requires intact afferent fibres of the vagus nerve. All three mediate their effect by the blood stream. •  All three amino acids induce gastric distension by delaying gastric emptying and inducing secretion. However, the gastric phenotype does not mediate the anorectic response. •  These results unravel amino acid‐specific mechanisms regulating digestion and eating behaviour and thereby contribute to the understanding of nutrient sensing in vivo. Abstract  To maintain nutrient homeostasis the central nervous system integrates signals that promote or inhibit eating. The supply of vital amino acids is tuned by adjusting food intake according to its dietary protein content. We hypothesized that this effect is based on the sensing of individual amino acids as a signal to control food intake. Here, we show that food intake was most potently reduced by oral l‐arginine (Arg), l‐lysine (Lys) and l‐glutamic acid (Glu) compared to all other 17 proteogenic amino acids in rats. These three amino acids induced neuronal activity in the area postrema and the nucleus of the solitary tract. Surgical lesion of the area postrema abolished the anorectic response to Arg and Glu, whereas vagal afferent lesion prevented the response to Lys. These three amino acids also provoked gastric distension by differentially altering gastric secretion and/or emptying. Importantly, these peripheral mechanical vagal stimuli were dissociated from the amino acids’ effect on food intake. Thus, Arg, Lys and Glu had a selective impact on food processing and intake suggesting them as direct sensory input to assess dietary protein content and quality in vivo. Overall, this study reveals novel amino acid‐specific mechanisms for the control of food intake and of gastrointestinal function.

  September 16, 2013   doi: 10.1113/jphysiol.2013.258947   open full text
• Properties of myenteric neurones and mucosal functions in the distal colon of diet‐induced obese mice.
  François Reichardt, Charlotte Baudry, Lisa Gruber, Gemma Mazzuoli, Raphaël Moriez, Christian Scherling, Patrick Kollmann, Hannelore Daniel, Sigrid Kisling, Dirk Haller, Michel Neunlist, Michael Schemann.
  The Journal of Physiology. September 16, 2013
  •  We investigated altered colonic functions in high fat diet‐induced obesity in pre‐diabetic mice. •  After feeding an adipogenic diet for 12 weeks, accelerated colonic transit was associated with upregulation and enhanced signalling of acetylcholine and serotonin, two key mediators in the enteric nervous system (ENS). Importantly, these changes occurred without signs of impaired mucosal integrity or immune cell infiltration in the gut wall. Neuronal sensitization was not observed in obese mice fed for 4 weeks. •  Weight gain correlated positively with the level of adipocyte markers and the degree of neuronal sensitization. •  We conclude that enhanced neural excitation in the colon by acetylcholine and serotonin is a key feature of a later phase of obesity and is involved in altered ENS functions and abnormal colonic transit. •  Furthermore, the results suggest that the occurrence of altered gut functions in obesity is independent of inflammation in the gut wall. Abstract  Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet‐induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2‐methyl‐5‐hydroxytryptamine (2‐methyl‐5‐HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2‐methyl‐5‐HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5‐hydroxytryptamine (5‐HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5‐HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin‐6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5‐HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.

  September 16, 2013   doi: 10.1113/jphysiol.2013.262733   open full text
• AMP‐activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle.
  Josef Brandauer, Sara G. Vienberg, Marianne A. Andersen, Stine Ringholm, Steve Risis, Per S. Larsen, Jonas M. Kristensen, Christian Frøsig, Lotte Leick, Joachim Fentz, Sebastian Jørgensen, Bente Kiens, Jørgen F. P. Wojtaszewski, Erik A. Richter, Juleen R. Zierath, Laurie J. Goodyear, Henriette Pilegaard, Jonas T. Treebak.
  The Journal of Physiology. September 13, 2013
  •  NAD is a substrate for sirtuins (SIRTs), which regulate gene transcription in response to specific metabolic stresses. •  Nicotinamide phosphoribosyl transferase (Nampt) is the rate‐limiting enzyme in the NAD salvage pathway. •  Using transgenic mouse models, we tested the hypothesis that skeletal muscle Nampt protein abundance would increase in response to metabolic stress in a manner dependent on the cellular nucleotide sensor, AMP‐activated protein kinase (AMPK). •  Exercise training, as well as repeated pharmacological activation of AMPK by 5‐amino‐1‐β‐d‐ribofuranosyl‐imidazole‐4‐carboxamide (AICAR), increased Nampt protein abundance. However, only the AICAR‐mediated increase in Nampt protein abundance was dependent on AMPK. •  Our results suggest that cellular energy charge and nutrient sensing by SIRTs may be mechanistically related, and that Nampt may play a key role for cellular adaptation to metabolic stress. Abstract  Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate‐limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP‐activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training‐ or 5‐amino‐1‐β‐d‐ribofuranosyl‐imidazole‐4‐carboxamide (AICAR)‐mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One‐legged knee‐extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild‐type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2‐containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post‐translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.

  September 13, 2013   doi: 10.1113/jphysiol.2013.259515   open full text
• Spinal TNFα is necessary for inactivity‐induced phrenic motor facilitation.
  Oleg Broytman, Nathan A. Baertsch, Tracy L. Baker‐Herman.
  The Journal of Physiology. September 13, 2013
  •  A central neural apnoea in the absence of hypoxia elicits a form of respiratory plasticity known as inactivity‐induced phrenic motor facilitation (iPMF), a rebound increase in phrenic burst amplitude when central respiratory neural activity is restored. •  iPMF requires spinal atypical protein kinase C (aPKC) activity in spinal segments encompassing the phrenic motor nucleus. •  Here, we report novel findings that tumour necrosis factor‐α (TNFα) signalling in or near the phrenic motor pool is necessary and sufficient for iPMF as: (1) spinal TNFα inhibition inhibits iPMF; and (2) spinal TNFα elicits long‐lasting increases in phrenic burst amplitude via an aPKC‐dependent mechanism. •  These data are consistent with the hypothesis that local mechanisms operating within or near the phrenic motor pool sense and respond to reduced respiratory neural activity, and suggest that TNFα‐induced activation of aPKC near phrenic motor neurons forms part of the core cellular pathway giving rise to iPMF. Abstract  A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity‐induced phrenic motor facilitation (iPMF), a ‘rebound’ increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long‐lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor‐α (TNFα) is necessary for iPMF via an aPKC‐dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long‐lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long‐lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long‐lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα‐induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ‐pseudosubstrate inhibitory peptide (PKCζ‐PS) 35 min following intrathecal TNFα arrested TNFα‐induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC‐dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα‐dependent increase in spinal aPKC activity.

  September 13, 2013   doi: 10.1113/jphysiol.2013.256644   open full text
• Modulation of synaptic depression of the calyx of Held synapse by GABAB receptors and spontaneous activity.
  Tiantian Wang, Silviu I. Rusu, Bohdana Hruskova, Rostislav Turecek, J. Gerard G. Borst.
  The Journal of Physiology. September 13, 2013
  •  Spontaneous activity contributes to low synaptic depression of the calyx of Held synapse in vivo. •  Application of a reversible blocker in combination with juxtacellular recordings allows a relatively good estimate of local drug concentrations reached during microiontophoresis. •  Activation of the GABAB receptor on the young‐adult calyx of Held can reduce short‐term depression, both in vivo and in slices. •  The ambient concentration of GABA in the auditory brainstem is low. Abstract  The calyx of Held synapse of the medial nucleus of the trapezoid body is a giant axosomatic synapse in the auditory brainstem, which acts as a relay synapse showing little dependence of its synaptic strength on firing frequency. The main mechanism that is responsible for its resistance to synaptic depression is its large number of release sites with low release probability. Here, we investigated the contribution of presynaptic GABAB receptors and spontaneous activity to release probability both in vivo and in vitro in young‐adult mice. Maximal activation of presynaptic GABAB receptors by baclofen reduced synaptic output by about 45% in whole‐cell voltage clamp slice recordings, which was accompanied by a reduction in short‐term depression. A similar reduction in transmission was observed when baclofen was applied in vivo by microiontophoresis during juxtacellular recordings using piggyback electrodes. No significant change in synaptic transmission was observed during application of the GABAB receptor antagonist CGP54626 both during in vivo and slice recordings, suggesting a low ambient GABA concentration. Interestingly, we observed that synapses with a high spontaneous frequency showed almost no synaptic depression during auditory stimulation, whereas synapses with a low spontaneous frequency did depress during noise bursts. Our data thus suggest that spontaneous firing can tonically reduce release probability in vivo. In addition, our data show that the ambient GABA concentration in the auditory brainstem is too low to activate the GABAB receptor at the calyx of Held significantly, but that activation of GABAB receptors can reduce sound‐evoked synaptic depression.

  September 13, 2013   doi: 10.1113/jphysiol.2013.256875   open full text
• Antioxidant treatment improves neonatal survival and prevents impaired cardiac function at adulthood following neonatal glucocorticoid therapy.
  Youguo Niu, Emilio A. Herrera, Rhys D. Evans, Dino A. Giussani.
  The Journal of Physiology. September 11, 2013
  •  Although neonatal glucocorticoid therapy is an effective measure to prevent and treat chronic lung disease in premature infants, it can cause long‐term adverse effects on the cardiovascular system secondary to oxidative stress and reduced nitric oxide (NO) bioavailability. •  Here, we report that neonatal dexamethasone therapy using human clinically relevant doses resulted in increased mortality, and that surviving offspring had significantly lower NO bioavailability and impaired cardiac function at adulthood. Combined neonatal treatment of dexamethasone with antioxidant vitamins prevented these adverse side‐effects in offspring. •  The data give insight into the mechanisms underlying the adverse effects of neonatal dexamethasone on the cardiovascular system. Further, the findings are of significant clinical importance in helping to modify current perinatal practice to minimise adverse side‐effects while maintaining the benefits of potent neonatal steroid therapy. Abstract  Glucocorticoids are widely used to treat chronic lung disease in premature infants but their longer‐term adverse effects on the cardiovascular system raise concerns. We reported that neonatal dexamethasone treatment in rats induced in the short term molecular indices of cardiac oxidative stress and cardiovascular tissue remodelling at weaning, and that neonatal combined antioxidant and dexamethasone treatment was protective at this time. In this study, we investigated whether such effects of neonatal dexamethasone have adverse consequences for NO bioavailability and cardiovascular function at adulthood, and whether neonatal combined antioxidant and dexamethasone treatment is protective in the adult. Newborn rat pups received daily i.p. injections of a human‐relevant tapering dose of dexamethasone (D; n= 8; 0.5, 0.3, 0.1 μg g−1) or D with vitamins C and E (DCE; n= 8; 200 and 100 mg kg−1, respectively) on postnatal days 1–3 (P1–3); vitamins were continued from P4 to P6. Controls received equal volumes of vehicle from P1 to P6 (C; n= 8). A fourth group received vitamins alone (CCE; n= 8). At P100, plasma NO metabolites (NOx) was measured and isolated hearts were assessed under both Working and Langendorff preparations. Relative to controls, neonatal dexamethasone therapy increased mortality by 18% (P < 0.05). Surviving D pups at adulthood had lower plasma NOx concentrations (10.6 ± 0.8 vs. 28.0 ± 1.5 μm), an increased relative left ventricular (LV) mass (70 ± 2 vs. 63 ± 1%), enhanced LV end‐diastolic pressure (14 ± 2 vs. 8 ± 1 mmHg) and these hearts failed to adapt output with increased preload (Δcardiac output: 2.9 ± 2.0 vs. 10.6 ± 1.2 ml min−1) or afterload (Δcardiac output: −5.3 ± 2.0 vs.1.4 ± 1.2 ml min−1); all P < 0.05. Combined neonatal dexamethasone with antioxidant vitamins improved postnatal survival, restored plasma NOx and protected against cardiac dysfunction at adulthood. In conclusion, neonatal dexamethasone therapy promotes cardiac dysfunction at adulthood. Combined neonatal treatment with antioxidant vitamins is an effective intervention.

  September 11, 2013   doi: 10.1113/jphysiol.2013.258210   open full text
• Burst generation mediated by cholinergic input in terminal nerve‐gonadotrophin releasing hormone neurones of the goldfish.
  Takafumi Kawai, Hideki Abe, Yoshitaka Oka.
  The Journal of Physiology. September 11, 2013
  •  Burst firing activities are effective for the release of neuropeptides from peptidergic neurones. •  A peptidergic neurone, the terminal nerve (TN)‐gonadotrophin releasing hormone (GnRH) neurone, shows spontaneous burst firing activities only infrequently. •  Only a single pulse electrical stimulation of the neuropil surrounding the TN‐GnRH neurones induces transient burst activities in TN‐GnRH neurones via cholinergic mechanisms. •  The activation of muscarinic acetylcholine receptors results in a long‐lasting hyperpolarisation, inducing rebound burst activities in TN‐GnRH neurones. •  These new findings suggest a novel type of cholinergic regulation of burst activities in peptidergic neurones, which should contribute to the release of neuropeptides. Abstract  Peptidergic neurones play a pivotal role in the neuromodulation of widespread areas in the nervous system. Generally, it has been accepted that the peptide release from these neurones is regulated by their firing activities. The terminal nerve (TN)‐gonadotrophin releasing hormone (GnRH) neurones, which are one of the well‐studied peptidergic neurones in vertebrate brains, are characterised by their spontaneous regular pacemaker activities, and GnRH has been suggested to modulate the sensory responsiveness of animals. Although many peptidergic neurones are known to exhibit burst firing activities when they release the peptides, TN‐GnRH neurones show spontaneous burst firing activities only infrequently. Thus, it remains to be elucidated whether the TN‐GnRH neurones show burst activities and, if so, how the mode switching between the regular pacemaking and bursting modes is regulated in these neurones. In this study, we found that only a single pulse electrical stimulation of the neuropil surrounding the TN‐GnRH neurones reproducibly induces transient burst activities in TN‐GnRH neurones. Our combined physiological and morphological data suggest that this phenomenon occurs following slow inhibitory postsynaptic potentials mediated by cholinergic terminals surrounding the TN‐GnRH neurones. We also found that the activation of muscarinic acetylcholine receptors induces persistent opening of potassium channels, resulting in a long‐lasting hyperpolarisation. This long hyperpolarisation induces sustained rebound depolarisation that has been suggested to be generated by a combination of persistent voltage‐gated Na+ channels and low‐voltage‐activated Ca2+ channels. These new findings suggest a novel type of cholinergic regulation of burst activities in peptidergic neurones, which should contribute to the release of neuropeptides.

  September 11, 2013   doi: 10.1113/jphysiol.2013.258343   open full text
• Calcium signalling of human pluripotent stem cell‐derived cardiomyocytes.
  Sen Li, Gaopeng Chen, Ronald A. Li.
  The Journal of Physiology. September 11, 2013
  Abstract  Loss of cardiomyocytes (CMs), which lack the innate ability to regenerate, due to aging or pathophysiological conditions (e.g. myocardial infarction or MI) is generally considered irreversible, and can lead to conditions from cardiac arrhythmias to heart failure. Human (h) pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), can self‐renew while maintaining their pluripotency to differentiate into all cell types, including CMs. Therefore, hPSCs provide a potential unlimited ex vivo source of human CMs for disease modelling, drug discovery, cardiotoxicity screening and cell‐based heart therapies. As a fundamental property of working CMs, Ca2+ signalling and its role in excitation‐contraction coupling are well described. However, the biology of these processes in hPSC‐CMs is just becoming understood. Here we review what are known about the immature Ca2+‐handling properties of hPSC‐CMs, at the levels of global transients and sparks, and the underlying molecular basis in relation to the development of various in vitro approaches to drive their maturation. This article is protected by copyright. All rights reserved

  September 11, 2013   doi: 10.1113/jphysiol.2013.256495   open full text
• Are skeletal muscle FNDC5 gene expression and irisin release regulated by exercise and related to health?
  Satu Pekkala, Petri K. Wiklund, Juha J. Hulmi, Juha P. Ahtiainen, Mia Horttanainen, Eija Pöllänen, Kari A. Mäkelä, Heikki Kainulainen, Keijo Häkkinen, Kai Nyman, Markku Alén, Karl‐Heinz Herzig, Sulin Cheng.
  The Journal of Physiology. September 11, 2013
  Abstract  Recently contradictory findings concerning the function of irisin and its precursor gene, skeletal muscle FNDC5 in energy homeostasis and the regulatory role of exercise and PGC‐1α therein have been reported. We therefore evaluated whether muscle FNDC5 mRNA and serum irisin are exercise‐responsive and whether PGC‐1α expression is associated with FNDC5 expression. The male study subjects performed single exercises either 1‐hour low‐intensity aerobic exercise (AE) (middle‐aged, n = 17) or a heavy‐intensity resistance exercise (RE) bout (young n = 10, old n = 11), or long‐term 21‐weeks endurance exercise (EE) training alone (twice/week, middle‐aged, n = 9) or combined EE and RE training (both twice/week, middle‐aged, n = 9). Skeletal muscle mRNA expression was analyzed by quantitative PCR and serum irisin by ELISA. No significant changes were observed in skeletal muscle PGC‐1α, FNDC5 and serum irisin after AE, EE training or combined EE+RE training. However, a single RE bout increased PGC‐1α by 4‐fold in young and by 2‐fold in old men, while FNDC5 mRNA increased by 1.4‐fold post‐RE only in young men. Changes in PGC‐1α or serum irisin were not consistently accompanied with changes in FNDC5. In conclusion, for the most part, neither longer‐term nor single exercise markedly increases skeletal muscle FNDC5 expression or serum irisin. Therefore their changes in response to exercise are likely random and not general excluding the confirmation of any definitive link between exercise and FNDC5 expression and irisin release in humans. Moreover, irisin and FNDC5 did not associate with glucose tolerance and overweight or metabolic disturbances, respectively. Finally, factor(s) other than PGC‐1α and transcription may regulate FNDC5 expression. This article is protected by copyright. All rights reserved

  September 11, 2013   doi: 10.1113/jphysiol.2013.263707   open full text
• Ba2+‐ and bupivacaine‐sensitive background K+ conductances mediate rapid EPSP attenuation in oligodendrocyte precursor cells.
  Chu‐Fang Chan, Tzu‐Wei Kuo, Ju‐Yun Weng, Yen‐Chu Lin, Ting‐Yu Chen, Jen‐Kun Cheng, Cheng‐Chang Lien.
  The Journal of Physiology. September 10, 2013
  •  We developed detailed passive cable models of rat oligodendrocyte precursor cells (OPCs) based on dual somatic recordings and complete morphological reconstructions. •  Both specific membrane capacitance and specific axial resistivity are comparable to those of central neurons, but the average specific membrane resistance (Rm∼4.1 kΩ cm2) is substantially lower in OPCs. •  Large Ba2+‐ and bupivacaine‐sensitive background K+ conductances contribute to the low Rm. •  Simultaneous dual soma and process whole‐cell recordings reveal powerful voltage attenuation along OPC processes, indicating that OPC processes are a strong voltage attenuator. •  The low Rm also sharpens EPSPs and thus narrows the temporal window for EPSP integration. Abstract  Glutamatergic transmission onto oligodendrocyte precursor cells (OPCs) may regulate OPC proliferation, migration and differentiation. Dendritic integration of excitatory postsynaptic potentials (EPSPs) is critical for neuronal functions, and mechanisms regulating dendritic propagation and summation of EPSPs are well understood. However, little is known about EPSP attenuation and integration in OPCs. We developed realistic OPC models for synaptic integration, based on passive membrane responses of OPCs obtained by simultaneous dual whole‐cell patch‐pipette recordings. Compared with neurons, OPCs have a very low value of membrane resistivity, which is largely mediated by Ba2+‐ and bupivacaine‐sensitive background K+ conductances. The very low membrane resistivity not only leads to rapid EPSP attenuation along OPC processes but also sharpens EPSPs and narrows the temporal window for EPSP summation. Thus, background K+ conductances regulate synaptic responses and integration in OPCs, thereby affecting activity‐dependent neuronal control of OPC development and function.

  September 10, 2013   doi: 10.1113/jphysiol.2013.257113   open full text
• Short‐term exercise training augments α2‐adrenoreceptor‐mediated sympathetic vasoconstriction in resting and contracting skeletal muscle.
  Nicholas G. Jendzjowsky, Darren S. DeLorey.
  The Journal of Physiology. September 10, 2013
  •  Postsynaptic α1‐ and α2‐adrenoreceptors produce tonic vasoconstriction in resting and contracting skeletal muscle. •  Muscular contraction attenuates sympathetic vasoconstriction (functional sympatholysis). The blunting of sympathetic vasoconstriction during contraction has been mechanistically linked to a reduction in postsynaptic α1‐ and α2‐adrenoreceptor responsiveness and nitric oxide. •  We recently demonstrated that exercise training augmented sympathetic vasoconstrictor responsiveness and functional sympatholysis. Whether these vascular adaptations were mediated by changes to postsynaptic adrenoreceptors was not investigated. •  The present findings demonstrate that exercise training augmented α2‐adrenoreceptor‐ mediated vasoconstriction in resting and contracting skeletal muscle. •  These data indicate that exercise training alters the relative contributions of α‐adrenoreceptors to sympathetic vasoconstriction in resting and contracting skeletal muscle and that the regulation of sympathetic vasoconstriction becomes more complex following exercise training. Abstract  We hypothesized that exercise training (ET) would alter α2‐adrenoreceptor‐mediated sympathetic vasoconstriction. Sprague‐Dawley rats (n= 30) were randomized to sedentary (S), mild‐ (M) or heavy‐intensity (H) treadmill ET groups (5 days per week for 4 weeks). Following the ET component of the study, rats were anaesthetized, and instrumented for lumbar sympathetic chain stimulation, triceps surae muscle contraction and measurement of femoral vascular conductance (FVC). The percentage change of FVC in response to sympathetic stimulation was determined at rest and during contraction in control, α2 blockade (yohimbine) and combined α2+ nitric oxide (NO) synthase (NOS) blockade (Nω‐nitro‐l‐arginine methyl ester hydrochloride, l‐NAME) conditions. ET augmented (P < 0.05) sympathetic vasoconstrictor responses at rest and during contraction. Yohimbine reduced (P < 0.05) the vasoconstrictor response in ET rats at rest (M: 2 Hz: 8 ± 2%, 5 Hz: 9 ± 4%; H: 2 Hz: 14 ± 5%, 5 Hz: 11 ± 6%) and during contraction (M: 2 Hz: 9 ± 2%, 5 Hz: 9 ± 5%; H: 2 Hz: 8 ± 3%, 5 Hz: 6 ± 6%) but did not change the response in S rats. The addition of l‐NAME caused a larger increase (P < 0.05) in the vasoconstrictor response in ET than in S rats at rest (2 Hz: S: 8 ± 2%, M: 15 ± 3%, H: 23 ± 7%; 5 Hz: S: 8 ± 5%, M: 15 ± 3%, H: 17 ± 5%) and during contraction (2 Hz: S: 9 ± 3%, M: 18 ± 3%, H: 22 ± 6%; 5 Hz: S: 9 ± 5%, M: 22 ± 4%, H:26 ± 9%). Sympatholysis was greater (P < 0.05) in ET than in S rats. Blockade of α2‐adrenoreceptors and NOS reduced (P < 0.05) sympatholysis in ET rats, but had no effect on sympatholysis in S rats. In conclusion, ET increased α2‐mediated vasoconstriction at rest and during contraction.

  September 10, 2013   doi: 10.1113/jphysiol.2013.257626   open full text
• Are type III – IV muscle afferents required for a normal steady state exercise hyperpnea in humans?
  Jerome A. Dempsey, Gregory Blain, Markus Amann.
  The Journal of Physiology. September 03, 2013
  Abstract  When tested in isolation, stimuli associated with respiratory CO2 exchange, feedforward central command and type III‐IV muscle afferent feedback have each been shown to be capable of eliciting exercise‐like cardio‐ventilatory responses – but their relative contributions in a setting of physiologic exercise remains controversial. We reasoned that in order to determine whether any of these regulators are obligatory to the exercise hyperpnea requires that each be removed or significantly diminished in a setting of physiologic steady state exercise, during which all recognized stimuli (and other potential modulators) are normally operative. In the past few years we and others have used intrathecal fentanyl, a μ‐opiate receptor agonist, in humans to reduce the input from type III‐IV opiate sensitive muscle afferents. During various types of intensities and durations of exercise a sustained hypoventilation as well as reduced systemic pressure and cardioacceleration were consistently observed with this blockade. These data provide the basis for the hypothesis that type III‐IV muscle afferents are obligatory to the hyperpnea of mild through moderate intensity rhythmic, large muscle, steady‐state exercise. We discuss the limitations of these studies, the reasons for their disagreement with previous negative findings, the nature of the muscle afferent feedback stimulus and the need for future investigations. This article is protected by copyright. All rights reserved

  September 03, 2013   doi: 10.1113/jphysiol.2013.261925   open full text
• Recent data do not provide evidence that resveratrol causes “mainly negative” or “adverse” effects on exercise training in humans.
  James M. Smoliga, Otis L. Blanchard.
  The Journal of Physiology. September 03, 2013
  Abstract  It was with great interest that we read the article by Gliemann et al. (2013). The authors should be commended for using a solid study design to determine if resveratrol enhances the exercise response in humans as previously demonstrated in animal models. The authors concluded “exercise training effectively improves several cardiovascular health parameters in aged men, [but] resveratrol supplementation blunts most of these effects.” This article is protected by copyright. All rights reserved

  September 03, 2013   doi: 10.1113/jphysiol.2013.262956   open full text
• Response to Letter to the Editor.
  Lasse Gliemann, Jakob Schmidt, Jesper Olesen, Rasmus Sjørup Biensø, Sebastian Louis Peronard, Simon Udsen Grandjean, Stefan Peter Mortensen, Michael Nyberg, Jens Bangsbo, Henriette Pilegaard, Ylva Hellsten.
  The Journal of Physiology. September 03, 2013
  Abstract  We would like to thank the authors of the Letter to the Editor for the interest in our study and for the kind positive remarks regarding the study per se. Our understanding of the Letter is that there is a concern regarding over interpretation of the data. This article is protected by copyright. All rights reserved

  September 03, 2013   doi: 10.1113/jphysiol.2013.264093   open full text
• Interlimb communication to the knee flexors during walking in humans.
  Andrew J. T. Stevenson, Svend S. Geertsen, Jacob B. Andersen, Thomas Sinkjær, Jens B. Nielsen, Natalie Mrachacz‐Kersting.
  The Journal of Physiology. September 02, 2013
  •  Following unexpected ipsilateral knee extension joint rotations applied during the late stance phase of the gait cycle in humans, a crossed reflex response was observed in the contralateral biceps femoris (cBF) muscle with a mean onset latency of 76 ms. •  Transcranial magnetic and electrical stimulation applied to the primary motor cortex revealed that a transcortical pathway probably contributes to the cBF response. •  We hypothesize that the cBF response signifies a preparation of the contralateral leg for early load bearing, helping the body to maintain dynamic stability during walking. •  This is the first study to show that a transcortical pathway contributes to an interlimb reflex in upper leg muscles. The transcortical nature of the response may allow for more adaptable responses than purely spinally mediated reflexes due to integration with other sensory information. Abstract  A strong coordination between the two legs is important for maintaining a symmetric gait pattern and adapting to changes in the external environment. In humans as well as animals, receptors arising from the quadriceps muscle group influence the activation of ipsilateral muscles. Moreover, strong contralateral spinal connections arising from quadriceps and hamstring afferents have been shown in animal models. Therefore, the aims of the present study were to assess if such connections also exist in humans and to elucidate on the possible pathways. Contralateral reflex responses were investigated in the right leg following unexpected unilateral knee joint rotations during locomotion in either the flexion or extension direction. Strong reflex responses in the contralateral biceps femoris (cBF) muscle with a mean onset latency of 76 ± 6 ms were evoked only from ipsilateral knee extension joint rotations in the late stance phase. To investigate the contribution of a transcortical pathway to this response, transcranial magnetic and electrical stimulation were applied. Motor evoked potentials elicited by transcranial magnetic stimulation, but not transcranial electrical stimulation, were facilitated when elicited at the time of the cBF response to a greater extent than the algebraic sum of the cBF reflex and motor evoked potentials elicited separately, indicating that a transcortical pathway probably contributes to this interlimb reflex. The cBF reflex response may therefore be integrated with other sensory input, allowing for responses that are more flexible. We hypothesize that the cBF reflex response may be a preparation of the contralateral leg for early load bearing, slowing the forward progression of the body to maintain dynamic equilibrium during walking.

  September 02, 2013   doi: 10.1113/jphysiol.2013.257949   open full text
• Exposure to cocaine regulates inhibitory synaptic transmission from the ventral tegmental area to the nucleus accumbens.
  Masago Ishikawa, Mami Otaka, Peter A. Neumann, Zhijian Wang, James M. Cook, Oliver M. Schlüter, Yan Dong, Yanhua H. Huang.
  The Journal of Physiology. September 02, 2013
  •  Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway; in addition to the well‐known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, but the cellular nature of VTA‐to‐NAc fast synaptic transmission and its roles in drug‐induced adaptations are not well understood. •  Using optogenetic approaches, the present study profiled fast excitatory synaptic projection from dopaminergic neurons and inhibitory synaptic projection from GABAergic neurons in the VTA to NAc core (NAcCo) medium spiny neurons. •  We further identified that, following repeated non‐contingent exposure to cocaine, VTA‐to‐NAcCo inhibitory synaptic transmission appears to be enhanced by an increase in the presynaptic release probability. •  No postsynaptic alterations were detected at either excitatory or inhibitory synapses within the VTA‐to‐NAcCo projection. Abstract  Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway, a neural circuit that mediates behavioural responses elicited by natural rewards as well as by cocaine and other drugs of abuse. In addition to the well‐known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, directly regulating NAc medium spiny neurons (MSNs). However, the cellular nature of VTA‐to‐NAc fast synaptic transmission and its roles in drug‐induced adaptations are not well understood. Using viral‐mediated in vivo expression of channelrhodopsin 2, the present study dissected fast excitatory and inhibitory synaptic transmission from the VTA to NAc MSNs in rats. Our results suggest that, following repeated exposure to cocaine (15 mg kg−1 day−1× 5 days, i.p., 1 or 21 day withdrawal), a presynaptic enhancement of excitatory transmission and suppression of inhibitory transmission occurred at different withdrawal time points at VTA‐to‐NAc core synapses. In contrast, no postsynaptic alterations were detected at either type of synapse. These results suggest that changes in VTA‐to‐NAc fast excitatory and inhibitory synaptic transmissions may contribute to cocaine‐induced alteration of the brain reward circuitry.

  September 02, 2013   doi: 10.1113/jphysiol.2013.262915   open full text
• Online correction of licking‐induced brain motion during two‐photon imaging with a tunable lens.
  Jerry L. Chen, Oliver A. Pfäffli, Fabian F. Voigt, David J. Margolis, Fritjof Helmchen.
  The Journal of Physiology. September 02, 2013
  •  In order to understand the underlying behaviour of neuronal circuit dynamics, it is necessary to monitor brain activity in the awake, behaving animal. •  Licking to obtain water reward is an approach that is often used to measure an animal's decision during reward‐based behaviour tasks. •  In head‐fixed mice, licking produces stereotyped brain motion that interferes with two‐photon calcium imaging of neuronal activity. •  We describe a method to provide online optical correction of licking‐induced brain motion during two‐photon imaging using refocusing with an electrically tunable lens. •  Online correction of licking‐induced brain motion improves the measurement of neuronal activity during reward‐based behaviour. Abstract  Two‐photon calcium imaging in awake, head‐fixed animals enables the measurement of neuronal activity during behaviour. Often, licking for the retrieval of water reward is used as a measurable report of the animal's decision during reward‐driven behaviour. However, licking behaviour can induce severe motion artifacts that interfere with two‐photon imaging of cellular activity. Here, we describe a simple method for the online correction of licking‐induced focus shifts for two‐photon calcium imaging of neocortical neurons in the head‐fixed mouse. We found that licking causes a stereotyped drop of neocortical tissue, shifting neurons up to 20 μm out of focus. Based on the measurement of licking with a piezo film sensor, we developed a feedback model, which provides a corrective signal for fast optical focus adjustments with an electrically tunable lens. Using online correction with this feedback model, we demonstrate a reduction of licking‐related focus changes below 3 μm, minimizing motion artifact contamination of cellular calcium signals. Focus correction with a tunable lens is a simple and effective method to improve the ability to monitor neuronal activity during reward‐based behaviour.

  September 02, 2013   doi: 10.1113/jphysiol.2013.259804   open full text
• Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.
  Juan José Arnáiz‐Cot, Brooke James Damon, Xiao‐Hua Zhang, Lars Cleemann, Naohiro Yamaguchi, Gerhard Meissner, Martin Morad.
  The Journal of Physiology. August 30, 2013
  Abstract  Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca2+ concentrations. Point mutations in the CaM‐binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2ADA) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca2+ signalling properties of enzymatically isolated, Fluo‐4 dialysed whole cell clamped cardiac myocytes from 10–15‐day‐old wild‐type (WT) and homozygous Ryr2ADA/ADA mice. Spontaneously occurring Ca2+ spark frequency, measured at −80 mV, was 14‐fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca2+ transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine‐triggered Ca2+ transients were about three times larger in mutant myocytes, generating three‐ to four‐fold bigger Na+‐Ca2+ exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca2+ transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2ADA mutation produces significant reduction in ICa density and ICa‐triggered Ca2+ release gain, longer but infrequently occurring Ca2+ sparks, larger sarcoplasmic reticulum Ca2+ loads, and spontaneous Ca2+ releases accompanied by activation of large and potentially arrhythmogenic inward INCX.

  August 30, 2013   doi: 10.1113/jphysiol.2013.256123   open full text
• Action potential wavelength restitution predicts alternans and arrhythmia in murine Scn5a+/− hearts.
  Gareth D. K. Matthews, Laila Guzadhur, Ian N. Sabir, Andrew A. Grace, Christopher L.‐H. Huang.
  The Journal of Physiology. August 30, 2013
  •  Mice which are haploinsufficient in the Scn5a+/− gene have reduced cardiac sodium channel (Nav1.5) density and are used to model the Brugada syndrome. •  Conduction velocity restitution showed lower initial values and earlier points of failure during incremental pacing in the murine Scn5a+/− right ventricle (RV) epicardium particularly when treated with flecainide. •  The broadness of the conduction velocity restitution function was a poor indicator of arrhythmia or alternans. Conduction velocity alternans occurred abruptly and was more marked in the flecainide‐treated Scn5a+/− RV epicardium. •  Introduction of wavelength restitution yielded functions that converged to a common instability condition in contrast to action potential duration (APD) or conduction velocity restitution. •  This occurred at significantly lower heart rates in Scn5a+/− RV epicardium following flecainide or quinidine challenge, corresponding to a smaller total wavelength (basic cycle distance) resulting from a reduction in conduction velocity. •  Wavelength restitution was superior at predicting alternans than either APD or conduction velocity restitution. Abstract  Reductions in cardiac action potential wavelength, and the consequent wavebreak, have been implicated in arrhythmogenesis. Tachyarrhythmias are more common in the Brugada syndrome, particularly following pharmacological challenge, previously modelled using Scn5a+/− murine hearts. Propagation latencies and action potential durations (APDs) from monophasic action potential recordings were used to assess wavelength changes with heart rate in Langendorff‐perfused wild‐type (WT) and Scn5a+/− hearts. Recordings were obtained from right (RV) and left (LV) ventricular, epicardial and endocardial surfaces during incremental pacing, before and following flecainide or quinidine challenge. Conduction velocities (θ′), action potential wavelengths (λ′= APD ×θ′), and their corresponding alternans depended non‐linearly upon diastolic interval (DI). Maximum θ′ was lower in Scn5a+/− RV epicardium than endocardium. Flecainide further reduced θ′, accentuating this RV conduction block. Quinidine reduced maximum θ′ in WT and caused earlier conduction failure in the RV of both Scn5a+/− and WT. Use of recovery wavelengths (λ′0= DI ×θ′) rather than DI, provided novel λ restitution plots of λ′ against λ′0, which sum to a basic cycle distance permitting feedback analysis. λ′ restitution gradient better correlated with alternans magnitude than either APD or θ restitution gradient. The large differences in θ′ and APD restitution contrasted with minor differences in maximum λ′ between epi‐ and endocardia of untreated hearts, and quinidine‐treated WT hearts. Strikingly, all regions and conditions converged to a common instability point, implying a conserved relationship. Flecainide or quinidine decreased the pacing rates at which this occurred, through reducing basic cycle distance, in the Scn5a+/− RV epicardium, directly predictive of its arrhythmic phenotype.

  August 30, 2013   doi: 10.1113/jphysiol.2013.254938   open full text
• Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.
  Norbert Jost, László Virág, Philippe Comtois, Balázs Ördög, Viktória Szuts, György Seprényi, Miklós Bitay, Zsófia Kohajda, István Koncz, Norbert Nagy, Tamás Szél, János Magyar, Mária Kovács, László G. Puskás, Csaba Lengyel, Erich Wettwer, Ursula Ravens, Péter P. Nánási, Julius Gy. Papp, András Varró, Stanley Nattel.
  The Journal of Physiology. August 30, 2013
  •  Cardiac repolarization, through which heart‐cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical conditions. •  Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man. •  Here, we studied the contribution of various ion‐currents to cardiac repolarization in canine and human ventricle. •  Humans showed much greater repolarization‐impairing effects of drugs blocking the rapid delayed‐rectifier current IKr than dogs, because of lower repolarization‐reserve contributions from two other important repolarizing currents (the inward‐rectifier IK1 and slow delayed‐rectifier IKs). •  Our findings clarify differences in cardiac repolarization‐processes among species, highlighting the importance of caution when extrapolating results from animal models to man. Abstract  The species‐specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole‐cell patch‐clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50–100 nmol l−1 dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3‐fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l−1 BaCl2) and IKs block (1 μmol l−1 HMR‐1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3‐ and 4.5‐fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ∼30% larger and ∼29% smaller in human, and Na+–Ca2+ exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKsα‐subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD‐prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human–canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization‐delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species‐specific determinants of repolarization and the limitations of animal models for human disease.

  August 30, 2013   doi: 10.1113/jphysiol.2013.261198   open full text
• Evoked centripetal Ca2+ mobilization in cardiac Purkinje cells: insight from a model of three Ca2+ release regions.
  Kazi T. Haq, Rebecca E. Daniels, Lawson S. Miller, Masahito Miura, Henk E. D. J. ter Keurs, Sharene D. Bungay, Bruno D. Stuyvers.
  The Journal of Physiology. August 30, 2013
  •  Abnormal oscillations of calcium (Ca2+) concentration in cardiac Purkinje cells (P‐cells) have been associated with life‐threatening arrhythmias, but the mechanism by which these cells control their Ca2+ level in normal conditions remains unknown. •  We modelled our previous hypothesis that the principal intracellular Ca2+ compartment (endoplasmic reticulum; ER) which governs intracellular Ca2+ concentration, formed, in P‐cells, three concentric and adjacent layers, each including a distinct Ca2+ release channel. We then tested the model against typical Ca2+ variations observed in stimulated P‐cells. •  We found in swine P‐cells, as in the rabbit and dog, that stimulation evokes an elevation of Ca2+ concentration first under the membrane, which then propagates to the interior of the cell. •  Our mathematical model could reproduce accurately this typical ‘centripetal’ Ca2+ spread, hence supporting (1) the existence of the ‘3 layered’ Ca2+ compartment, and (2) its central role in the regulation of Ca2+ concentration in P‐cells. •  To model the ‘centripetal’ Ca2+ spread, local variations of Ca2+ concentration were calculated for a virtual cell environment encompassing three different regions that mimicked the three layers of ER in P‐cells. Various tests of the model revealed that the second intermediate layer was essential for ‘forwarding’ the Ca2+ elevation from the periphery to the cell centre. •  This novel finding suggests that a thin intermediate layer of specific ER Ca2+ channels controls the entire Ca2+ signalling of P‐cells. Because Ca2+ plays a role in the electric properties of P‐cells, any abnormality affecting this intermediate region is likely to be pro‐arrhythmic and could explain the origin of serious cardiac arrhythmias known to start in the Purkinje fibres. Abstract  Despite strong suspicion that abnormal Ca2+ handling in Purkinje cells (P‐cells) is implicated in life‐threatening forms of ventricular tachycardias, the mechanism underlying the Ca2+ cycling of these cells under normal conditions is still unclear. There is mounting evidence that P‐cells have a unique Ca2+ handling system. Notably complex spontaneous Ca2+ activity was previously recorded in canine P‐cells and was explained by a mechanistic hypothesis involving a triple layered system of Ca2+ release channels. Here we examined the validity of this hypothesis for the electrically evoked Ca2+ transient which was shown, in the dog and rabbit, to occur progressively from the periphery to the interior of the cell. To do so, the hypothesis was incorporated in a model of intracellular Ca2+ dynamics which was then used to reproduce numerically the Ca2+ activity of P‐cells under stimulated conditions. The modelling was thus performed through a 2D computational array that encompassed three distinct Ca2+ release nodes arranged, respectively, into three consecutive adjacent regions. A system of partial differential equations (PDEs) expressed numerically the principal cellular functions that modulate the local cytosolic Ca2+ concentration (Cai). The apparent node‐to‐node progression of elevated Cai was obtained by combining Ca2+ diffusion and ‘Ca2+‐induced Ca2+ release’. To provide the modelling with a reliable experimental reference, we first re‐examined the Ca2+ mobilization in swine stimulated P‐cells by 2D confocal microscopy. As reported earlier for the dog and rabbit, a centripetal Ca2+ transient was readily visible in 22 stimulated P‐cells from six adult Yucatan swine hearts (pacing rate: 0.1 Hz; pulse duration: 25 ms, pulse amplitude: 10% above threshold; 1 mm Ca2+; 35°C; pH 7.3). An accurate replication of the observed centripetal Ca2+ propagation was generated by the model for four representative cell examples and confirmed by statistical comparisons of simulations against cell data. Selective inactivation of Ca2+ release regions of the computational array showed that an intermediate layer of Ca2+ release nodes with an ∼30–40% lower Ca2+ activation threshold was required to reproduce the phenomenon. Our computational analysis was therefore fully consistent with the activation of a triple layered system of Ca2+ release channels as a mechanism of centripetal Ca2+ signalling in P‐cells. Moreover, the model clearly indicated that the intermediate Ca2+ release layer with increased sensitivity for Ca2+ plays an important role in the specific intracellular Ca2+ mobilization of Purkinje fibres and could therefore be a relevant determinant of cardiac conduction.

  August 30, 2013   doi: 10.1113/jphysiol.2013.253583   open full text
• Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex.
  Dongxu Guan, William E. Armstrong, Robert C. Foehring.
  The Journal of Physiology. August 29, 2013
  •  Neurons express many types of potassium channels that are activated by voltage but relatively little is known concerning the division of labour between different channel types in a given cell. •  Our understanding of the functional roles of Kv2 channels has been hindered by the lack of selective pharmacological agents for these channels. •  We manipulated Kv2 channel expression by transfecting pyramidal neurons with wild‐type and pore mutant channels. •  We found that reduction in functional Kv2 channels led to slower firing rates, reduced gain of firing and increased spike frequency adaptation. •  We hypothesize that Kv2 channels regulate firing by controlling membrane potential during the inter‐spike interval, which in turn regulates availability of voltage‐gated sodium channels. Abstract  The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7‐P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold ‘bullets’ coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non‐transfected cell from the same layer and area. Whole‐cell voltage‐clamp recordings obtained after 3–7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from –78 to –2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current‐clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady‐state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na+ channels from inactivation. Depolarization and the number of inactivated Na+ channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

  August 29, 2013   doi: 10.1113/jphysiol.2013.257253   open full text
• The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site.
  Marco Caremani, Luca Melli, Mario Dolfi, Vincenzo Lombardi, Marco Linari.
  The Journal of Physiology. August 29, 2013
  •  Force and shortening in muscle are caused by ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies indicate that the working stroke consists in an interdomain movement of the myosin motor associated with the release of inorganic phosphate (Pi). •  Here the coupling of the working stroke with the release of Pi is studied in situ using fast half‐sarcomere mechanics on skinned fibres from rabbit psoas. •  The isotonic velocity transient following stepwise force reductions superimposed on isometric contraction measures the mechanical manifestation of the working stroke and its rate of regeneration. •  The results indicate that the release of Pi from the catalytic site of an actin‐attached myosin motor can occur at any stage of the working stroke, and a myosin motor uses two consecutive actin monomers to maximize the power during shortening. Abstract  Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light‐chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half‐sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo‐mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half‐sarcomere.

  August 29, 2013   doi: 10.1113/jphysiol.2013.257410   open full text
• Permeability and contractile responses of collecting lymphatic vessels elicited by atrial and brain natriuretic peptides.
  Joshua P. Scallan, Michael J. Davis, Virginia H. Huxley.
  The Journal of Physiology. August 29, 2013
  •  Atrial and brain natriuretic peptides (ANP and BNP, respectively) are hormones released into the bloodstream when heart muscle is stretched (e.g. zero‐gravity, hypertension, congestive heart failure) and serve to reduce the blood volume. •  One way that these peptides relieve blood volume is to increase the permeability of the smallest blood vessels, facilitating fluid and protein distribution into the tissue spaces. •  Whether these peptides target lymphatic vessel function to participate in fluid distribution is currently unknown. •  ANP and BNP (100 nm) both elicited significant increases in lymphatic vessel permeability, but altered contractile function differentially in vivo. •  A likely consequence is that more fluid leaks from the lymphatics into the tissues, which represents a novel compensation for volume overload. This work demonstrates for the first time that lymphatic vessel permeability can be regulated in vivo. Abstract  Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones released into the bloodstream in response to hypervolaemia or fluid shifts to the central circulation. The actions of both peptides include natriuresis and diuresis, a decrease in systemic blood pressure, and inhibition of the renin–angiotensin–aldosterone system. Further, ANP and BNP elicit increases in blood microvessel permeability sufficient to cause protein and fluid extravasation into the interstitium to reduce the vascular volume. Given the importance of the lymphatic vasculature in maintaining fluid balance, we tested the hypothesis that ANP or BNP (100 nm) would likewise elevate lymphatic permeability (Ps) to serum albumin. Using a microfluorometric technique adapted to in vivo lymphatic vessels, we determined that rat mesenteric collecting lymphatic Ps to rat serum albumin increased by 2.0 ± 0.4‐fold (P= 0.01, n= 7) and 2.7 ± 0.8‐fold (P= 0.07, n= 7) with ANP and BNP, respectively. In addition to measuring Ps responses, we observed changes in spontaneous contraction amplitude and frequency from the albumin flux tracings in vivo. Notably, ANP abolished spontaneous contraction amplitude (P= 0.005) and frequency (P= 0.006), while BNP augmented both parameters by ∼2‐fold (P < 0.01 each). These effects of ANP and BNP on contractile function were examined further by using an in vitro assay. In aggregate, these data support the theory that an increase in collecting lymphatic permeability opposes the absorptive function of the lymphatic capillaries, and aids in the retention of protein and fluid in the interstitial space to counteract volume expansion.

  August 29, 2013   doi: 10.1113/jphysiol.2013.260042   open full text
• Teleost fish models in membrane transport research: the PEPT1(SLC15A1) H+/oligopeptide transporter as a case study†.
  Alessandro Romano, Amilcare Barca, Carlo Storelli, Tiziano Verri.
  The Journal of Physiology. August 27, 2013
  Abstract  Human genes for passive, ion‐coupled transporters and exchangers are included in the so‐called Solute Carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC‐like form in 50 families and at least 338 genes after systematic GenBank database mining (December 2010‐March 2011). When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary “molecular diversity” with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+/oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g. the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g. the adaptation of icefish PEPT1 to sub‐zero temperatures) and/or substrate specificity (e.g. the species‐specificity of PEPT1 for the uptake of L‐lysine‐containing peptides). The keen estimation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g. molecular structure‐function analyses) and applied research (e.g. optimising diets to enhance growth of commercially valuable fish). This article is protected by copyright. All rights reserved

  August 27, 2013   doi: 10.1113/jphysiol.2013.259622   open full text
• Laryngeal and tracheal afferent nerve stimulation evokes swallowing in anaesthetized guinea pigs.
  Takanori Tsujimura, Chioma Udemgba, Makoto Inoue, Brendan J. Canning.
  The Journal of Physiology. August 23, 2013
  •  Activation of vagal afferent nerves innervating the tracheal and laryngeal mucosa evokes swallowing in anaesthetized guinea pigs. •  The swallowing evoked by airway stimulation was probably initiated by the activation of the same afferent nerves that regulate coughing but occurred at lower stimulus intensities and was less susceptible to desensitization than the cough reflex. •  We speculate that the swallowing associated with airway sensory nerve activation is a key component of airways protection from aspiration. Abstract  We describe swallowing reflexes evoked by laryngeal and tracheal vagal afferent nerve stimulation in anaesthetized guinea pigs. The swallowing reflexes evoked by laryngeal citric acid challenges were abolished by recurrent laryngeal nerve (RLN) transection and mimicked by electrical stimulation of the central cut ends of an RLN. By contrast, the number of swallows evoked by upper airway/pharyngeal distensions was not significantly reduced by RLN transection but they were virtually abolished by superior laryngeal nerve transection. Laryngeal citric acid‐evoked swallowing was mimicked by laryngeal capsaicin challenges, implicating transient receptor potential vanilloid 1 (TRPV1)‐expressing laryngeal afferent nerves arising from the jugular ganglia. The swallowing evoked by citric acid and capsaicin and evoked by electrical stimulation of either the tracheal or the laryngeal mucosa occurred at stimulation intensities that were typically subthreshold for evoking cough in these animals. Swallowing evoked by airway afferent nerve stimulation also desensitized at a much slower rate than cough. We speculate that swallowing is an essential component of airway protection from aspiration associated with laryngeal and tracheal afferent nerve activation.

  August 23, 2013   doi: 10.1113/jphysiol.2013.256024   open full text
• A membrane glucocorticoid receptor mediates the rapid/non‐genomic actions of glucocorticoids in mammalian skeletal muscle fibres.
  María Hernández‐Alcalá Pérez, Jonathan Cormack, David Mallinson, Gabriel Mutungi.
  The Journal of Physiology. August 23, 2013
  •  Glucocorticoids are stress hormones used in the treatment of many chronic inflammatory diseases including asthma. They exert most of their physiological/pharmacological actions by regulating the activity of genes involved in the inflammatory response. However, they also have rapid/non‐genomic effects whose functions are poorly understood. •  In this study we used two widely prescribed glucocorticoids, beclomethasone dipropionate and prednisolone acetate, to investigate whether these hormones have rapid/non‐genomic effects in mammalian skeletal muscles. •  Both glucocorticoids increased maximum force in slow‐twitch muscle fibres/cells without significantly affecting that of fast‐twitch muscle fibres. •  The increase in force occurred within 10 min and was blocked by an inhibitor of the glucocorticoid receptor and a protein (antibody) that binds the receptor. •  These findings suggest that these hormones/drugs have rapid/non‐genomic effects in mammalian skeletal muscles; these effects are mediated by a membrane glucocorticoid receptor and are physiologically/pharmacologically beneficial, especially in slow muscles. Abstract  Glucocorticoids (GCs) are steroid hormones released from the adrenal gland in response to stress. They are also some of the most potent anti‐inflammatory and immunosuppressive drugs currently in clinical use. They exert most of their physiological and pharmacological actions through the classical/genomic pathway. However, they also have rapid/non‐genomic actions whose physiological and pharmacological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the rapid/non‐genomic effects of two widely prescribed glucocorticoids, beclomethasone dipropionate (BDP) and prednisolone acetate (PDNA), on force production in isolated, intact, mouse skeletal muscle fibre bundles. The results show that the effects of both GCs on maximum isometric force (Po) were fibre‐type dependent. Thus, they increased Po in the slow‐twitch fibre bundles without significantly affecting that of the fast‐twitch fibre bundles. The increase in Po occurred within 10 min and was insensitive to the transcriptional inhibitor actinomycin D. Also, it was maximal at ∼250 nm and was blocked by the glucocorticoid receptor (GCR) inhibitor RU486 and a monoclonal anti‐GCR, suggesting that it was mediated by a membrane (m) GCR. Both muscle fibre types expressed a cytosolic GCR. However, a mGCR was present only in the slow‐twitch fibres. The receptor was more abundant in oxidative than in glycolytic fibres and was confined mainly to the periphery of the fibres where it co‐localised with laminin. From these findings we conclude that the rapid/non‐genomic actions of GCs are mediated by a mGCR and that they are physiologically/therapeutically beneficial, especially in slow‐twitch muscle fibres.

  August 23, 2013   doi: 10.1113/jphysiol.2013.256586   open full text
• Neuronal major histocompatibility complex class I molecules are implicated in the generation of asymmetries in hippocampal circuitry.
  Aiko Kawahara, Shotaro Kurauchi, Yuko Fukata, José Martínez‐Hernández, Terumi Yagihashi, Yuya Itadani, Rui Sho, Taiichi Kajiyama, Nao Shinzato, Kenji Narusuye, Masaki Fukata, Rafael Luján, Ryuichi Shigemoto, Isao Ito.
  The Journal of Physiology. August 23, 2013
  •  The molecular basis of left–right asymmetries in brain structure and function is a central question in neuroscience. •  We have previously demonstrated that the neuronal circuitry composed of hippocampal pyramidal neurones is asymmetrical depending on the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. •  In this study, we analysed the hippocampus of β2‐microglobulin (β2m)‐deficient mice lacking stable cell surface expression of major histocompatibility complex class I (MHCI), which is known to be important in cellular immunity. •  We found that β2m‐deficient mice lacked structural and functional asymmetries in hippocampal circuitry, suggesting that MHCI is critical for the generation of hippocampal asymmetry. •  Our results provide a first step in elucidating the cellular process that generates brain asymmetries. Abstract  Left–right asymmetry is a fundamental feature of higher‐order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluRɛ2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of ɛ2 subunits affects the properties of NMDARs and generates two populations of synapses, ‘ɛ2‐dominant’ and ‘ɛ2‐non‐dominant’ synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of β2‐microglobulin (β2m)‐deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity‐dependent refinement of neuronal connections and normal synaptic plasticity. We found that β2m proteins were localised in hippocampal synapses in wild‐type mice. NMDA EPSCs in β2m‐deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25‐6981, an ɛ2 subunit‐selective antagonist, with those in ‘ɛ2‐dominant’ synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the β2m‐deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of ‘ɛ2‐dominant’ synapses. These observations indicate that the β2m‐deficient hippocampus lacks ‘ɛ2‐non‐dominant’ synapses and circuit asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.

  August 23, 2013   doi: 10.1113/jphysiol.2013.252122   open full text
• Mechanisms of retroaxonal barrage firing in hippocampal interneurons.
  Mark E. J. Sheffield, Gabrielle B. Edgerton, Robert J. Heuermann, Tara Deemyad, Brett D. Mensh, Nelson Spruston.
  The Journal of Physiology. August 23, 2013
  •  Persistent firing can be triggered in a population of inhibitory interneurons found in the hippocampus and neocortex. Repeated stimulation eventually triggers an autonomous barrage of spikes that is generated and maintained in the axon, followed by antidromic propagation to the soma. •  This barrage of spikes is generated and maintained in the axon, followed by antidromic propagation to the soma. The mechanisms underlying this ‘retroaxonal barrage firing’ are unknown. •  We find that retroaxonal barrage firing is Ca2+ dependent, is inhibited by the L‐type Ca2+ channel blockers cadmium, nifedipine and verapamil, and does not require synaptic transmission. Loading the stimulated interneuron with BAPTA did not block barrage firing, suggesting that the required Ca2+ entry may occur in other cells. •  Retroaxonal barrage firing was observed in mice lacking the Cx36 isoform (most common neuronal isoform), indicating that this particular isoform is not required. Abstract  We recently described a new form of neural integration and firing in a subset of interneurons, in which evoking hundreds of action potentials over tens of seconds to minutes produces a sudden barrage of action potentials lasting about a minute beyond the inciting stimulation. During this persistent firing, action potentials are generated in the distal axon and propagate retrogradely to the soma. To distinguish this from other forms of persistent firing, we refer to it here as ‘retroaxonal barrage firing’, or ‘barrage firing’ for short. Its induction is blocked by chemical inhibitors of gap junctions and curiously, stimulation of one interneuron in some cases triggers barrage firing in a nearby, unstimulated interneuron. Beyond these clues, the mechanisms of barrage firing are unknown. Here we report new results related to these mechanisms. Induction of barrage firing was blocked by lowering extracellular calcium, as long as normal action potential threshold was maintained, and it was inhibited by blocking L‐type voltage‐gated calcium channels. Despite its calcium dependence, barrage firing was not prevented by inhibiting chemical synaptic transmission. Furthermore, loading the stimulated/recorded interneuron with BAPTA did not block barrage firing, suggesting that the required calcium entry occurs in other cells. Finally, barrage firing was normal in mice with deletion of the primary gene for neuronal gap junctions (connexin36), suggesting that non‐neuronal gap junctions may be involved. Together, these findings suggest that barrage firing is probably triggered by a multicellular mechanism involving calcium signalling and gap junctions, but operating independently of chemical synaptic transmission.

  August 23, 2013   doi: 10.1113/jphysiol.2013.258418   open full text
• Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice.
  Sara A. Olenich, Navarre Gutierrez‐Reed, Gerald N. Audet, I. Mark Olfert.
  The Journal of Physiology. August 20, 2013
  •  Angiogenic regulators respond to acute exercise with different temporal expression patterns (e.g. 2–4 h versus 12–24 h) creating a complex multifaceted response that must be considered in studies using a single time point for post‐exercise analyses. •  In response to chronic training there appears to be a complex coordination in the proteomic responses of both positive and negative angiogenic factors that correspond with training‐induced muscle capillary adaptation, such that altered basal expression and acute responses to exercise appear to withdraw or reduce the stimulus of angiogenic regulators in an expanding capillary bed with active angiogenesis. •  These are the first data to show that nucleolin (a protein responsible for transcriptional processing and transportation of proteins from the cytoplasm to the nucleus) is responsive to acute exercise. We speculate that nucleolin may work in concert with vascular endothelial growth factor‐A (VEGF) and endostatin. •  Temporal responses observed in mice, particularly for VEGF, MMP‐2 and MMP‐9, may not be directly comparable to humans. Abstract  Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro‐angiogenic and anti‐angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor‐A (VEGF), endostatin and nucleolin were increased at 2–4 h (P < 0.05), whereas matrix metalloproteinase (MMP)‐2 was elevated within a 12–24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP‐2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP‐1) was ∼900% greater in 14d‐ and 28d‐trained mice compared to either 5d‐ and 7d‐trained mice (P < 0.05), and tended to increase by ∼180–258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly‐coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.

  August 20, 2013   doi: 10.1113/jphysiol.2013.254979   open full text
• Intrauterine inflammation alters fetal cardiopulmonary and cerebral haemodynamics in sheep.
  Robert Galinsky, Stuart B. Hooper, Graeme R. Polglase, Timothy J. M. Moss.
  The Journal of Physiology. August 20, 2013
  •  Intrauterine inflammation impairs fetal pulmonary vascular development and increases cerebral metabolism in fetal sheep. Whether these structural and metabolic changes have functional consequences for fetal cardiopulmonary and cerebral haemodynamics is presently unknown. •  We demonstrated that intra‐amniotic administration of lipopolysaccharide increased fetal pulmonary vascular resistance, and reduced pulmonary blood flow and carotid arterial oxygen content in the fetus and caused a transient increase in fetal cerebral blood flow. These effects occurred over a time course consistent with previously observed effects on pulmonary vascular development and cerebral metabolism. •  Our data suggest that pathophysiological changes in cardiopulmonary and cerebral haemodynamics observed in fetuses exposed to intrauterine inflammation may be present and detectable in human pregnancies, offering potential for detecting fetuses affected by intrauterine inflammation. Abstract  Intrauterine inflammation impairs fetal pulmonary vascular development and increases cerebral metabolic rate in fetal sheep. We hypothesized that these structural and metabolic effects of intrauterine inflammation would be accompanied by reduced fetal pulmonary blood flow and increased cerebral perfusion. Fetal sheep were instrumented at 112 days of gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days ewes were randomly assigned to receive intra‐amniotic lipopolysaccharide (LPS, 20 mg from Escherichia coli; n= 7) or saline (control, 4 ml; n= 6). Fetal haemodynamic data were recorded continually from 1 h before intra‐amniotic LPS or saline, until 144 h after. Fetal arterial blood was sampled before, and periodically after, intra‐amniotic LPS or saline. End‐diastolic and mean pulmonary blood flows were significantly lower than control from 48 and 96 h after LPS exposure, respectively, until the end of the experiment. Carotid blood flow was transiently increased at 96 and 120 h after LPS exposure. Carotid arterial oxygen content was lower than control from 48 h after intra‐amniotic LPS. Fetal arterial lactate concentration was higher than control between 4 and 12 h after intra‐amniotic LPS. Experimental intrauterine inflammation reduces pulmonary blood flow in fetal sheep, over a time course consistent with impaired pulmonary vascular development. Increased carotid blood flow after LPS administration may reflect an inflammation‐induced increase in cerebral metabolic demand.

  August 20, 2013   doi: 10.1113/jphysiol.2013.259119   open full text
• Transcriptional and functional regulation of the intestinal peptide transporter PEPT1.
  Britta Spanier.
  The Journal of Physiology. August 20, 2013
  Abstract  Dietary proteins are cleaved within the intestinal lumen to oligopeptides which are further processed to small peptides (di‐ and tripeptides) and free amino acids. While the transport of amino acids is mediated by several specific amino acid transporters, the proton‐coupled uptake of the more than 8000 different di‐ and tripeptides is performed by the high‐capacity/low‐affinity peptide transporter isoform PEPT1 (SLC15A1). Its wide substrate tolerance also allows the transport of a repertoire of structurally closely related compounds and drugs, which explains their high oral bioavailability and brings PEPT1 into focus of medical and pharmaceutical approaches. Although first evidence for the interplay of nutrient supply and PEPT1 expression and function had been described over 20 years ago, many aspects of the molecular processes controlling its transcription and translation and modifying its transporter properties are still awaiting discovery. The present review summarizes the recent knowledge about factors modulating PEPT1 expression and function in C. elegans, D. rerio, M. musculus and H. sapiens with focus on dietary ingredients, transcription factors and functional modulators like the sodium‐proton exchanger NHE3 and selected scaffold proteins. This article is protected by copyright. All rights reserved

  August 20, 2013   doi: 10.1113/jphysiol.2013.258889   open full text
• Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men.
  Lasse Gliemann, Jakob Friis Schmidt, Jesper Olesen, Rasmus Sjørup Biensø, Sebastian Louis Peronard, Simon Udsen Grandjean, Stefan Peter Mortensen, Michael Nyberg, Jens Bangsbo, Henriette Pilegaard, Ylva Hellsten.
  The Journal of Physiology. August 16, 2013
  •  In rodents, resveratrol has been shown to enhance training‐induced changes in cardiovascular function, exercise performance and the retardation of atherosclerosis. We examined the effect of 8 weeks of exercise training with and without concomitant resveratrol supplementation in aged men. •  Exercise training potently improved blood pressure, blood cholesterol, maximal oxygen uptake and the plasma lipid profile. •  Resveratrol supplementation reduced the positive effect of exercise training on blood pressure, blood cholesterol and maximal oxygen uptake and did not affect the retardation of atherosclerosis. •  Whereas exercise training improved formation of the vasodilator prostacyclin, concomitant resveratrol supplementation caused a shift in vasoactive systems favouring vasoconstriction. •  The present study is the first to demonstrate negative effects of resveratrol on training‐induced improvements in cardiovascular health parameters in humans and adds to the growing body of evidence questioning the positive effects of resveratrol supplementation in humans. Abstract  Ageing is thought to be associated with decreased vascular function partly due to oxidative stress. Resveratrol is a polyphenol, which in animal studies has been shown to decrease atherosclerosis, and improve cardiovascular health and physical capacity, in part through its effects on Sirtuin 1 signalling and through an improved antioxidant capacity. We tested the hypothesis that resveratrol supplementation enhances training‐induced improvements in cardiovascular health parameters in aged men. Twenty‐seven healthy physically inactive aged men (age: 65 ± 1 years; body mass index: 25.4 ± 0.7 kg m−2; mean arterial pressure (MAP): 95.8 ± 2.2 mmHg; maximal oxygen uptake: 2488 ± 72 ml O2 min−1) were randomized into 8 weeks of either daily intake of either 250 mg trans‐resveratrol (n= 14) or of placebo (n= 13) concomitant with high‐intensity exercise training. Exercise training led to a 45% greater (P < 0.05) increase in maximal oxygen uptake in the placebo group than in the resveratrol group and to a decrease in MAP in the placebo group only (−4.8 ± 1.7 mmHg; P < 0.05). The interstitial level of vasodilator prostacyclin was lower in the resveratrol than in the placebo group after training (980 ± 90 vs. 1174 ± 121 pg ml−1; P < 0.02) and muscle thromboxane synthase was higher in the resveratrol group after training (P < 0.05). Resveratrol administration also abolished the positive effects of exercise on low‐density lipoprotein, total cholesterol/high‐density lipoprotein ratio and triglyceride concentrations in blood (P < 0.05). Resveratrol did not alter the effect of exercise training on the atherosclerosis marker vascular cell adhesion molecule 1 (VCAM‐1). Sirtuin 1 protein levels were not affected by resveratrol supplementation. These findings indicate that, whereas exercise training effectively improves several cardiovascular health parameters in aged men, concomitant resveratrol supplementation can blunt these effects.

  August 16, 2013   doi: 10.1113/jphysiol.2013.258061   open full text
• Contraction‐induced lipolysis is not impaired by inhibition of hormone‐sensitive lipase in skeletal muscle.
  Thomas J. Alsted, Thorkil Ploug, Clara Prats, Annette K. Serup, Louise Høeg, Peter Schjerling, Cecilia Holm, Robert Zimmermann, Christian Fledelius, Henrik Galbo, Bente Kiens.
  The Journal of Physiology. August 16, 2013
  •  In skeletal muscle hormone‐sensitive lipase (HSL) is considered the only enzyme responsible for breakdown of intramyocellular triacylglycerol (IMTG) during contractions. This notion is based on indirect measures in which important cellular events are not taken into account. •  Using two histochemical techniques to measure breakdown of IMTG during contractions in isolated skeletal muscles we found that IMTG was decreased (1) in rat muscles during acute pharmacological blockade of HSL, and (2) in muscles of HSL knockout mice. •  We demonstrated that adipose triglyceride lipase (ATGL) and HSL collectively account for at least 98% of the total TG lipase activity in mouse muscle, and other TG lipases accordingly seem of negligible importance for breakdown of IMTG. •  In conclusion, breakdown of IMTG occurs in the contracting muscle in the absence of HSL activity. Our data suggest that ATGL is activated during contractions and plays a major role in breakdown of IMTG. Abstract  In skeletal muscle hormone‐sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL‐KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction‐induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL‐KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction‐induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

  August 16, 2013   doi: 10.1113/jphysiol.2013.260794   open full text
• The fast exercise drive to breathe.
  James Duffin.
  The Journal of Physiology. August 16, 2013
  Abstract  This paper presents a personal view of research into the exercise drive to breathe that can be observed to act immediately to increase breathing at the start of rhythmic exercise. It is based on a talk given at the Experimental Biology 2013 meeting in a session entitled ‘Recent advances in understanding mechanisms regulating breathing during exercise’. This drive to breathe has its origin in a combination of central command, whereby voluntary motor commands to the exercising muscles produce a concurrent respiratory drive, and afferent feedback, whereby afferent information from the exercising muscles affects breathing. The drive at the start and end of rhythmic exercise is proportional to limb movement frequency, and its magnitude decays as exercise continues so that the immediate decrease of ventilation at the end of exercise is about 60% of the immediate increase at the start. With such evidence for the effect of this fast drive to breathe at the start and end of rhythmic exercise, its existence during exercise is hypothesised. Experiments to test this hypothesis have however provided debatable evidence. A fast drive to breathe during both ramp and sine wave changes in treadmill exercise speed and grade appears to be present some individuals, but not so evident in the general population. Recent sine‐wave cycling experiments show that when cadence is varied sinusoidally the ventilation response lags by about 10 s, whereas when pedal loading is varied ventilation lags by about 30 s. It therefore appears that limb movement frequency is effective in influencing ventilation during exercise as well as at the start and end of exercise. This article is protected by copyright. All rights reserved

  August 16, 2013   doi: 10.1113/jphysiol.2013.258897   open full text
• Defining the neuro‐circuitry of exercise hyperpnoea.
  David J. Paterson.
  The Journal of Physiology. August 16, 2013
  Abstract  One hundred years ago in this Journal, Krogh and Lindhard published a seminal paper highlighting the importance of the brain in the control of breathing during exercise. This symposium report reviews the historical developments that have taken place since 1913, and attempts to place the detailed neuro‐circuitry thought to underpin exercise hyperpnoea into context by focusing on key structures that might form the command network. With the advent of enhanced neuroimaging and functional neurosurgical techniques, a unique window of opportunity has recently arisen to target potential circuits in humans. Animal studies have identified a priori sites of interest in mid‐brain structures, in particular the sub‐thalamic locomotor region (STN) and the periaqueductal grey (PAG) that have now been recorded from in humans during exercise. When all data are viewed in an integrative manner, the PAG, in particular the lateral PAG, and aspects of the dorsal lateral PAG, appear to be key communicating circuitry for ‘central command’. Moreover, the PAG also fulfils many requirements of a command centre. It has functional connectivity to higher centres (dorsal lateral pre‐frontal cortex), the basal ganglia (in particular the STN), and receives a sensory input from contracting muscle, but importantly, it sends efferent information to brain stem nuclei involved in cardio‐respiratory control. This article is protected by copyright. All rights reserved

  August 16, 2013   doi: 10.1113/jphysiol.2013.261586   open full text
• The role of Ca2+ in the pathophysiology of pancreatitis.
  Julia V. Gerasimenko, Oleg V. Gerasimenko, Ole H. Petersen.
  The Journal of Physiology. August 16, 2013
  Abstract  Acute pancreatitis is a human disease in which the pancreatic pro‐enzymes, packaged into the zymogen granules of the acinar cells, become activated and cause auto‐digestion. The main causes of pancreatitis are alcohol abuse and biliary disease. A considerable body of evidence indicates that the primary event initiating the disease process is excessive release of Ca2+ from intracellular stores, followed by excessive entry of Ca2+ from the interstitial fluid. However, Ca2+ release and subsequent entry are also precisely the processes that control physiological secretion of the digestive enzymes in response to stimulation via the vagal nerve or the hormone cholecystokinin. The spatial and temporal Ca2+ signal patterns in physiology and pathology, as well as the contributions from different organelles in the different situations, are therefore critical issues. There has recently been significant progress in our understanding of both physiological stimulus‐secretion coupling and the pathophysiology of acute pancreatitis. Very recently, a promising potential therapeutic development has occurred with the demonstration that blockade of Ca2+ release‐activated Ca2+ currents in pancreatic acinar cells offers remarkable protection against Ca2+ overload, intracellular protease activation and necrosis evoked by a combination of alcohol and fatty acids, which is a major trigger of acute pancreatitis. This article is protected by copyright. All rights reserved

  August 16, 2013   doi: 10.1113/jphysiol.2013.261784   open full text
• Smooth muscle cell transient receptor potential polycystin‐2 (TRPP2) channels contribute to the myogenic response in cerebral arteries.
  Damodaran Narayanan, Simon Bulley, M. Dennis Leo, Sarah K. Burris, Kyle S. Gabrick, Frederick A. Boop, Jonathan H. Jaggar.
  The Journal of Physiology. August 15, 2013
  •  Intravascular pressure is reported to activate several mechanosensitive ion channels, leading to smooth muscle cell (SMC) depolarization, voltage‐dependent Ca2+ channel activation and vasoconstriction; a process known as the ‘myogenic response’. •  Polycystin‐1 and ‐2 (TRPP1 and ‐2) have been shown to differentially regulate the mesenteric artery myogenic response, with TRPP2 expression attenuating vasoconstriction. •  We show that TRPP2 is the major TRPP isoform expressed and that TRPP2 is located primarily in the plasma membrane in cerebral artery SMCs. •  Selective TRPP2 knockdown reduced swelling‐induced non‐selective cation currents (ICat) in SMCs and myogenic tone in cerebral arteries. •  These data indicate that TRPP2 activation contributes to the cerebral artery myogenic response and suggest that TRPP2 performs differential functions in different vascular beds. Abstract  Intravascular pressure‐induced vasoconstriction is a smooth muscle cell‐specific mechanism that controls systemic blood pressure and organ regional blood flow. Smooth muscle cell polycystin‐1 and ‐2 (TRPP1 and ‐2) proteins modulate the myogenic response in mesenteric arteries, but involvement in other vascular beds is unclear. Here, we examined TRPP2 expression, cellular distribution, cation currents (ICat), and physiological functions in smooth muscle cells of rat and human cerebral arteries. We demonstrate that TRPP2 is the major TRPP isoform expressed in cerebral artery smooth muscle cells, with message levels higher than those of TRPP1. Arterial biotinylation and immunofluorescence indicated that TRPP2 is located primarily (∼88%) in the smooth muscle cell plasma membrane. RNA interference reduced TRPP2 expression by ∼55% compared to control, but did not alter levels of TRPP1, TRPC1, TRPC3, TRPC6, TRPM4, ANO1/TMEM16A, or voltage‐dependent Ca2+ (CaV1.2) channels, other ion channel proteins that modulate myogenic tone. Cell swelling induced by hyposmotic (250 osmol (l solution)−1) bath solution stimulated Gd3+‐sensitive ICat in smooth muscle cells that were reduced by selective TRPP2 knockdown. TRPP2 knockdown did not alter myogenic tone at 20 mmHg but reduced tone between ∼28 and 39% over an intravascular pressure range between 40 and 100 mmHg. In contrast, TRPP2 knockdown did not alter depolarization‐induced (60 mmol l K+) vasoconstriction. In summary, we show that TRPP2 is expressed in smooth muscle cells of resistance‐size cerebral arteries, resides primarily in the plasma membrane, and contributes to the myogenic response. Data also suggest that TRPP2 differentially regulates the myogenic response in cerebral and mesenteric arteries.

  August 15, 2013   doi: 10.1113/jphysiol.2013.258319   open full text
• Microcircuit mechanisms involved in paired associative stimulation‐induced depression of corticospinal excitability.
  David Weise, Jakob Mann, Michael Ridding, Kevin Eskandar, Martin Huss, Jost‐Julian Rumpf, Vincenzo Di Lazzaro, Paolo Mazzone, Federico Ranieri, Joseph Classen.
  The Journal of Physiology. August 14, 2013
  •  Repetitively pairing peripheral nerve stimulation with transcranial magnetic stimulation of the corresponding contralateral motor cortex at 10 ms (paired associative stimulation; PAS10) leads to centre‐depressant effects on corticospinal excitability in a short time window. •  PAS10‐induced centre‐depressant effects are due to weakening of excitatory synapses between principal cortical neurons, but not those located on corticospinal neurons, or inhibitory synapses. •  Inhibitory interneurons are gate‐keepers to producing centre‐depressant PAS effects. The same mechanisms appear to govern PAS10‐induced surround‐facilitatory effects. •  We propose a model specifying the composition and laminar location of the involved microcircuit of PAS‐induced plasticity that may enhance its utility as a model of spike‐timing‐ dependent plasticity in humans. Abstract  Synaptic weight changes induced by temporal correlations between the spikes of pre‐ and postsynaptic neurons are referred to as spike‐timing‐dependent plasticity (STDP). Transcranial magnetic stimulation (TMS) induces long‐lasting effects on corticospinal excitability, if it is repetitively paired with stimulation of afferents from a corresponding contralateral hand region at short intervals (paired associative stimulation, PAS). PAS‐induced plasticity has been linked with synaptic STDP. We aimed to investigate which elements of the cortical microcircuitry sustain and govern PAS‐induced depression of corticospinal excitability in the target muscle representation (and enhancement of excitability in its functional surround). We show that the time window during which the interaction between both stimulus‐induced cortical events leads to immediate post‐interventional depression is short (<4.5 ms). The depressant PAS effects at the target representation were completely blocked by applying a subthreshold magnetic pulse 3 ms before the principal TMS pulse, even when the strength of the latter was adjusted to generate a motor‐evoked potential of similar amplitude to that with the unconditioned magnetic pulse. Epidural recordings from the cervical cord of a patient showed that under this condition late TMS‐evoked I‐waves remain suppressed. When the intensity of the TMS component during PAS was lowered – sufficient to allow activation of inhibitory neurons, but insufficient to activate corticospinal neurons – excitability of short‐latency intracortical inhibition remained unchanged. PAS‐induced facilitation in the functional surround followed the same pattern as the centre‐depressant effects. These findings may suggest that excitability‐depressant PAS‐induced effects are due to weakening of excitatory synapses between upper cortical layer principal neurons, but not those located on the corticospinal neuron, or inhibitory synapses. Inhibitory interneurons involved in short‐latency intracortical inhibition are gate‐keepers to producing centre‐depressant/surround‐facilitatory PAS effects. Based on these and earlier findings we propose a model specifying the composition and laminar location of the involved microcircuit of PAS‐induced plasticity that may enhance its utility as a model of STDP in humans.

  August 14, 2013   doi: 10.1113/jphysiol.2013.253989   open full text
• Subcortical effects of transcranial direct current stimulation in the rat.
  F. Bolzoni, M. Bączyk, E. Jankowska.
  The Journal of Physiology. August 14, 2013
  •  Previously demonstrated facilitation of activation of subcortical neurons by transcranial direct current stimulation (tDCS) in acute experiments on deeply anaesthetized animals was fairly weak. It resulted in only small increases in the amplitude and in a slight shortening of latencies of subcortically initiated descending volleys. •  Here we show that despite weak effects on descending volleys, EMG responses evoked in neck muscles by reticulospinal and rubrospinal neurons in deeply anaesthetized non‐paralysed rats are potently facilitated by tDCS and that the facilitation outlasts tDCS. •  We further show that the facilitatory subcortical effects of tDCS in the rat are evoked by cathodal rather than anodal polarization, i.e. by a polarity that is the reverse of that most often found to be effective in humans and in the cat. Anodal polarization depressed activation of the same rat subcortical neurons. •  These findings should assist further studies of mechanisms of tDCS in vivo in rodents. Abstract  Transcranial direct current stimulation (tDCS) affects neurons at both cortical and subcortical levels. The subcortical effects involve several descending motor systems but appeared to be relatively weak, as only small increases in the amplitude of subcortically initiated descending volleys and a minute shortening of latencies of these volleys were found. The aim of the present study was therefore to evaluate the consequences of facilitation of these volleys on the ensuing muscle activation. The experiments were carried out on deeply anaesthetized rats without neuromuscular blockade. Effects of tDCS were tested on EMG potentials recorded from neck muscles evoked by weak (20–60 μA) single, double or triple stimuli applied in the medial longitudinal fascicle (MLF) or in the red nucleus (RN). Short latencies of these potentials were compatible with monosynaptic or disynaptic actions of reticulospinal and disynaptic or trisynaptic actions of rubrospinal neurons on neck motoneurons. Despite only weak effects on indirect descending volleys, the EMG responses from both the MLF and the RN were potently facilitated by cathodal tDCS and depressed by anodal tDCS. Both the facilitation and the depression developed relatively rapidly (within the first minute) but both outlasted tDCS and were present for up to 1 h after tDCS. The study thus demonstrates long‐lasting effects of tDCS on subcortical neurons in the rat, albeit evoked by an opposite polarity of tDCS to that found to be effective on subcortical neurons in the cat investigated in the preceding study, or for cortical neurons in the humans.

  August 14, 2013   doi: 10.1113/jphysiol.2013.257063   open full text
• Acute inhibition of diacylglycerol lipase blocks endocannabinoid‐mediated retrograde signalling: evidence for on‐demand biosynthesis of 2‐arachidonoylglycerol.
  Yuki Hashimotodani, Takako Ohno‐Shosaku, Asami Tanimura, Yoshihiro Kita, Yoshikazu Sano, Takao Shimizu, Vincenzo Di Marzo, Masanobu Kano.
  The Journal of Physiology. August 13, 2013
  •  2‐Arachidonoylglycerol (2‐AG), one of the best‐characterized retrograde messengers at central synapses, has been thought to be produced ‘on demand’ through a diacylglycerol lipase α (DGLα)‐dependent pathway upon activation of postsynaptic neurons (on‐demand synthesis hypothesis). •  However, recent studies propose an alternative hypothesis that 2‐AG is pre‐synthesized by DGLα, stored in neurons, and released from such ‘pre‐formed pools’ without the participation of DGLα (pre‐formed pool hypothesis). •  To test these hypotheses, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM‐188, on retrograde 2‐AG signalling. •  We found that 2‐AG‐mediated retrograde signalling was blocked after 1 h treatment with OMDM‐188 in acute slices from the hippocampus, striatum and cerebellum, and was blocked several minutes after OMDM‐188 application in cultured hippocampal neurons. •  These results fit well with the on‐demand synthesis hypothesis, rather than the pre‐formed pool hypothesis. Abstract  The endocannabinoid (eCB) 2‐arachidonoylglycerol (2‐AG) produced by diacylglycerol lipase α (DGLα) is one of the best‐characterized retrograde messengers at central synapses. It has been thought that 2‐AG is produced ‘on demand’ upon activation of postsynaptic neurons. However, recent studies propose that 2‐AG is pre‐synthesized by DGLα and stored in neurons, and that 2‐AG is released from such ‘pre‐formed pools’ without the participation of DGLα. To address whether the 2‐AG source for retrograde signalling is the on‐demand biosynthesis by DGLα or the mobilization from pre‐formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM‐188, on retrograde eCB signalling triggered by Ca2+ elevation, Gq/11 protein‐coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM‐188 effectively blocked depolarization‐induced suppression of inhibition (DSI), a purely Ca2+‐dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre–Purkinje cell synapses in the cerebellum OMDM‐188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca2+ elevation and group I metabotropic glutamate receptor (I‐mGluR) activation. Moreover, brief OMDM‐188 treatments for several minutes were sufficient to suppress both DSI and the I‐mGluR‐induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2‐AG for synaptic retrograde signalling is supplied as a result of on‐demand biosynthesis by DGLα rather than mobilization from presumptive pre‐formed pools.

  August 13, 2013   doi: 10.1113/jphysiol.2013.254474   open full text
• Segmentation of the mouse fourth deep lumbrical muscle connectome reveals concentric organisation of motor units.
  Theodore C. Hirst, Richard R. Ribchester.
  The Journal of Physiology. August 13, 2013
  Abstract  Connectomic analysis of the nervous system aims to discover and establish principles that underpin normal and abnormal neural connectivity and function. Here we performed image analysis of motor unit connectivity in the fourth deep lumbrical muscle (4DL) of mice, using transgenic expression of fluorescent protein in motor neurones as a morphological reporter. We developed a method that accelerated segmentation of confocal image projections of 4DL motor units, by applying high resolution (63x 1.4NA objective) imaging or deconvolution only where either proved necessary, in order to resolve axon crossings that produced ambiguities in the correct assignment of axon terminals to identified motor units imaged at lower optical resolution (40x, 1.3NA). The 4DL muscles contained between 4 and 9 motor units and motor unit sizes ranged in distribution from 3 to 111 motor nerve terminals per unit. Several structural properties of the motor units were consistent with those reported in other muscles, including suboptimal wiring length and distribution of motor unit size. Surprisingly, however, small motor units were confined to a region of the muscle near the nerve entry point, whereas their larger counterparts were progressively more widely dispersed, suggesting a previously unrecognised form of segregated motor innervation in this muscle. We also found small but significant differences in variance of motor endplate length in motor units, which correlated weakly with their motor unit size. Thus, our connectomic analysis has revealed a pattern of concentric innervation that may perhaps also exist in other, cylindrical muscles that have not previously thought to show segregated motor unit organisation. This organisation may be the outcome of competition during postnatal development based on intrinsic neuronal differences in synaptic size or synaptic strength that generates a territorial hierarchy in motor unit size and disposition. This article is protected by copyright. All rights reserved

  August 13, 2013   doi: 10.1113/jphysiol.2013.258087   open full text
• Aldosterone increases cardiac vagal tone via G protein‐coupled oestrogen receptor activation.
  G. Cristina Brailoiu, Khalid Benamar, Jeffrey B. Arterburn, Erhe Gao, Joseph E. Rabinowitz, Walter J. Koch, Eugen Brailoiu.
  The Journal of Physiology. August 12, 2013
  •  Faster cellular effects of aldosterone incompatible with the genomic effects mediated by mineralocorticoid receptors have been proposed for 40 years but the receptors remained elusive. •  Recently, aldosterone has been shown to activate the G protein‐coupled oestrogen receptor (GPER) in the vasculature. •  Our results indicate that aldosterone activates the GPER in cardiac vagal neurons of nucleus ambiguus leading to an increase in cytosolic Ca2+ concentration and depolarization; in addition, in vivo studies indicate that microinjection of aldosterone in nucleus ambiguus produces bradycardia in conscious rats. •  In summary, our results identified a new role for aldosterone in the modulation of cardiac vagal tone via GPER activation in nucleus ambiguus. Abstract  In addition to acting on mineralocorticoid receptors, aldosterone has been recently shown to activate the G protein‐coupled oestrogen receptor (GPER) in vascular cells. In light of the newly identified role for GPER in vagal cardiac control, we examined whether or not aldosterone activates GPER in rat nucleus ambiguus. Aldosterone produced a dose‐dependent increase in cytosolic Ca2+ concentration in retrogradely labelled cardiac vagal neurons of nucleus ambiguus; the response was abolished by pretreatment with the GPER antagonist G‐36, but was not affected by the mineralocorticoid receptor antagonists, spironolactone and eplerenone. In Ca2+‐free saline, the response to aldosterone was insensitive to blockade of the Ca2+ release from lysosomes, while it was reduced by blocking the Ca2+ release via ryanodine receptors and abolished by blocking the IP3 receptors. Aldosterone induced Ca2+ influx via P/Q‐type Ca2+ channels, but not via L‐type and N‐type Ca2+ channels. Aldosterone induced depolarization of cardiac vagal neurons of nucleus ambiguus that was sensitive to antagonism of GPER but not of mineralocorticoid receptor. in vivo studies, using telemetric measurement of heart rate, indicate that microinjection of aldosterone into the nucleus ambiguus produced a dose‐dependent bradycardia in conscious, freely moving rats. Aldosterone‐induced bradycardia was blocked by the GPER antagonist, but not by the mineralocorticoid receptor antagonists. In summary, we report for the first time that aldosterone decreases heart rate by activating GPER in cardiac vagal neurons of nucleus ambiguus.

  August 12, 2013   doi: 10.1113/jphysiol.2013.257204   open full text
• Errata.
  Hong Song, Scott M. Thompson, Mordecai P. Blaustein.
  The Journal of Physiology. August 12, 2013
  Abstract  Erratum: Page 1682, Figure 9 legend, lines 2 and 5: The dose of SEA0400 was 300 nM (not 300 μM). This article is protected by copyright. All rights reserved

  August 12, 2013   doi: 10.1113/jphysiol.2013.263285   open full text
• Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release.
  Michelle J. Connolly, Jesus Prieto‐Lloret, Silke Becker, Jeremy P. T. Ward, Philip I. Aaronson.
  The Journal of Physiology. August 09, 2013
  •  Hypoxic pulmonary vasoconstriction (HPV) is a mechanism by which pulmonary arteries maintain blood oxygenation during alveolar hypoxia. •  HPV is generally studied using a vasoconstricting co‐stimulus that amplifies the HPV but may also distort its properties; we therefore characterised HPV in isolated rat intrapulmonary arteries during 40 min hypoxic challenges in the absence of any such stimulus. •  Immediate (phase 1) and sustained (phase 2) components of HPV were unaffected by blocking voltage‐gated Ca2+ channels but were abolished by depletion of sarcoplasmic reticulum Ca2+. Phase 2 was attenuated by blockade of store‐operated Ca2+ entry (SOCE), although it largely persisted in Ca2+‐free physiological saline solution. •  HPV was associated with an increase in the intrapulmonary artery ratio of oxidised to reduced glutathione and was inhibited by antioxidants. •  HPV resulted primarily from intracellular Ca2+ release, with SOCE making a contribution, particularly to phase 2. Sustained HPV involves oxidation of the pulmonary artery redox state. Abstract  Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist‐induced preconstriction (‘pretone’). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L‐type Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store‐operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+‐free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+‐ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+‐free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two‐pore channel blocker NED‐19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+‐depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8‐bromo‐cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca2+‐sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y‐27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage‐gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP‐dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

  August 09, 2013   doi: 10.1113/jphysiol.2013.253682   open full text
• Length dependence of striated muscle force generation is controlled by phosphorylation of cTnI at serines 23/24.
  Laurin M. Hanft, Brandon J. Biesiadecki, Kerry S. McDonald.
  The Journal of Physiology. August 09, 2013
  •  An important mechanism in beat‐to‐beat optimization of heart performance is matching ventricular output with end‐diastolic volume, which is known as the Frank–Starling Relationship. •  The cellular basis for this regulation involves myofilament length–tension relationships. •  We previously showed two populations of length–tension relationships in mammalian left ventricular cardiac myocytes, one steep like fast‐twitch skeletal muscle fibres and the other shallow like slow‐twitch skeletal muscle fibres, and cardiac myocytes with shallow length–tension relationships shift to steep relationships by protein kinase A‐induced myofilament phosphorylation. •  The current study investigated the molecular and amino acid residue mechanisms that control length–tension relationships. •  The single muscle cell experiments demonstrated that cardiac troponin I phosphorylation at serines 23/24 control length–tension relationships in striated muscle. •  This study provides: (i) a mechanism to explain a length dependence of force generation in striated muscle; and (ii) an important target to potentially treat heart disease associated with compromised Frank–Starling relationships. Abstract  According to the Frank–Starling relationship, greater end‐diastolic volume increases ventricular output. The Frank–Starling relationship is based, in part, on the length–tension relationship in cardiac myocytes. Recently, we identified a dichotomy in the steepness of length–tension relationships in mammalian cardiac myocytes that was dependent upon protein kinase A (PKA)‐induced myofibrillar phosphorylation. Because PKA has multiple myofibrillar substrates including titin, myosin‐binding protein‐C and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules could control length–tension relationships. We focused on cTnI as troponin can be exchanged in permeabilized striated muscle cell preparations, and tested the hypothesis that phosphorylation of cTnI modulates length dependence of force generation. For these experiments, we exchanged unphosphorylated recombinant cTn into either a rat cardiac myocyte preparation or a skinned slow‐twitch skeletal muscle fibre. In all cases unphosphorylated cTn yielded a shallow length–tension relationship, which was shifted to a steep relationship after PKA treatment. Furthermore, exchange with cTn having cTnI serines 23/24 mutated to aspartic acids to mimic phosphorylation always shifted a shallow length–tension relationship to a steep relationship. Overall, these results indicate that phosphorylation of cTnI serines 23/24 is a key regulator of length dependence of force generation in striated muscle.

  August 09, 2013   doi: 10.1113/jphysiol.2013.258400   open full text
• ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.
  Scott M. O'Grady, Nandadavi Patil, Tamene Melkamu, Peter J. Maniak, Cheryl Lancto, Hirohito Kita.
  The Journal of Physiology. August 09, 2013
  •  Exposure of human bronchial epithelial (HBE) cells to fungal aeroallergens derived from Alternaria alternata stimulates Ca2+‐dependent and Ca2+‐independent ATP release across the apical membrane. •  The Ca2+‐dependent component was blocked by inhibitors of both ATP uptake and transport of exocytotic vesicles to the plasma membrane. •  Treatment with inhibitors that target cysteine proteases significantly blocked Ca2+‐dependent ATP release evoked by Alternaria in normal HBE cells, but not in cells derived from asthmatic patients. •  The magnitude of ATP release and associated intracellular Ca2+ mobilization was significantly greater in bronchial epithelial cells obtained from patients with asthma. •  These findings establish a novel role for ATP release as a mechanism underlying Alternaria aeroallergen activation of airway mucosal immunity and that cells derived from patients with asthma exhibit greater responsiveness to these allergens. Abstract  Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell‐permeable Ca2+‐chelating compound (BAPTA‐AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria‐evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA‐AM significantly blocked Alternaria‐stimulated incorporation of fluorescent lipid (FM1‐43)‐labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA‐AM. These results indicate that an important mechanism for Alternaria‐induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria‐induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease‐activated receptor (PAR2) stimulation.

  August 09, 2013   doi: 10.1113/jphysiol.2013.254649   open full text
• Skeletal muscle carnitine loading increases energy expenditure, modulates fuel metabolism gene networks and prevents body fat accumulation in humans.
  Francis B. Stephens, Benjamin T. Wall, Kanagaraj Marimuthu, Chris E. Shannon, Dumitru Constantin‐Teodosiu, Ian A. Macdonald, Paul L. Greenhaff.
  The Journal of Physiology. August 09, 2013
  •  Carnitine is a substrate for the carnitine palmitoyltransferase 1 enzyme, a rate‐limiting step in fatty acid oxidation within skeletal muscle. •  Insulin stimulates carnitine transport into skeletal muscle. •  A 20% increase in muscle carnitine content, achieved via 12 weeks of twice daily supplementation of a beverage containing 1.36 g of l‐carnitine and 80 g of carbohydrate (in order to stimulate insulin‐mediated muscle carnitine transport), prevented an 18% increase in body fat mass associated with carbohydrate supplementation alone in healthy young men. •  A novel finding of the present study was that this prevention of fat gain was associated with a greater energy expenditure and fat oxidation during low‐intensity physical activity, and an adaptive increase in expression of gene networks involved in muscle insulin signalling and fatty acid metabolism. •  Implications to health warrant further investigation, particularly in obese individuals who have a reduced reliance on muscle fat oxidation during exercise. Abstract  Twelve weeks of daily l‐carnitine and carbohydrate feeding in humans increases skeletal muscle total carnitine content, and prevents body mass accrual associated with carbohydrate feeding alone. Here we determined the influence of l‐carnitine and carbohydrate feeding on energy metabolism, body fat mass and muscle expression of fuel metabolism genes. Twelve males exercised at 50% maximal oxygen consumption for 30 min once before and once after 12 weeks of twice daily feeding of 80 g carbohydrate (Control, n= 6) or 1.36 g l‐carnitine + 80 g carbohydrate (Carnitine, n= 6). Maximal carnitine palmitolytransferase 1 (CPT1) activity remained similar in both groups over 12 weeks. However, whereas muscle total carnitine, long‐chain acyl‐CoA and whole‐body energy expenditure did not change over 12 weeks in Control, they increased in Carnitine by 20%, 200% and 6%, respectively (P < 0.05). Moreover, body mass and whole‐body fat mass (dual‐energy X‐ray absorptiometry) increased over 12 weeks in Control by 1.9 and 1.8 kg, respectively (P < 0.05), but did not change in Carnitine. Seventy‐three of 187 genes relating to fuel metabolism were upregulated in Carnitine vs. Control after 12 weeks, with ‘insulin signalling’, ‘peroxisome proliferator‐activated receptor signalling’ and ‘fatty acid metabolism’ as the three most enriched pathways in gene functional analysis. In conclusion, increasing muscle total carnitine in healthy humans can modulate muscle metabolism, energy expenditure and body composition over a prolonged period, which is entirely consistent with a carnitine‐mediated increase in muscle long‐chain acyl‐group translocation via CPT1. Implications to health warrant further investigation, particularly in obese individuals who have a reduced reliance on muscle fat oxidation during low‐intensity exercise.

  August 09, 2013   doi: 10.1113/jphysiol.2013.255364   open full text
• Cyclooxygenase‐2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine‐induced presynaptic enhancement at the vertebrate neuromuscular junction.
  Clark A. Lindgren, Zachary L. Newman, Jamie J. Morford, Steven B. Ryan, Kathryn A. Battani, Zheng Su.
  The Journal of Physiology. August 09, 2013
  •  The synapse between a nerve and muscle, called the neuromuscular junction (NMJ), undergoes a biphasic modulation, a decrease followed by an increase, when muscarinic acetylcholine receptors are continuously activated. •  The initial depression is caused by the endocannabinoid 2‐arachidonylglycerol (2‐AG), which is synthesized in and released from the muscle; 2‐AG then activates cannabinoid receptors on the presynaptic nerve. •  In the work presented here, we explored the mechanism responsible for the delayed enhancement, uncovering a role for the enzyme cyclooxygenase‐2 and locating it in the glial cells at the NMJ called perisynaptic Schwann cells (PSCs) where it converts 2‐AG into the glycerol ester of prostaglandin E2. •  These results reveal a complex mechanism for regulating neurotransmitter release that involves the nerve, muscle and PSCs (i.e. the tripartite synapse) and may serve to ensure reliable neuromuscular transmission during periods of intense or long‐term activity. Abstract  Previous work has demonstrated that activation of muscarinic acetylcholine receptors at the lizard neuromuscular junction (NMJ) induces a biphasic modulation of evoked neurotransmitter release: an initial depression followed by a delayed enhancement. The depression is mediated by the release of the endocannabinoid 2‐arachidonylglycerol (2‐AG) from the muscle and its binding to cannabinoid type 1 receptors on the motor nerve terminal. The work presented here suggests that the delayed enhancement of neurotransmitter release is mediated by cyclooxygenase‐2 (COX‐2) as it converts 2‐AG to the glycerol ester of prostaglandin E2 (PGE2‐G). Using immunofluorescence, COX‐2 was detected in the perisynaptic Schwann cells (PSCs) surrounding the NMJ. Pretreatment with either of the selective COX‐2 inhibitors, nimesulide or DuP 697, prevents the delayed increase in endplate potential (EPP) amplitude normally produced by muscarine. In keeping with its putative role as a mediator of the delayed muscarinic effect, PGE2‐G enhances evoked neurotransmitter release. Specifically, PGE2‐G increases the amplitude of EPPs without altering that of spontaneous miniature EPPs. As shown previously for the muscarinic effect, the enhancement of evoked neurotransmitter release by PGE2‐G depends on nitric oxide (NO) as the response is abolished by application of either NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of NO synthesis, or carboxy‐PTIO, a chelator of NO. Intriguingly, the enhancement is not prevented by AH6809, a prostaglandin receptor antagonist, but is blocked by capsazepine, a TRPV1 and TRPM8 receptor antagonist. Taken together, these results suggest that the conversion of 2‐AG to PGE2‐G by COX‐2 underlies the muscarine‐induced enhancement of neurotransmitter release at the vertebrate NMJ.

  August 09, 2013   doi: 10.1113/jphysiol.2013.256727   open full text
• The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.
  Naoko Adachi, Takamasa Yoshida, Fumiaki Nin, Genki Ogata, Soichiro Yamaguchi, Toshihiro Suzuki, Sizuo Komune, Yasuo Hisa, Hiroshi Hibino, Yoshihisa Kurachi.
  The Journal of Physiology. August 09, 2013
  •  The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K+ transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells. •  The EP depends upon the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium. •  We examined by electrophysiological approaches an involvement of Na+,K+‐ATPase, which occurs at the syncytium's basolateral surface comprising the fibrocytes’ membranes and would mediate K+ transport across the lateral wall, in maintenance of the EP. •  We show that the Na+,K+‐ATPase sustains the syncytium's high [K+] that is crucial for the K+ gradient across the apical surface of the syncytium. •  The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus. Abstract  The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+‐ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+‐ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+‐ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

  August 09, 2013   doi: 10.1113/jphysiol.2013.258046   open full text
• Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease?
  Carey A. Hobbs, Chong Da Tan, Robert Tarran.
  The Journal of Physiology. August 07, 2013
  Abstract  Airway epithelia absorb Na+ through the Epithelial Na+ Channel (ENaC) and secrete Cl− through the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. This balance maintains sufficient airway surface liquid (ASL) hydration to permits efficient mucus clearance, which is needed to maintain sterility of the lung. Cystic fibrosis (CF) is a common autosomal recessive inherited disease caused by mutations in the CFTR gene that lead to the reduction or elimination of the CFTR protein. CF is a multi‐organ disease that affects epithelia lining the intestines, lungs, pancreas, sweat ducts, and vas deferens, among others. CF lungs are characterized by viscous, dehydrated mucus, persistent neutrophilia, and chronic infections. ENaC is negatively regulated by CFTR and in patients with CF, CFTR's absence results in a double hit of reduced Cl−/HCO3− and H2O secretion as well as ENaC hyperactivity and increased Na+ and H2O absorption. Together, these effects are hypothesized to trigger mucus dehydration, resulting in a failure to clear mucus. Rehydrating CF mucus has become a recent clinical focus and yields important end points for clinical trials. However, while ENaC hyperactivity in CF airways has been detected in vivo and in vitro, recent data have brought the role of ENaC in CF lung disease pathogenesis into question. This review will focus on our current understanding of the contribution of ENaC to CF pathogenesis. This article is protected by copyright. All rights reserved

  August 07, 2013   doi: 10.1113/jphysiol.2012.240861   open full text
• Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes.
  Xinran Li, Abigail G. Garrity, Haoxing Xu.
  The Journal of Physiology. August 07, 2013
  Abstract  Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP‐dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane‐bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment‐specific manner to mediate transport and signalling. Finally, Ca2+, a locally‐acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of Lysosome Storage Diseases (LSDs). This article is protected by copyright. All rights reserved

  August 07, 2013   doi: 10.1113/jphysiol.2013.258301   open full text
• Long and short term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury.
  Keith K. Fenrich, Pascal Weber, Geneviève Rougon, Franck Debarbieux.
  The Journal of Physiology. August 07, 2013
  Abstract  After spinal cord injury (SCI) resident and peripheral myeloid cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two‐photon microscopy of adult mice with SCI, here we show that infiltrating LysM(+) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles and exhibit changes in morphology, motility, phagocytic activity, and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI. This article is protected by copyright. All rights reserved

  August 07, 2013   doi: 10.1113/jphysiol.2013.256388   open full text
• Ca2+ and cAMP cross‐talk in mitochondria.
  Giulietta Di Benedetto, Diana Pendin, Elisa Greotti, Paola Pizzo, Tullio Pozzan.
  The Journal of Physiology. August 07, 2013
  Abstract  While mitochondrial Ca2+ homeostasis has been studied for several decades and many of the functional roles of Ca2+ accumulation within the matrix, at least in part, clarified, the possibility of modulation of the organelle functions by cAMP remains largely unknown. In this contribution we briefly summarize the key aspects of Ca2+ and cAMP signalling pathways in mitochondria. In particular, we focus on recent findings concerning the discovery of an autonomous cAMP toolkit within the mitochondrial matrix and its role in controlling mitochondrial ATP synthesis. A description of the main methods presently available to measure Ca2+ and cAMP in mitochondria of living cells with genetically encoded probes is also presented. This article is protected by copyright. All rights reserved

  August 07, 2013   doi: 10.1113/jphysiol.2013.259135   open full text
• CrossTalk: Mechanical ventilation‐induced diaphragm atrophy is primarily due to inactivity.
  Scott K. Powers, Ashley J. Smuder, David Fuller, Sanford Levine.
  The Journal of Physiology. August 02, 2013
  Abstract  Drs. Sieck and Mantilla have presented an eloquent argument that diaphragm muscle fibre atrophy following unilateral phrenicotomy, tetrodotoxin (TTX) nerve blockade or high cervical spinal hemisection injury does not result from muscle inactivity alone. Nonetheless, we retain our position that when diaphragmatic contractile work is diminished during prolonged mechanical ventilation (MV), the ensuing diaphragm fibre atrophy occurs primarily due to decreased diaphragm contractile activity. In our response to Dr. Sieck and Mantilla's position, we highlight several fundamental differences between the unilateral denervation model of diaphragm inactivity and the reduced diaphragmatic contractile work that occurs during prolonged MV. This article is protected by copyright. All rights reserved

  August 02, 2013   doi: 10.1113/jphysiol.2013.261552   open full text
• CrossTalk rebuttal.
  Gary C. Sieck.
  The Journal of Physiology. August 02, 2013
  Abstract  Powers et al. (2013) propose that mechanical ventilation (MV) imposes rapid diaphragm muscle atrophy as a result of inactivity. Their argument is essentially one of exclusion, rather than direct evidence. They argue that MV does not influence circulating inflammatory cytokines or induce sepsis that may be responsible for atrophy. This article is protected by copyright. All rights reserved

  August 02, 2013   doi: 10.1113/jphysiol.2013.261560   open full text
• Network interactions within the canine intrinsic cardiac nervous system: implications for reflex control of regional cardiac function.
  Eric Beaumont, Siamak Salavatian, E. Marie Southerland, Alain Vinet, Vincent Jacquemet, J. Andrew Armour, Jeffrey L. Ardell.
  The Journal of Physiology. August 01, 2013
  •  Control of regional cardiac function, as mediated by the intrinsic cardiac (IC) nervous system, is dependent upon its cardiac afferent neuronal inputs, changes in its central neuronal drive and interactions mediated within via local circuit neurons. •  The majority of it's local circuit neurons receive indirect central (sympathetic and parasympathetic) inputs, lesser proportions transducing the cardiac milieu. •  Fifty per cent of IC neurons exhibit cardiac cycle‐related periodicity that is primarily related to direct cardiac mechano‐sensory afferent inputs and, secondarily, to indirect central autonomic efferent inputs. •  In response to mediastinal nerve stimulation, most IC neurons became excessively activated in the induction of atrial arrhythmias such that their stochastic interactivity precedes and persists throughout neuronally induced atrial fibrillation. •  Modulation of such stochastic IC local circuit neuronal recruitment may represent a novel target for the treatment of select cardiac disease, including atrial arrhythmias. Abstract  The aims of the study were to determine how aggregates of intrinsic cardiac (IC) neurons transduce the cardiovascular milieu versus responding to changes in central neuronal drive and to determine IC network interactions subsequent to induced neural imbalances in the genesis of atrial fibrillation (AF). Activity from multiple IC neurons in the right atrial ganglionated plexus was recorded in eight anaesthetized canines using a 16‐channel linear microelectrode array. Induced changes in IC neuronal activity were evaluated in response to: (1) focal cardiac mechanical distortion; (2) electrical activation of cervical vagi or stellate ganglia; (3) occlusion of the inferior vena cava or thoracic aorta; (4) transient ventricular ischaemia, and (5) neurally induced AF. Low level activity (ranging from 0 to 2.7 Hz) generated by 92 neurons was identified in basal states, activities that displayed functional interconnectivity. The majority (56%) of IC neurons so identified received indirect central inputs (vagus alone: 25%; stellate ganglion alone: 27%; both: 48%). Fifty per cent transduced the cardiac milieu responding to multimodal stressors applied to the great vessels or heart. Fifty per cent of IC neurons exhibited cardiac cycle periodicity, with activity occurring primarily in late diastole into isovolumetric contraction. Cardiac‐related activity in IC neurons was primarily related to direct cardiac mechano‐sensory inputs and indirect autonomic efferent inputs. In response to mediastinal nerve stimulation, most IC neurons became excessively activated; such network behaviour preceded and persisted throughout AF. It was concluded that stochastic interactions occur among IC local circuit neuronal populations in the control of regional cardiac function. Modulation of IC local circuit neuronal recruitment may represent a novel approach for the treatment of cardiac disease, including atrial arrhythmias.

  August 01, 2013   doi: 10.1113/jphysiol.2013.259382   open full text
• Spike timing‐dependent plasticity at GABAergic synapses in the Ventral tegmental area.
  Jayaraj N. Kodangattil, Matthieu Dacher, Michael E. Authement, Fereshteh S. Nugent.
  The Journal of Physiology. July 31, 2013
  Abstract  Persistent changes in excitatory and inhibitory synaptic strengths to the ventral tegmental area (VTA) dopamine (DA) neurons in response to addictive drugs may underlie the transition from casual to compulsive drug use. While enormous amount of work has been done in the area of glutamatergic plasticity of the VTA, little is known regarding the learning rules governing GABAergic plasticity in the VTA. Spike‐timing‐dependent plasticity, STDP, has attracted considerable attention primarily due to its potential roles in processing and storage of information in the brain and there is emerging evidence for the existence of STDP at inhibitory synapses. We therefore used whole‐cell recordings in rat midbrain slices to investigate whether near coincident pre‐ and postsynaptic firing induces a lasting change in synaptic efficacy of VTA GABAergic synapses. We found that a Hebbian form of STDP including LTP and LTD can be induced at GABAergic synapses onto VTA DA neurons and relies on the precise temporal order of pre‐and postsynaptic spiking. Importantly, GABAergic STDP is heterosynaptic (NMDR‐dependent): triggered by correlated activities of the presynaptic glutamatergic input and postsynaptic DA cells. GABAergic STDP is postsynaptic and has an associative component since pre‐ or postsynaptic spiking per se did not induce STDP. STDP of GABAergic synapses in the VTA provides physiologically relevant forms of inhibitory plasticity that may underlie natural reinforcement of reward‐related behaviors. Moreover, this form of inhibitory plasticity may mediate some of the reinforcing, aversive and addictive properties of drugs of abuse. This article is protected by copyright. All rights reserved

  July 31, 2013   doi: 10.1113/jphysiol.2013.257873   open full text
• Acetazolamide attenuates transvascular fluid flux in equine lungs during intense exercise.
  Modest Vengust, Henry Staempfli, Laurent Viel, Erik R. Swenson, George Heigenhauser.
  The Journal of Physiology. July 29, 2013
  •  During high intensity exercise approximately 4% of the cardiac output leaves the pulmonary circulation into the interstitium. This fluid flux has been attributed to an increase in pulmonary transmural hydrostatic (Starling) forces. •  Fluid efflux from erythrocytes may account for a considerable fraction of fluid exiting the pulmonary circulation. Transcapillary erythrocyte volume changes are largely determined by the Jacobs–Stewart cycle, a series of intracellular and extracellular diffusion and chemical reaction events of carbon dioxide, water, bicarbonate, hydrogen ions and chloride that are initiated when blood is exposed to a gradient such as when blood enters and traverses systemic and pulmonary capillaries. •  We tested the hypothesis that the Jacobs–Stewart cycle contributes to pulmonary transvascular fluid fluxes during exercise by inhibiting red cell carbonic anhydrase, the activity of which is critical to rapid completion of the Jacobs–Stewart cycle during capillary transit. •  Our results indicate that during exercise in horses, transvascular fluid fluxes in the lung appear to be dependent on the Jacobs–Stewart cycle and much less dependent upon transmural hydrostatic (Starling) forces. It also appears that pulmonary circulation transvascular fluid fluxes are mediated by chloride and water egress from erythrocytes directly into the interstitium without transit through plasma, which is likely the result of functional apposition of the erythrocyte and vascular endothelial membranes occurring during capillary transit. Abstract  During intense exercise in horses the transvascular fluid flux in the pulmonary circulation (Jv‐a) represents 4% of cardiac output (). This fluid flux has been attributed to an increase in pulmonary transmural hydrostatic forces, increases in perfused microvascular surface area, and reversible alterations in capillary permeability under conditions of high flow and pressure. Erythrocyte fluid efflux, however, accounts for a significant fraction of Jv‐a. In the lung the Jacobs–Stewart cycle occurs with diffusion of CO2 into alveolar space with possible accompanying chloride (Cl−) and water movement from the erythrocyte directly into the pulmonary interstitium. We hypothesised that inhibition of carbonic anhydrase in erythrocytes inhibits the Jacobs–Stewart cycle and attenuates Jv‐a. Five horses were exercised on a treadmill until fatigue without (control) and with acetazolamide treatment (30 mg kg−1 30 min before exercise). Erythrocyte fluid efflux, plasma fluid flux across the lung and Jv‐a were calculated using haemoglobin, haematocrit, plasma protein and Q. Fluid fluxes were used to calculate erythrocyte, plasma and whole blood Cl− fluxes across the lung. Cardiac output was not different between control and acetazolamide treatment. During exercise erythrocyte fluid efflux and Jv‐a increased in control (9.3 ± 3.3 and 11.0 ± 4.4 l min−1, respectively) and was higher than after acetazolamide treatment (3.8 ± 1.6 and 1.2 ± 1.2 l min−1, respectively) (P < 0.05). Plasma fluid flux did not change from rest in control and decreased after acetazolamide treatment (−4.5 ± 1.5 l min−1) (P < 0.05). Erythrocyte Cl− flux increased during exercise in control and after acetazolamide treatment (P < 0.05). During exercise plasma Cl− flux across the lung did not change in control; however, it increased with acetazolamide treatment (P= 0.0001). During exercise whole blood Cl− flux increased across the lung in control (P < 0.05) but not after acetazolamide treatment. The results indicate that Jv‐a in the lung is dependent on the Jacobs–Stewart cycle and mostly independent of transmural hydrostatic forces. It also appears that Jv‐a is mediated by Cl− and water egress from erythrocytes directly into the interstitium without transit through plasma.

  July 29, 2013   doi: 10.1113/jphysiol.2013.257956   open full text
• Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia.
  Carlo Manno, Lourdes Figueroa, Leandro Royer, Sandrine Pouvreau, Chang Seok Lee, Pompeo Volpe, Alessandra Nori, Jingsong Zhou, Gerhard Meissner, Susan L. Hamilton, Eduardo Ríos.
  The Journal of Physiology. July 29, 2013
  •  Malignant Hyperthermia (MH) affects the Ca2+ movements that control muscle contraction. We measured Ca2+ movements in skeletal muscle of “Y522S” mice, with a tyrosine‐to‐serine mutation in the RyR channel that causes MH in mice and humans. •  In YS cells, [Ca2+] inside the Ca2+ store (sarcoplasmic reticulum, SR) was 45% of that in the wild type (WT), but the SR membrane permeability increased 2‐fold, resulting in Ca2+ release of initially normal value. •  During Ca2+release, cytosolic [Ca2+] and SR Ca2+ buffering power evolved differently in YS and WT. These variables became similar in WT exposed to BAPTA, an inhibitor of Ca2+‐dependent inactivation (CDI) of the RyR, suggesting that tyrosine 522 is involved in CDI. •  Similar paradoxical observations in YS and WT cells with reduced content of the SR protein calsequestrin, revealed the importance of balance between SR Ca permeability (increased in YS) and storage capability (decreased when calsequestrin is low). Abstract  Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell ‘cores’. Imaging cytosolic and intra‐SR [Ca2+] in muscle cells of heterozygous YS mice we determined Ca2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca2+] gradient) and SR Ca2+ buffering power (B). In YS cells resting [Ca2+]SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca2+]SR(t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca2+‐dependent inactivation (CDI). The [Ca2+] transients showed abnormal ‘breaks’, decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibres with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca2+]SR and alters SR Ca2+ buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca2+ control that defines a fulminant MH event.

  July 29, 2013   doi: 10.1113/jphysiol.2013.259572   open full text
• Cholinergic modulation of neuronal excitability and recurrent excitation‐inhibition in prefrontal cortex circuits: implications for gamma oscillations.
  Diego E. Pafundo, Takeaki Miyamae, David A. Lewis, Guillermo Gonzalez‐Burgos.
  The Journal of Physiology. July 29, 2013
  •  Previous studies indicate that cholinergic neuromodulation is required for cognitive processes and for gamma oscillatory activity in neocortical networks in vivo. The cholinergic agonist carbachol (CCh) induces gamma oscillations in vitro, via mechanisms that may be shared with those mediating in vivo gamma oscillations. •  Here, we studied the effects of CCh on cortical circuit components thought to be critical for gamma oscillations, and found that CCh stimulated firing of pyramidal cells (PCs) and increased excitatory synaptic input onto fast‐spiking interneurons (FSNs). •  CCh also modulated synaptic transmission between FSNs and PCs, decreasing synaptic depression during repetitive presynaptic firing, while simultaneously reducing the unitary synaptic currents. •  CCh increased the probability of neuron firing per oscillation cycle when PCs and FSNs fired in response to oscillatory input at gamma frequency. •  Combined, these effects of CCh may help explain the contribution of cholinergic modulation to gamma oscillations. Abstract  Cholinergic neuromodulation in neocortical networks is required for gamma oscillatory activity associated with working memory and other cognitive processes. Importantly, the cholinergic agonist carbachol (CCh) induces gamma oscillations in vitro, via mechanisms that may be shared with in vivo gamma oscillations and that are consistent with the pyramidal interneuron network gamma (PING) model. In PING oscillations, pyramidal cells (PCs), driven by asynchronous excitatory input, recruit parvalbumin‐positive fast‐spiking interneurons (FSNs), which then synchronize the PCs via feedback inhibition. Whereas the PING model is favoured by current data, how cholinergic neuromodulation contributes to gamma oscillation production is poorly understood. We thus studied the effects of cholinergic modulation on circuit components of the PING model in mouse medial prefrontal cortex (mPFC) brain slices. CCh depolarized and evoked action potential firing in a fraction of PCs and increased excitatory synaptic input onto FSNs. In synaptically connected pairs, CCh reduced the short‐term depression at FSN–PC and PC–FSN synapses, equalizing synaptic strength during repetitive presynaptic firing while simultaneously increasing the failure probability. Interestingly, when PCs or FSNs fired in response to gamma frequency oscillatory inputs, CCh increased the firing probability per cycle. Combined with the equalization of synaptic strength, an increase by CCh in the fraction of neurons recruited per oscillation cycle may support oscillatory synchrony of similar strength during relatively long oscillation episodes such as those observed during working memory tasks, suggesting a significant functional impact of cholinergic modulation of mPFC circuit components crucial for the PING model.

  July 29, 2013   doi: 10.1113/jphysiol.2013.253823   open full text
• How LeuT shapes our understanding of the mechanisms of sodium‐coupled neurotransmitter transporters.
  Aravind Penmatsa, Eric Gouaux.
  The Journal of Physiology. July 29, 2013
  Abstract  Neurotransmitter transporters are ion‐coupled symporters that drive the uptake of neurotransmitters from the synapse. In the past decade, the structure of a bacterial amino acid transporter, LeuT has given valuable insights into understanding the architecture and mechanism of mammalian neurotransmitter transporters. Different conformations of LeuT that include a substrate‐free state, inward‐open state, competitive and non‐competitive inhibitor bound states have revealed a mechanistic frame work for the transport and transport inhibition of neurotransmitters. The current review integrates our understanding of the mechanistic and pharmacological properties of eukaryotic neurotransmitter transporters obtained through structural snapshots of LeuT. This article is protected by copyright. All rights reserved

  July 29, 2013   doi: 10.1113/jphysiol.2013.259051   open full text
• Normal mucus formation requires cAMP‐dependent HCO3− secretion and Ca2+‐mediated mucin exocytosis.
  Ning Yang, Mary Abigail S. Garcia, Paul M. Quinton.
  The Journal of Physiology. July 25, 2013
  •  HCO3− is required for gel‐forming mucins to form normal mucus, but how? •  Two apparently separate signalling pathways are activated concurrently to bring mucus formation to completion: a Ca2+‐mediated pathway mainly directs goblet cell exocytosis, and an independent cAMP‐mediated pathway stimulates HCO3− secretion to help discharge exocytosed mucus. •  cAMP‐dependent HCO3− secretion fails, disrupting the normal formation and discharge of mucins in cystic fibrosis (CF) leading to pathologically viscous and tenacious mucus in affected organs. •  This work advances our understanding of the role of cAMP (CFTR)‐dependent HCO3− secretion in forming normal mucus and underscores a new importance of addressing the defect in HCO3− secretion as a critical new therapeutic target in CF. Abstract  Evidence from the pathology in cystic fibrosis (CF) and recent results in vitro indicate that HCO3− is required for gel‐forming mucins to form the mucus that protects epithelial surfaces. Mucus formation and release is a complex process that begins with an initial intracellular phase of synthesis, packaging and apical granule exocytosis that is followed by an extracellular phase of mucin swelling, transport and discharge into a lumen. Exactly where HCO3− becomes crucial in these processes is unknown, but we observed that in the presence of HCO3−, stimulating dissected segments of native mouse intestine with 5‐hydroxytryptamine (5–HT) and prostaglandin E2 (PGE2) induced goblet cell exocytosis followed by normal mucin discharge in wild‐type (WT) intestines. CF intestines that inherently lack cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent HCO3− secretion also demonstrated apparently normal goblet cell exocytosis, but in contrast, this was not followed by similar mucin discharge. Moreover, we found that even in the presence of HCO3−, when WT intestines were stimulated only with a Ca2+‐mediated agonist (carbachol), exocytosis was followed by poor discharge as with CF intestines. However, when the Ca2+‐mediated agonist was combined with a cAMP‐mediated agonist (isoproterenol (isoprenaline) or vasoactive intestinal peptide) in the presence of HCO3− both normal exocytosis and normal discharge was observed. These results indicate that normal mucus formation requires concurrent activation of a Ca2+‐mediated exocytosis of mucin granules and an independent cAMP‐mediated, CFTR‐dependent, HCO3− secretion that appears to mainly enhance the extracellular phases of mucus excretion.

  July 25, 2013   doi: 10.1113/jphysiol.2013.257436   open full text
• Aggressive behaviour and physiological responses to pheromones are strongly impaired in mice deficient for the olfactory G‐protein γ‐subunit Gγ8.
  Giorgia Montani, Simone Tonelli, Valentina Sanghez, Pier Francesco Ferrari, Paola Palanza, Andreas Zimmer, Roberto Tirindelli.
  The Journal of Physiology. July 25, 2013
  •  Pheromones are intraspecies chemical signals that take part in the sexual recognition and choice of appropriate mating partners. •  In the vomeronasal organ (VNO), pheromone responses are probably triggered by two distinct neuronal populations, respectively expressing the heterotrimeric G‐proteins Gαi2β2γ2 and Gαoβ2γ8 that, in turn, coexpress with two pheromone receptor families, V1R and V2R. •  We demonstrate that the olfactory‐specific G‐protein γ8 subunit (Gγ8) plays an important role in pheromone‐dependent socio‐sexual recognition. •  Deficient mice for Gγ8 show a marked reduction in the pheromone‐mediated aggressive behaviour in both females and males that corresponds with a failure to activate V2R targets in the brain. These effects occur in combination with a consistent loss of vomeronasal neurons. •  Thus, Gγ8 is essential for maintenance of the neuronal population of the VNO and for correct transduction of the pheromonal signal. Abstract  Heterotrimeric G‐proteins are critical players in the transduction mechanisms underlying odorant and pheromonal signalling. In the vomeronasal organ (VNO) of the adult mouse, two different G‐protein complexes have been identified. Gαoβ2γ8 is preferentially expressed in the basal neurons and coexpresses with type‐2 vomeronasal pheromone receptors (V2Rs) whereas Gαi2β2γ2 is found in the apical neurons and coexpresses with type‐1 vomeronasal pheromone receptors (V1Rs). V2R‐expressing neurons project to the posterior accessory olfactory bulb (AOB) whereas neurons expressing V1Rs send their axon to the anterior AOB. Gγ8 is also expressed in developing olfactory neurons where this protein is probably associated with Go. Here, we generated mice with a targeted deletion of the Gγ8 gene and investigated the behavioural effects and the physiological consequences of this mutation. Gγ8−/− mice show a normal development of the main olfactory epithelium; moreover, they do not display major deficits in odour perception. In contrast, the VNO undergoes a slow but remarkable loss of basal neurons starting from the fourth postnatal week, with a 40% reduction of cells at 2 months and 70% at 1 year. This loss is associated with a reduced early‐gene expression in the posterior AOB of mice stimulated with pheromones. More interestingly, the Gγ8 deletion specifically leads to a reduced pheromone‐mediated aggressiveness in both males and females, all other socio‐sexual behaviours remaining unaltered. This study defines a specific role for Gγ8 in maintenance of the neuronal population of the VNO and in the mechanisms of pheromonal signalling that involve the aggressive behaviour towards conspecifics.

  July 25, 2013   doi: 10.1113/jphysiol.2012.247528   open full text
• Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short‐term endurance training.
  Aaron P. Russell, Severine Lamon, Hanneke Boon, Shogo Wada, Isabelle Güller, Erin L. Brown, Alexander V. Chibalin, Juleen R. Zierath, Rod J. Snow, Nigel Stepto, Glenn D. Wadley, Takayuki Akimoto.
  The Journal of Physiology. July 23, 2013
  •  The discovery of microRNAs (miRNAs) has established new mechanisms that control health, but little is known about the regulation of skeletal muscle miRNAs in response to exercise. •  This study investigated components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin‐5), muscle enriched miRNAs, (miR‐1, ‐133a, ‐133b and 206), and several miRNAs dysregulated in muscle myopathies, and showed that 3 h following an acute exercise bout, Drosha, Dicer and Exportin‐5, as well as miR‐1, ‐133a, ‐133‐b and miR‐181a were all increased, while miR‐9, ‐23a, ‐23b and ‐31 were decreased. •  Short‐term training increased miR‐1 and miR‐29b, while miR‐31 remained decreased. •  Negative correlations were observed between miR‐9 and HDAC4 protein, miR‐31 and HDAC4 protein and between miR‐31 and NRF1 protein, 3 h after exercise. •  miR‐31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. •  Exercise rapidly and transiently regulates several miRNA species potentially involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Abstract  The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin‐5), muscle enriched miRNAs, (miR‐1, ‐133a, ‐133b and ‐206), and several miRNAs dysregulated in muscle myopathies (miR‐9, ‐23, ‐29, ‐31 and ‐181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate‐intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin‐5, as well as miR‐1, ‐133a, ‐133‐b and ‐181a were all increased. In contrast miR‐9, ‐23a, ‐23b and ‐31 were decreased. Short‐term training increased miR‐1 and ‐29b, while miR‐31 remained decreased. Negative correlations were observed between miR‐9 and HDAC4 protein (r=−0.71; P= 0.04), miR‐31 and HDAC4 protein (r =−0.87; P= 0.026) and miR‐31 and NRF1 protein (r =−0.77; P= 0.01) 3 h following exercise. miR‐31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise‐mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health.

  July 23, 2013   doi: 10.1113/jphysiol.2013.255695   open full text
• Placement of implantable cardioverter‐defibrillators in paediatric and congenital heart defect patients: a pipeline for model generation and simulation prediction of optimal configurations.
  Lukas J. Rantner, Fijoy Vadakkumpadan, Philip J. Spevak, Jane E. Crosson, Natalia A. Trayanova.
  The Journal of Physiology. July 23, 2013
  •  Implantable cardioverter‐defibrillators (ICDs) with transvenous leads often cannot be implanted in a standard manner in paediatric and congenital heart defect (CHD) patients. Currently, there is no reliable approach to predict the optimal ICD placement in these patients. •  A pipeline for constructing personalized, electrophysiological heart–torso models from clinical magnetic resonance imaging scans was developed and applied to a paediatric CHD patient. •  Optimal ICD placement was determined using patient‐specific simulations of the defibrillation process. In a patient with tricuspid valve atresia, two configurations with epicardial leads were found to have the lowest defibrillation threshold. •  We demonstrated that determining extracellular potential (Φe) gradients during the shock – without actually simulating defibrillation – was not sufficient to predict defibrillation success or failure. •  Using the proposed methodology, the optimal ICD placement in paediatric/CHD patients can be predicted computationally, which could reduce defibrillation energy if the pipeline is used as part of ICD implantation planning. Abstract  There is currently no reliable way of predicting the optimal implantable cardioverter‐defibrillator (ICD) placement in paediatric and congenital heart defect (CHD) patients. This study aimed to: (1) develop a new image processing pipeline for constructing patient‐specific heart–torso models from clinical magnetic resonance images (MRIs); (2) use the pipeline to determine the optimal ICD configuration in a paediatric tricuspid valve atresia patient; (3) establish whether the widely used criterion of shock‐induced extracellular potential (Φe) gradients ≥5 V cm−1 in ≥95% of ventricular volume predicts defibrillation success. A biophysically detailed heart–torso model was generated from patient MRIs. Because transvenous access was impossible, three subcutaneous and three epicardial lead placement sites were identified along with five ICD scan locations. Ventricular fibrillation was induced, and defibrillation shocks were applied from 11 ICD configurations to determine defibrillation thresholds (DFTs). Two configurations with epicardial leads resulted in the lowest DFTs overall and were thus considered optimal. Three configurations shared the lowest DFT among subcutaneous lead ICDs. The Φe gradient criterion was an inadequate predictor of defibrillation success, as defibrillation failed in numerous instances even when 100% of the myocardium experienced such gradients. In conclusion, we have developed a new image processing pipeline and applied it to a CHD patient to construct the first active heart–torso model from clinical MRIs.

  July 23, 2013   doi: 10.1113/jphysiol.2013.255109   open full text
• Ranolazine recruits muscle microvasculature and enhances insulin action in rats.
  Zhuo Fu, Lina Zhao, Weidong Chai, Zhenhua Dong, Wenhong Cao, Zhenqi Liu.
  The Journal of Physiology. July 19, 2013
  •  Ranolazine, an anti‐anginal compound, improves glycaemic control in clinical trials and increases myocardial perfusion via a direct vasodilatatory effect on coronary arteries. •  Skeletal muscle microvasculature controls the delivery of nutrient and hormones into muscle and their exchanges between plasma and muscle interstitium by providing microvascular exchange surface area. •  In this study we examined whether ranolazine improves glycaemic control via exerting vasodilatatory action on the pre‐capillary arterioles to recruit muscle microvasculature. •  We demonstrate that ranolazine potently recruits muscle microvasculature, which expands microvascular endothelial surface area in muscle and results in increased muscle delivery and action of insulin. •  The results help us better understand the physiological mechanism by which ranolazine improves glycaemic control and its potential as an insulin‐sensitizing agent. Abstract  Ranolazine, an anti‐anginal compound, has been shown to significantly improve glycaemic control in large‐scale clinical trials, and short‐term ranolazine treatment is associated with an improvement in myocardial blood flow. As microvascular perfusion plays critical roles in insulin delivery and action, we aimed to determine if ranolazine could improve muscle microvascular blood flow, thereby increasing muscle insulin delivery and glucose use. Overnight‐fasted, anaesthetized Sprague‐Dawley rats were used to determine the effects of ranolazine on microvascular recruitment using contrast‐enhanced ultrasound, insulin action with euglycaemic hyperinsulinaemic clamp, and muscle insulin uptake using 125I‐insulin. Ranolazine's effects on endothelial nitric oxide synthase (eNOS) phosphorylation, cAMP generation and endothelial insulin uptake were determined in cultured endothelial cells. Ranolazine‐induced myographical changes in tension were determined in isolated distal saphenous artery. Ranolazine at therapeutically effective dose significantly recruited muscle microvasculature by increasing muscle microvascular blood volume (∼2‐fold, P < 0.05) and increased insulin‐mediated whole body glucose disposal (∼30%, P= 0.02). These were associated with an increased insulin delivery into the muscle (P < 0.04). In cultured endothelial cells, ranolazine increased eNOS phosphorylation and cAMP production without affecting endothelial insulin uptake. In ex vivo studies, ranolazine exerted a potent vasodilatatory effect on phenylephrine pre‐constricted arterial rings, which was partially abolished by endothelium denudement. In conclusion, ranolazine treatment vasodilatates pre‐capillary arterioles and increases microvascular perfusion, which are partially mediated by endothelium, leading to expanded microvascular endothelial surface area available for nutrient and hormone exchanges and resulting in increased muscle delivery and action of insulin. Whether these actions contribute to improved glycaemic control in patients with insulin resistance warrants further investigation.

  July 19, 2013   doi: 10.1113/jphysiol.2013.257246   open full text
• TRPV3: time to decipher a poorly understood family member!
  Bernd Nilius, Tamás Bíró, Grzegorz Owsianik.
  The Journal of Physiology. July 19, 2013
  Abstract  The vanilloid transient receptor potential channel TRPV3 differs in several aspects from other members of the TRPV subfamily. This Ca2+‐, ATP‐ and calmodulin‐regulated channel constitutes a target for many natural compounds and has a unique expression pattern as the most prominent and important TRP channel in keratinocytes of the skin. Although TRPV3 is considered as a thermosensitive channel, its function as a thermosensor in the skin is challenged. Nevertheless, it plays important roles in other skin functions such as cutaneous sensations, hair development and barrier function. More recently, mutations in TRPV3 were linked with a rare genodermatosis known as the Olmsted syndrome. This review gives an overview on properties of TRPV3 and its functions in the skin and skin diseases.

  July 19, 2013   doi: 10.1113/jphysiol.2013.255968   open full text
• Specificity in monosynaptic and disynaptic bulbospinal connections to thoracic motoneurones in the rat.
  Anoushka T. R. de Almeida, Peter A. Kirkwood.
  The Journal of Physiology. July 15, 2013
  •  In the rat, unlike in other species, motoneurones of both the internal intercostal nerve and the external intercostal nerve show a phase of excitation in expiration. •  This study investigated the pathways transmitting this excitation from the medulla. •  Direct (monosynaptic) excitation was found from individual expiratory neurones in the medulla to internal intercostal nerve motoneurones, but only indirect (disynaptic) excitation was found from the same neurones to the motoneurones of the external intercostal nerve. •  This is the first demonstration of two separate pathways from individual long descending fibres specific to two different sets of motoneurones. •  This specificity could be useful in studying plasticity or regeneration in thoracic segments in investigations of mechanisms involved in spinal cord injury and repair. Abstract  The respiratory activity in the intercostal nerves of the rat is unusual, in that motoneurones of both branches of the intercostal nerves, internal and external, are activated during expiration. Here, the pathways involved in that activation were investigated in anaesthetised and in decerebrate rats by cross‐correlation and by intracellular spike‐triggered averaging from expiratory bulbospinal neurones (EBSNs), with a view to revealing specific connections that could be used in studies of experimental spinal cord injury. Decerebrate preparations, which showed the strongest expiratory activity, were found to be the most suitable for these measurements. Cross‐correlations in these preparations showed monosynaptic connections from 16/19 (84%) of EBSNs, but only to internal intercostal nerve motoneurones (24/37, 65% of EBSN/nerve pairs), whereas disynaptic connections were seen for external intercostal nerve motoneurones (4/19, 21% of EBSNs or 7/25, 28% of EBSN/nerve pairs). There was evidence for additional disynaptic connections to internal intercostal nerve motoneurones. Intracellular spike‐triggered averaging revealed excitatory postsynaptic potentials, which confirmed these connections. This is believed to be the first report of single descending fibres that participate in two different pathways to two different groups of motoneurones. It is of interest compared with the cat, where only one group of motoneurones is activated during expiration and only one of the pathways has been detected. The specificity of the connections could be valuable in studies of plasticity in pathological situations, but care will be needed in studying connections in such situations, because their strength was found here to be relatively weak.

  July 15, 2013   doi: 10.1113/jphysiol.2013.256503   open full text
• Physical activity is associated with retained muscle metabolism in human myotubes challenged with palmitate.
  C. J. Green, T. Bunprajun, B. K. Pedersen, C. Scheele.
  The Journal of Physiology. July 15, 2013
  •  It is known that saturated fatty acids play a role in the progression of insulin resistance in skeletal muscle while physical activity promotes insulin sensitivity. •  The effect of diet and exercise on muscle satellite/stem cells is not well defined: we found that differentiated human muscle satellite cells exhibit metabolic differences. These differences were associated with physical activity level and may reflect a memory of the in vivo environment. •  Differentiated muscle satellite cells from physically active individuals have a higher tolerance to saturated fatty acids reflected by a partial protection from fatty acid‐induced insulin resistance. •  Physical activity and diet have significant effects on the physiological function of differentiated human muscle satellite cells. As these cells exhibit some phenotypes associated with in vivo adaptations and are involved in muscle maintenance, dysregulatory function could have profound effects on health. Abstract  The aim of this study was to investigate whether physical activity is associated with preserved muscle metabolism in human myotubes challenged with saturated fatty acids. Human muscle satellite cells were isolated from sedentary or active individuals and differentiated into myocytes in culture. Metabolic differences were then investigated in the basal state or after chronic palmitate treatment. At basal, myocytes from sedentary individuals exhibited higher CD36 and HSP70 protein expression as well as elevated phosphorylation of c‐Jun NH2‐terminal kinase (JNK) and insulin receptor substrate 1 (IRS1) serine307 compared to myocytes from active individuals. Despite equal lipid accumulation following palmitate treatment, myocytes from sedentary individuals exhibited delayed acetyl coenzyme A carboxylase phosphorylation compared to the active group. Myocytes from sedentary individuals had significantly higher basal glucose uptake and palmitate promoted insulin resistance in sedentary myocytes. Importantly, myocytes from active individuals were partially protected from palmitate‐induced insulin resistance. Palmitate treatment enhanced IRS1 serine307 phosphorylation in myocytes from sedentary individuals and correlated positively to JNK phosphorylation. In conclusion, muscle satellite cells retain metabolic differences associated with physical activity. Physical activity partially protects myocytes from fatty acid‐induced insulin resistance and inactivity is associated with dysregulation of metabolism in satellite cells challenged with palmitate. Although the benefits of physical activity on whole body physiology have been well investigated, this paper presents novel findings that both diet and exercise impact satellite cells directly. Given the fact that satellite cells are important for muscle maintenance, a dysregulated function could have profound effects on health. Therefore the effects of lifestyle on satellite cells needs to be delineated.

  July 15, 2013   doi: 10.1113/jphysiol.2013.251421   open full text
• cGMP/PKG‐mediated regulation of lymphatic contractility in rat thoracic duct.
  Olga Yu. Gasheva, Anatoliy A. Gashev, David C. Zawieja.
  The Journal of Physiology. July 12, 2013
  Abstract  We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear‐dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cGMP/PKG (cyclic guanosine monophosphate/cGMP‐dependent protein kinase) is central to the intrinsic and extrinsic flow‐dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1 to 5 cm H2O) and imposed flow (1 to 5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: 1. inhibition of guanylate cyclase (GC) with and without a NO donor, 2. application of stable cGMP analog and 3. inhibition of the cGMP activation of PKG. Additionally, western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor SNAP. The cGMP analog – 8pCPTcGMP mimicked the extrinsic flow‐induced relaxation in a dose‐dependent manner, whereas treatment with the cGMP/PKG inhibitor – Rp‐8‐Br‐PET‐cGMPS eliminated the intrinsic flow‐dependent relaxation, and largely inhibited the extrinsic flow‐dependent relaxation. Western blotting demonstrated that both PKG‐Iα and Iβ isoforms are found in TD, with ∼10‐times greater expression of the PKG‐Iα protein in TD compared with aorta and vena cava. The PKG‐Iβ isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labelling of PKG‐Iα protein in the wall of rat thoracic duct confirmed it's localization inside the TD muscle cells. These findings demonstrate that cGMP is critical to the flow‐dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG‐Iα in these processes and identifies PKG protein as a potential therapeutic target. This article is protected by copyright. All rights reserved.

  July 12, 2013   doi: 10.1113/jphysiol.2013.258681   open full text
• An adaptive‐filter model of cerebellar zone c3 as a basis for safe limb control?
  Paul Dean, Sean Anderson, John Porrill, Henrik Jörntell.
  The Journal of Physiology. July 12, 2013
  Abstract  The review asks how the adaptive‐filter model of the cerebellum might be relevant to experimental work on zone C3, one of the most extensively studied regions of cerebellar cortex. As far as features of the cerebellar microcircuit are concerned, the model appears to fit very well with electrophysiological discoveries concerning the importance of molecular layer interneurons and their plasticity, the significance of LTP, and the striking number of silent parallel‐fibre synapses. Regarding external connectivity and functionality, a key feature of the adaptive‐filter model is its use of the decorrelation algorithm, which renders it uniquely suited to problems of sensory noise‐cancellation. However, this capacity can be extended to the avoidance of sensory interference, by appropriate movements of e.g. the eyes in the vestibulo‐ocular reflex. Avoidance becomes particularly important when painful signals are involved, and since the climbing‐fibre input to zone C3 is extremely responsive to nociceptive stimuli, it is proposed that one function of this zone is the avoidance of pain, by for example adjusting movements of the body to avoid self‐harm. This hypothesis appears consistent with evidence from humans and animals concerning the role of the intermediate cerebellum in classically conditioned withdrawal reflexes, but further experiments focussing on conditioned avoidance are required to test the hypothesis more stringently. The proposed architecture may also be useful for automatic self‐adjusting damage‐avoidance in robots, an important consideration for next generation ‘soft’ robots designed to interact with people. This article is protected by copyright. All rights reserved.

  July 12, 2013   doi: 10.1113/jphysiol.2013.261545   open full text
• Classification of frequency response areas in the inferior colliculus reveals continua not discrete classes.
  Alan R. Palmer, Trevor M. Shackleton, Christian J. Sumner, Oliver Zobay, Adrian Rees.
  The Journal of Physiology. July 12, 2013
  •  Neurons in the auditory midbrain, the inferior colliculus, are selectively sensitive to combinations of sound frequency and level as illustrated by their frequency/level receptive fields. Different receptive field shapes have been described, but we do not know if these represent discrete classes reflecting afferent inputs from individual sources, or a more complex pattern of integration. •  In this study we used objective methods to analyse the receptive fields of over 2000 neurons in the guinea pig inferior colliculus. •  Subjectively we identified seven different receptive field classes, but objectively these classes formed continua with many neurons having receptive field shapes intermediate to these extremes. •  These findings are consistent with neurons receiving inhibitory inputs of different strength and frequency disposition but not consistent with neurons reflecting inputs only from individual brainstem nuclei. •  These results are important for understanding the functional organisation of the inferior colliculus and its role in auditory processing. Abstract  A differential response to sound frequency is a fundamental property of auditory neurons. Frequency analysis in the cochlea gives rise to V‐shaped tuning functions in auditory nerve fibres, but by the level of the inferior colliculus (IC), the midbrain nucleus of the auditory pathway, neuronal receptive fields display diverse shapes that reflect the interplay of excitation and inhibition. The origin and nature of these frequency receptive field types is still open to question. One proposed hypothesis is that the frequency response class of any given neuron in the IC is predominantly inherited from one of three major afferent pathways projecting to the IC, giving rise to three distinct receptive field classes. Here, we applied subjective classification, principal component analysis, cluster analysis, and other objective statistical measures, to a large population (2826) of frequency response areas from single neurons recorded in the IC of the anaesthetised guinea pig. Subjectively, we recognised seven frequency response classes (V‐shaped, non‐monotonic Vs, narrow, closed, tilt down, tilt up and double‐peaked), that were represented at all frequencies. We could identify similar classes using our objective classification tools. Importantly, however, many neurons exhibited properties intermediate between these classes, and none of the objective methods used here showed evidence of discrete response classes. Thus receptive field shapes in the IC form continua rather than discrete classes, a finding consistent with the integration of afferent inputs in the generation of frequency response areas. The frequency disposition of inhibition in the response areas of some neurons suggests that across‐frequency inputs originating at or below the level of the IC are involved in their generation.

  July 12, 2013   doi: 10.1113/jphysiol.2013.255943   open full text
• Muscle metabolism and fatigue – in memory of Eric Hultman (10th October 1925 to 9th March 2011).
  Paul Greenhaff, Roger Harris.
  The Journal of Physiology. July 08, 2013
  Abstract  With the opening day of the 15th International Biochemistry of Exercise Congress in Stockholm, June 2012, it was appropriate that the scientific organising committee welcomed delegates to a symposium dedicated to the memory of Professor Eric Hultman, who spent the whole of his scientific career at the Karolinska Institute, Stockholm. The symposium, entitled muscle metabolism and fatigue, highlighted the outstanding research contributions of Professor Hultman over a career spanning more than 4 decades, and also emphasized new research findings stemming from his original research contributions. This edition of the Journal of Physiology includes review articles from 2 of the speakers from the symposium, Niels Ørtenblad and Arend Bonen, which focus on muscle glycogen availability and fatigue (Niels Ørtenblad, Häkan Westerblad, and Joachim Nielsen. Muscle glycogen stores and fatigue), and the regulation of muscle fatty acid oxidation during exercise and training (Yuko Yoshida, Swati S. Jain, Jay T McFarlan, Laelie A. Snook, Adrian Chabowski, and Arend Bonen. Exercise‐ and training‐induced upregulation of skeletal muscle fatty acid oxidation are not solely dependent on mitochondrial machinery and biogenesis). This article is protected by copyright. All rights reserved

  July 08, 2013   doi: 10.1113/jphysiol.2013.261354   open full text
• SNARE proteins are essential in the potentiation of NMDA receptors by group II metabotropic glutamate receptors.
  Jia Cheng, Wenhua Liu, Lara J. Duffney, Zhen Yan.
  The Journal of Physiology. July 08, 2013
  •  Activation of group II metabotropic glutamate receptors (mGluRs) enhances NMDA receptor (NMDAR)‐mediated currents in cortical pyramidal neurons. •  In this study, we found that group II mGluR‐induced enhancement of NMDAR currents was associated with increased NMDAR surface expression and synaptic localization. •  Inhibition of SNAP‐25 or knockdown of syntaxin 4 blocked the enhancement of NMDAR currents by group II mGluRs. •  Group II mGluRs increase the activity of Rab4 small GTPase. Rab4 knockdown or dominant negative Rab4 abolished the enhancing effect of Group II mGluRs on NMDAR currents. •  These results suggest that SNARE proteins and Rab4 are key molecules involved in the enhancement of NMDAR exocytosis and function by group II mGluRs. Identification of key molecules involved in NMDAR up‐regulation could provide novel drug targets for schizophrenia treatment. Abstract  The group II metabotropic glutamate receptors (group II mGluRs) have emerged as the new drug targets for the treatment of mental disorders like schizophrenia. To understand the potential mechanisms underlying the antipsychotic effects of group II mGluRs, we examined their impact on NMDA receptors (NMDARs), since NMDAR hypofunction has been implicated in schizophrenia. The activation of group II mGluRs caused a significant enhancement of NMDAR currents in cortical pyramidal neurons, which was associated with increased NMDAR surface expression and synaptic localization. We further examined whether these effects of group II mGluRs are through the regulation of NMDAR exocytosis via SNARE proteins, a family of proteins involved in vesicle fusion. We found that the enhancing effect of APDC, a selective agonist of group II mGluRs, on NMDAR currents was abolished when botulinum toxin was delivered into the recorded neurons to disrupt the SNARE complex. Inhibiting the function of two key SNARE proteins, SNAP‐25 and syntaxin 4, also eliminated the effect of APDC on NMDAR currents. Moreover, the application of APDC increased the activity of Rab4, a small Rab GTPase mediating fast recycling from early endosomes to the plasma membrane, and enhanced the interaction between syntaxin 4 and Rab4. Knockdown of Rab4 or expression of dominant‐negative Rab4 attenuated the effect of APDC on NMDAR currents. Taken together, these results have identified key molecules involved in the group II mGluR‐induced potentiation of NMDAR exocytosis and function.

  July 08, 2013   doi: 10.1113/jphysiol.2013.255075   open full text
• Cl− homeodynamics in gap junction‐coupled astrocytic networks on activation of GABAergic synapses.
  Kiyoshi Egawa, Junko Yamada, Tomonori Furukawa, Yuchio Yanagawa, Atsuo Fukuda.
  The Journal of Physiology. July 08, 2013
  •  Astrocytes encapsulate GABAergic synapses and express GABAA receptors and GABA transporters. They are tightly coupled by gap junctions, and are referred to as the gap junction‐coupled astrocytic network. •  With higher [Cl−]i, GABA application can mediate bidirectional Cl− fluxes in astrocytes, Cl− efflux via GABAA receptors, and Cl− influx along with GABA uptake via GABA transporters. •  We focused on the Cl− dynamics of the astrocytic network under GABAergic synapse transmission. Spillover of GABA predominantly induced Cl− efflux via GABAA receptors, presumably because they are localized more closely to the synaptic cleft. •  GABAA receptor‐mediated currents were propagated via gap junctions within the astrocytic network. These results indicate that Cl− efflux from astrocytes mediated by GABAergic transmission is homeostatically maintained within gap junction‐coupled astrocytic networks. •  Blockage of gap junctional coupling by octanol promoted the collapse of the driving force for neuronal inhibitory transmission during intense activation of GABAergic synapses. Thus, the astrocytic network may play a role in maintaining GABAergic transmission by regulating [Cl−]o. Abstract  The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction‐coupled astrocytic network are still unclear. GABA‐induced astrocytic Cl− flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl−]o. Thus, revealing the properties of GABA‐mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl− dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl− imaging during GABA application, and whole cell recordings from interneuron–astrocyte pairs in the stratum lacunosum‐moleculare. Astrocytic [Cl−]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl− flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor‐mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing‐induced currents and induced bicuculline‐insensitive, mGAT4 inhibitor‐sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl− efflux because GABAA receptors are localized near the synaptic clefts. This GABA‐induced Cl− efflux was accompanied by Cl− siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing‐induced currents. Thus, Cl− efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity‐dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl− conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl−]o.

  July 08, 2013   doi: 10.1113/jphysiol.2013.257162   open full text
• Pro‐arrhythmogenic effects of atrial fibrillation‐induced electrical remodelling: insights from the three‐dimensional virtual human atria.
  Michael A. Colman, Oleg V. Aslanidi, Sanjay Kharche, Mark R. Boyett, Clifford Garratt, Jules C. Hancox, Henggui Zhang.
  The Journal of Physiology. July 08, 2013
  •  Previous studies have shown that atrial electrical properties are altered (remodelled) by atrial fibrillation (AF) and that the recurrence of AF is high following remodelling. However, demonstrating a causal link between atrial remodelling in experimental models and the increased risk of AF is a challenge. •  AF‐induced electrical remodelling abbreviated atrial action potential duration (APD) non‐uniformly across the atria; this resulted in relatively short APDs co‐existing with marked regional differences in the APD at junctions of the crista terminalis/pectinate muscle, pulmonary veins/left atrium. •  It increases tissue vulnerability to re‐entry initiation and maintenance at these tissue junctions. •  The AF‐induced electrical remodelling also stabilized and accelerated re‐entrant excitation waves, leading to rapid and sustained re‐entry. •  This study provides novel insights towards understanding the mechanisms underlying the pro‐arrhythmic effects of the AF‐induced electrical remodelling in atrial tissue. Abstract  Chronic atrial fibrillation (AF) is associated with structural and electrical remodelling in the atria, which are associated with a high recurrence of AF. Through biophysically detailed computer modelling, this study investigated mechanisms by which AF‐induced electrical remodelling promotes and perpetuates AF. A family of Courtemanche–Ramirez–Nattel variant models of human atrial cell action potentials (APs), taking into account of intrinsic atrial electrophysiological properties, was modified to incorporate various experimental data sets on AF‐induced changes of major ionic channel currents (ICaL, IKur, Ito, IK1, IKs, INaCa) and on intracellular Ca2+ handling. The single cell models for control and AF‐remodelled conditions were incorporated into multicellular three‐dimensional (3D) atrial tissue models. Effects of the AF‐induced electrical remodelling were quantified as the changes of AP profile, AP duration (APD) and its dispersion across the atria, and the vulnerability of atrial tissue to the initiation of re‐entry. The dynamic behaviour of re‐entrant excitation waves in the 3D models was characterised. In our simulations, AF‐induced electrical remodelling abbreviated atrial APD non‐uniformly across the atria; this resulted in relatively short APDs co‐existing with marked regional differences in the APD at junctions of the crista terminalis/pectinate muscle, pulmonary veins/left atrium. As a result, the measured tissue vulnerability to re‐entry initiation at these tissue junctions was increased. The AF‐induced electrical remodelling also stabilized and accelerated re‐entrant excitation waves, leading to rapid and sustained re‐entry. Under the AF‐remodelled condition, re‐entrant scroll waves in the 3D model degenerated into persistent and erratic wavelets, leading to fibrillation. In conclusion, realistic 3D atrial tissue models indicate that AF‐induced electrical remodelling produces regionally heterogeneous and shortened APD; these respectively facilitate initiation and maintenance of re‐entrant excitation waves.

  July 08, 2013   doi: 10.1113/jphysiol.2013.254987   open full text
• Rebuttal to “Prolonged Intense Exercise Training Does Not Lead to Myocardial Damage”.
  Eduard Guasch, Stanley Nattel.
  The Journal of Physiology. July 04, 2013
  Abstract  In their interesting article, Ruiz et al. (2013) raise a number of points in disputing our contention that excess long‐term exercise‐training can be harmful. First, they question the relevance of animal models. Our animal work, which exposed cardiac risks of high‐level exercise‐training and defined underlying mechanisms, showed cardiac remodeling very similar to changes seen in man (Benito et al., 20011; Guasch et al., 2013). This article is protected by copyright. All rights reserved

  July 04, 2013   doi: 10.1113/jphysiol.2013.260000   open full text
• Rebuttal to: Prolonged Intense Exercise Training Does Lead to Myocardial Damage, by Guasch and Nattel.
  Jonatan R. Ruiz, Michael Joyner, Alejandro Lucia.
  The Journal of Physiology. July 04, 2013
  Abstract  Guash and Nattel argue a ‘safety threshold’ exists for endurance training, above which the risk of developing pathological cardiovascular conditions increases (Guash and Nattel, 2013). This article is protected by copyright. All rights reserved

  July 04, 2013   doi: 10.1113/jphysiol.2013.260026   open full text
• Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca2+‐dependent interactions via its EF‐hand 5 domain.
  Oliver Arendt, Beat Schwaller, Edward B. Brown, Jens Eilers, Hartmut Schmidt.
  The Journal of Physiology. July 03, 2013
  •  The dynamics of the second messenger Ca2+ are tightly controlled by Ca2+‐binding proteins (CaBPs). •  The diffusional mobility of a given CaBP, such as calretinin (CR), defines how it affects the range‐of‐action of Ca2+ but, if unexpectedly low, may also indicate that the CaBP acts as a Ca2+ sensor, undergoing specific protein interactions. •  Here we quantified the diffusional mobility of CR in dendrites of cerebellar granule cells using microscopic methods. •  We find that CR diffuses unexpectedly slow, that its mobility is further reduced when Ca2+ levels are elevated and that a distinct region of CR interacts with specific targets in a Ca2+‐dependent manner. •  Our findings indicate a new ‘sensor’ role for CR, which may allow for Ca2+‐dependent feedback control of neuronal excitability. Abstract  Ca2+‐binding proteins (CaBPs) are important regulators of neuronal Ca2+ signalling, acting either as buffers that shape Ca2+ transients and Ca2+ diffusion and/or as Ca2+ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range‐of‐action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye‐labelled CR in mouse granule cells using two‐photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca2+ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF‐hand 5 of CR. Our results suggest long‐lasting, Ca2+‐dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca2+ buffer and point towards a Ca2+ sensor function of CR.

  July 03, 2013   doi: 10.1113/jphysiol.2013.256628   open full text
• Crosslinking the ligand‐binding domain dimer interface locks kainate receptors out of the main open state.
  Bryan A. Daniels, Elizabeth D. Andrews, Mark R. P. Aurousseau, Michael V. Accardi, Derek Bowie.
  The Journal of Physiology. July 02, 2013
  •  This study identifies the gating structure responsible for controlling ion‐channel subconductance behaviour at a major neurotransmitter receptor, namely kainate‐type ionotropic glutamate receptor. •  Evidence is provided that the activation process may be made up of two clearly distinct conductance phases. •  The study speculates that functional diversity amongst ionotropic glutamate receptors emerged during evolution by re‐deploying the same structures to carry out different tasks. Abstract  Kainate‐selective ionotropic glutamate receptors (iGluRs) fulfil key roles in the CNS, making them the subject of detailed structural and functional analyses. Although they are known to gate a channel pore with high and low ion‐permeation rates, it is still not clear how switches between these gating modes are achieved at the structural level. Here, we uncover an unexpected role for the ligand‐binding domain (LBD) dimer assembly in this process. Covalent crosslinking of the dimer interface keeps kainate receptors out of the main open state but permits access to lower conductance states suggesting that significant rearrangements of the dimer interface are required for the receptor to achieve full activation. These observations differ from NMDA‐selective iGluRs where constraining dimer movement reduces open‐channel probability. In contrast, our data show that restricting movement of the dimer interface interferes with conformational changes that underlie both activation and desensitization. Working within the limits of a common architectural design, we propose functionally diverse iGluR families were able to emerge during evolution by re‐deploying existing gating structures to fulfil different tasks.

  July 02, 2013   doi: 10.1113/jphysiol.2013.253666   open full text
• Heterogeneous responses of nucleus incertus neurons to corticotrophin‐releasing factor and coherent activity with hippocampal theta rhythm in the rat.
  Sherie Ma, Anna Blasiak, Francisco E. Olucha‐Bordonau, Anthony J. M. Verberne, Andrew L. Gundlach.
  The Journal of Physiology. July 01, 2013
  •  The nucleus incertus (NI) is a stress and arousal responsive, hindbrain region involved in ascending control of septohippocampal theta rhythm. •  NI neurons express high levels of the neuropeptide relaxin‐3 and corticotrophin‐releasing factor (CRF) receptor‐1 (CRF‐R1). •  We report the first in‐depth characterization of NI neurons, using in vivo and in vitro electrophysiological techniques, which reveal a population of relaxin‐3‐containing NI neurons activated by CRF via postsynaptic CRF‐R1 and a non‐relaxin‐3 neuron population inhibited or unaffected by CRF. •  Relaxin‐3 NI neurons exhibit strong phase‐locked firing with the ascending phase of hippocampal theta oscillations. •  These findings suggest the NI is a heterogeneous neuronal population and key site of CRF action with the capacity to modulate cognition in response to stress. Abstract  The nucleus incertus (NI) of the rat hindbrain is a putative node in the ascending control of the septohippocampal system and hippocampal theta rhythm and is stress and arousal responsive. NI contains GABA neurons that express multiple neuropeptides, including relaxin‐3 (RLN3) and neuropeptide receptors, including corticotrophin‐releasing factor receptor‐1 (CRF‐R1), but the precise anatomical and physiological characteristics of NI neurons are unclear. Therefore, we examined the firing properties of NI neurons and their responses to CRF, the correlation of these responses with occurrence of relaxin‐3, and NI neuron morphology in the rat. Most NI neurons excited by intracerebroventricular CRF infusion were RLN3‐positive (9 of 10), whereas all inhibited cells were RLN3‐negative (8 of 8). The spontaneous firing of RLN3 (n= 6) but not non‐RLN3 neurons (n= 6) was strongly modulated and phase‐locked with the initial ascending phase of hippocampal theta oscillations. In brain slices, the majority of recorded NI neurons (15 of 19) displayed excitatory responses to CRF, which uniformly increased action potential frequency and membrane potential depolarization in the presence of tetrodotoxin, indicating a direct, postsynaptic action of CRF on NI neurons. This excitation was associated with reduction in the slow component of afterhyperpolarization and a strong depolarization. Quantitative analysis in naïve rats of validated CRF‐R1, RLN3 and neuronal nuclear antigen (NeuN) immunoreactivity revealed 52% of NI neurons as CRF‐R1 positive, of which 53% were RLN3 positive, while 48% of NI neurons lacked CRF‐R1 and RLN3. All RLN3 neurons expressed CRF‐R1. CRF neurons that projected to the NI were identified in lateral preoptic hypothalamus, but not in paraventricular hypothalamus, bed nucleus of stria terminalis or central amygdala. Our findings suggest NI is an important site for CRF modulation of hippocampal theta rhythm via effects on GABA/RLN3 transmission.

  July 01, 2013   doi: 10.1113/jphysiol.2013.254300   open full text
• The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor‐mediated signalling.
  Xiaowei Wang, Benjamin J. Whalley, Gary J. Stephens.
  The Journal of Physiology. July 01, 2013
  •  Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. •  Mutant mice with deficits in Ca2+ channel auxiliary α2δ‐2 subunits are used as models of cerebellar ataxia. •  Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. •  We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter‐matched control mice. •  These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ‐2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract  Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’du2J mouse model of ataxia and absence epilepsy represents a clean knock‐out of the auxiliary Ca2+ channel subunit α2δ‐2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone–Purkinje cell (IN‐PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. The du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN‐PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)‐mediated modulation of spontaneous neuronal spike firing and CB1R‐mediated presynaptic inhibition of synaptic transmission at IN‐PC synapses in both +/du2J and du2J/du2J mutants, effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

  July 01, 2013   doi: 10.1113/jphysiol.2012.244947   open full text
• The coupling of plasma membrane calcium entry to calcium uptake by endoplasmic reticulum and mitochondria.
  Javier García‐Sancho.
  The Journal of Physiology. June 28, 2013
  Abstract  Cross‐talk between organelles and plasma membrane Ca2+ channels is essential for modulation of the cytosolic Ca2+ ([Ca2+]C) signals, but such modulation may differ among cells. In chromaffin cells Ca2+ entry through voltage‐operated channels (VOCC) induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca2+]C microdomains as high as 20–50 μM are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca2+ through the mitochondrial Ca2+ uniporter (MCU). Mitochondria confine the high‐Ca2+ microdomains (HCMD) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca2+]C is much smaller (1–2 μM). By acting as a Ca2+ sink, mitochondria stabilise the HCMD in space and time. In non‐excitable HEK293 cells, activation of store‐operated Ca2+ entry (SOCE), triggered by ER Ca2+ emptying, did also generate subplasmalemmal HCMD, but, in this case, most of the Ca2+ was taken up by the ER rather than by mitochondria. The smaller size of the [Ca2+]C peak in this case (about 2 μM) may contribute to this outcome, as the SERCA has much higher Ca2+ affinity than MCU. There is also possible that relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins play an important role. This article is protected by copyright. All rights reserved.

  June 28, 2013   doi: 10.1113/jphysiol.2013.255661   open full text
• Cardiac sodium channelopathy associated with SCN5A mutations: electrophysiological, molecular and genetic aspects.
  Carol Ann Remme.
  The Journal of Physiology. June 28, 2013
  Abstract  Over the last two decades, an increasing number of SCN5A mutations have been described in patients with long QT syndrome type 3 (LQT3), Brugada syndrome, (progressive) conduction disease, sick sinus syndrome, atrial standstill, atrial fibrillation, dilated cardiomyopathy, and sudden infant death syndrome (SIDS). Combined genetic, electrophysiological and molecular studies have provided insight into the dysfunction and dysregulation of the cardiac sodium channel in the setting of SCN5A mutations identified in patients with these inherited arrhythmia syndromes. However, risk stratification and patient management is hindered by the reduced penetrance and variable disease expressivity in sodium channelopathies. Furthermore, various SCN5A‐related arrhythmia syndromes are known to display mixed phenotypes, known as cardiac sodium channel overlap syndromes. Determinants of variable disease expressivity, including genetic background and environmental factors, are suspected but still largely unknown. Moreover, it has become increasingly clear that sodium channel function and regulation is more complicated than previously assumed, and the sodium channel may play additional, as of yet unrecognized roles in cardiac structure and function. Development of cardiac structural abnormalities secondary to SCN5A mutations has been reported, but the clinical relevance and underlying mechanisms are unclear. Increased insight into these issues would enable a major next step in research related to cardiac sodium channel disease, ultimately enabling improved diagnosis, risk stratification and treatment strategies. This article is protected by copyright. All rights reserved.

  June 28, 2013   doi: 10.1113/jphysiol.2013.256461   open full text
• Modulation of homomeric and heteromeric kainate receptors by the auxiliary subunit Neto1.
  Janet L. Fisher, David D. Mott.
  The Journal of Physiology. June 28, 2013
  Abstract  The ionotropic glutamate receptors are primary mediators of fast excitatory neurotransmission, and their properties are determined both by their subunit composition and their association with auxiliary subunits. The Neto1 and Neto2 proteins have been recently identified as auxiliary subunits for kainate‐type glutamate receptors. Heteromeric kainate receptors can be assembled from varying combinations of low affinity (GluK1–3) and high‐affinity (GluK4–5) subunits. To better understand the functional impact of auxiliary subunits on kainate receptors, we examined the effect of Neto1 on the responses of recombinant homomeric and heteromeric kainate receptors to varying concentrations of glutamate. We found that co‐expression of Neto1 with homomeric GluK2 receptors had a small effect on sensitivity of the receptors to glutamate, but decreased the onset of desensitization while speeding recovery from desensitization. In the absence of Neto1, addition of GluK5 subunits to form GluK2/K5 heteromeric receptors slowed the onset of desensitization at low glutamate concentrations, compared to GluK2 homomers. Co‐expression of Neto1 with GluK2/5 receptors further enhanced these effects, essentially eliminating desensitization at μM glutamate concentrations without altering the EC50 for activation by glutamate. In addition, a prominent rebound current was observed upon removal of the agonist. The rate of recovery from desensitization was increased to the same degree by Neto1 for both homomeric GluK2 and heteromeric GluK2/K5 receptors. Expression of Neto1 with GluK1/K5, GluK3/K5 or GluK2/K4 receptors produced qualitatively similar effects on whole‐cell currents, suggesting that the impact of Neto1 on the desensitization properties of heteromeric receptors was not subunit dependent. These results provide greater insight into the functional effects of the auxiliary subunit Neto1 on both homomeric and heteromeric kainate receptors. Alteration of the characteristics of desensitization at both sub‐maximal and saturating glutamate concentrations could influence the responsiveness of these receptors to repeated stimuli. As a result, assembly of kainate receptors with the Neto auxiliary subunits could change the kinetic properties of the neuronal response to glutamatergic input. This article is protected by copyright. All rights reserved.

  June 28, 2013   doi: 10.1113/jphysiol.2013.256776   open full text
• The bioelectrical basis and validity of gastrointestinal extracellular slow wave recordings.
  Timothy R. Angeli, Peng Du, Niranchan Paskaranandavadivel, Patrick W.M. Janssen, Arthur Beyder, Roger G. Lentle, Ian P. Bissett, Leo K. Cheng, Gregory O’Grady.
  The Journal of Physiology. June 28, 2013
  •  Extracellular recording techniques are commonly used to measure bioelectrical activity. However, the validity of gastrointestinal extracellular recordings has recently been challenged. •  In this joint experimental and modelling study, slow waves were recorded during contractile inhibition, biphasic and monophasic slow wave potentials were recorded simultaneously, and the biophysical basis of extracellular potentials was modelled with comparison to experimental data. •  The results showed that in vivo extracellular techniques reliably recorded slow waves in the absence of contractions, and potentials recorded using conventional serosal electrodes (biphasic) were concordant in phase and morphology with those recorded using suction electrodes (monophasic). •  Modelling further demonstrated that the morphology of experimental recordings is consistent with the biophysics underlying slow wave depolarisation. •  In total, these results demonstrate that gastrointestinal extracellular recordings are valid when performed and analysed correctly, reliably representing bioelectrical slow wave events. Motion suppression is not routinely required for in vivo extracellular studies. Abstract  Gastrointestinal extracellular recordings have been a core technique in motility research for a century. However, the bioelectrical basis of extracellular data has recently been challenged by claims that these techniques preferentially assay movement artifacts, cannot reproduce the underlying slow wave kinetics, and misrepresent the true slow wave frequency. These claims motivated this joint experimental–theoretical study, which aimed to define the sources and validity of extracellular potentials. In vivo extracellular recordings and video capture were performed in the porcine jejunum, before and after intra‐arterial nifedipine administration. Gastric extracellular recordings were recorded simultaneously using conventional serosal contact and suction electrodes, and biphasic and monophasic extracellular potentials were simulated in a biophysical model. Contractions were abolished by nifedipine, but extracellular slow waves persisted, with unchanged amplitude, downstroke rate, velocity, and downstroke width (P > 0.10 for all), at reduced frequency (24% lower; P= 0.03). Simultaneous suction and conventional serosal extracellular recordings were identical in phase (frequency and activation–recovery interval), but varied in morphology (monophasic vs. biphasic; downstroke rate and amplitude: P < 0.0001). Simulations demonstrated the field contribution of current flow to extracellular potential and quantified the effects of localised depolarisation due to suction pressure on extracellular potential morphology. In sum, these results demonstrate that gastrointestinal extracellular slow wave recordings cannot be explained by motion artifacts, and are of a bioelectrical origin that is highly consistent with the underlying biophysics of slow wave propagation. Motion suppression is shown to be unnecessary as a routine control in in vivo extracellular studies, supporting the validity of the extant gastrointestinal extracellular literature.

  June 28, 2013   doi: 10.1113/jphysiol.2013.254292   open full text
• Stabilization of Kv4 protein by the accessory K+ channel interacting protein 2 (KChIP2) subunit is required for the generation of native myocardial fast transient outward K+ currents.
  Nicholas C. Foeger, Wei Wang, Rebecca L. Mellor, Jeanne M. Nerbonne.
  The Journal of Physiology. June 28, 2013
  •  The cytosolic K+ channel accessory subunit, K+ channel interacting protein 2 (KChIP2), was previously suggested to be critical in the generation of cardiac fast transient outward current (Ito,f) channels. •  The experiments presented here revealed the novel finding that targeted deletion of KChIP2 results in the complete loss of the Kv4.2 protein, although Kcnd2 (Kv4.2) transcript expression is not decreased in KChIP2−/− ventricles. •  In contrast, the slow transient outward current, Ito,s, is increased in KChIP2−/− left ventricular apex myocytes and ventricular action potential waveforms in KChIP2−/− and WT mice are not significantly different. •  These results demonstrate the critical role of KChIP2 in the stabilization of native Kv4 proteins and that the loss of the Kv4.2 protein underlies the elimination of Ito,f in KChIP2−/− myocytes. •  Taken together, the results here demonstrate that electrical remodelling compensates for the elimination of Ito,f, maintaining physiological action potential repolarization in mouse myocardium. Abstract  The fast transient outward K+ current (Ito,f) underlies the early phase of myocardial action potential repolarization, contributing importantly to the coordinated propagation of activity in the heart and to the generation of normal cardiac rhythms. Native Ito,f channels reflect the tetrameric assembly of Kv4 pore‐forming (α) subunits, and previous studies suggest roles for accessory and regulatory proteins in controlling the cell surface expression and the biophysical properties of Kv4‐encoded Ito,f channels. Here, we demonstrate that the targeted deletion of the cytosolic accessory subunit, K+ channel interacting protein 2 (KChIP2), results in the complete loss of the Kv4.2 protein, the α subunit critical for the generation of mouse ventricular Ito,f. Expression of the Kcnd2 (Kv4.2) transcript in KChIP2−/− ventricles, however, is unaffected. The loss of the Kv4.2 protein results in the elimination of Ito,f in KChIP2−/− ventricular myocytes. In parallel with the elimination of Ito,f, the slow transient outward K+ current (Ito,s) is upregulated and voltage‐gated Ca2+ currents (ICa,L) are decreased. In addition, surface electrocardiograms and ventricular action potential waveforms in KChIP2−/− and wild‐type mice are not significantly different, suggesting that the upregulation of Ito,s and the reduction in ICa,L compensate for the loss of Ito,f. Additional experiments revealed that Ito,f is not ‘rescued’ by adenovirus‐mediated expression of KChIP2 in KChIP2−/− myocytes, although ICa,L densities are increased. Taken together, these results demonstrate that association with KChIP2 early in the biosynthetic pathway and KChIP2‐mediated stabilization of Kv4 protein are critical determinants of native cardiac Ito,f channel expression.

  June 28, 2013   doi: 10.1113/jphysiol.2013.255836   open full text
• CrossTalk: Peripheral and central chemoreceptors have hypoadditive effects on respiratory motor output.
  Richard J.A. Wilson, Trevor A. Day.
  The Journal of Physiology. June 24, 2013
  Breathing is all‐important for survival, yet key aspects of the control system remain hidden from the gaze of consensus, not least the issue of central and peripheral chemoreceptor interaction. Activation (or inactivation) of either chemoreceptor alone will increase (or decrease) ventilation, but it remains unclear how the activation state of one chemoreceptor modality affects the chemoreflex response of the other (i.e., how inputs interact). Recent investigations consider three possibilities (e.g., Blain et. al., 2010; Cui et. al., 2012; Day and Wilson, 2009; Forster and Smith, 2012; Smith et. al., 2010; Tin et. al., 2012). Below, we consider four possibilities, three of which implicate some degree of hypoadditive interaction. This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.256578   open full text
• Cross‐Talk: The peripheral and central chemoreflexes have additive effects on ventilation in humans.
  James Duffin, Jason H. Mateika.
  The Journal of Physiology. June 24, 2013
  In humans respiratory chemoreceptors are located centrally in the medulla (Nattie, 2010) and peripherally in the carotid bodies (Torrance, 1996; Kumar & Bin‐Jaliah, 2007). Does the interaction between hypoxia and CO2 occur in the medulla between the central and peripheral chemoreceptor signals or is it within the peripheral chemoreceptors? Several observations are pertinent to this question. This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.256800   open full text
• Peripheral and central chemoreceptors have hyperadditive effects on respiratory motor control.
  Luc J. Teppema, Curtis A. Smith.
  The Journal of Physiology. June 24, 2013
  Since the discovery of the O2 and CO2 respiratory chemoreceptors there has been a long debate as to their relative contributions to eupnea and the ventilatory responses to hypoxia and hypercapnia. Recent evidence suggests that attempting to assign relative contributions to the central and peripheral chemoreceptors may not be a useful approach (e.g., Teppema & Dahan, 2010; Smith et al., 2010) if the two sets of chemoreceptors interact in other than a simply additive way and are thus capable of modulating the responsiveness of one another. This means that neural signals arising from stimuli at both sets of chemoreceptors have the potential to interact; indeed, such interdependence is a pre‐condition for hypo‐ or hyperadditive interaction (Adams & Severns, 1982). In this pro/con debate three potential interaction modes are discussed: hypoadditive, additive and hyperadditive. The literature reports a broad spectrum of results and opinions between the extremes of hypo‐ and hyperaddition (reviewed in Teppema & Dahan, 2010; Blain et al., 2010; Smith et al., 2010). Here we focus on recent evidence that supports a hyperadditive or multiplicative (synergistic) interaction; we will not discuss the well‐known O2‐CO2 interaction at the level of the carotid bodies (Fitzgerald & Parks, 1971; Lahiri & DeLaney, 1975). This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.256818   open full text
• CrossTalk Rebuttal 1 (of 3).
  James Duffin, Jason H. Mateika.
  The Journal of Physiology. June 24, 2013
  Our comments in reply to the arguments presented in the CrossTalks by Teppema and Smith (2013) and Wilson and Day (2013) are confined to experiments on humans, and in their CrossTalk Teppema and Smith (2013) cite two studies that found evidence supporting a hyperadditive interaction. First, carotid body denervation or removal in man has consequences for the central chemoreception of CO2. We agree that such denervation removes the tonic drive from the carotid bodies (Dahan et al., 2007); evident from their involvement in sympathetic tone. This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.259853   open full text
• CrossTalk Rebuttal: Peripheral and central chemoreceptors have hypoadditive effects on respiratory motor output.
  Richard J.A. Wilson, Trevor A. Day.
  The Journal of Physiology. June 24, 2013
  Our colleagues skillfully addressed how peripheral chemoreceptors interact with systemic effects of CO2. Unfortunately, systemic CO2 affects oxyhemoglobin dissociation, sympathetic, endocrine and cardiovascular systems, and afferents from lungs and CO2‐sensitive upper airway receptors – all capable of having effects on ventilation independent of central chemoreceptors. Duffin's ingenious rebreathing studies add further concerns, namely psychological and physiological aspects of voluntary hyperventilation (e.g., Steinback et al. 2011). This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.259861   open full text
• Rebuttals to : “Cross‐Talk: The peripheral and central chemoreflexes have additive effects on ventilation”, by James Duffin and Jason H. Mateika, And: “Cross‐Talk: The peripheral and central chemoreflexes have hypoadditive effects on ventilation”, by Richard J.A. Wilson and Trevor A. Day.
  Luc J. Teppema, Curtis A. Smith.
  The Journal of Physiology. June 24, 2013
  Duffin and Mateika (Duffin J. & Mateika, 2013) state that their rebreathing data could also be fitted to a parabola. If they consider a parabolic shape an indication of hyper‐additive interaction, it is surprising that they did not compare the quality of the fits of linear regression vs. parabolic or hyperbolic fits in order to decide which interaction mode would fit their data best. Apart from this, we have doubts about several assumptions underlying the modified rebreathing technique : 1. the absence of carotid body activity in hyperoxia; 2) that the hypoxic response would be a modified acidic response; 3. due to variable changes in CBF during the manouever, the tissue‐arterial PCO2 relationship cannot be constant (Battisti‐Charbonney et al., 2011); and 4. absence of cortical influences on ventilation following five minutes of voluntary hyperventilation. This article is protected by copyright. All rights reserved.

  June 24, 2013   doi: 10.1113/jphysiol.2013.259879   open full text
• Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier‐like activation of ACh‐gated K+ current by choline in feline atrial myocytes.
  Ricardo A. Navarro‐Polanco, Iván A. Aréchiga‐Figueroa, Pedro D. Salazar‐Fajardo, Dora E. Benavides‐Haro, Julio C. Rodríguez‐Elías, Frank B. Sachse, Martin Tristani‐Firouzi, José A. Sánchez‐Chapula, Eloy G. Moreno‐Galindo.
  The Journal of Physiology. June 24, 2013
  •  Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). •  Previously, in cardiomyocytes Ch was shown to activate an outward K+ current in a delayed rectifier fashion, which has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the identity of this current remains elusive. •  Single‐channel recordings, biophysical profiles and specific pharmacological inhibition indicate that the current activated by Ch is the ACh‐activated K+ current (IKACh). •  Membrane depolarization increased the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K+ current at depolarized potentials. •  Our findings support the emerging concept that IKACh modulation is both voltage‐ and ligand‐specific and reinforce the importance of these properties in understanding cardiac physiology. Abstract  Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K+ current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand‐ and voltage‐dependent properties of muscarinic activation of ACh‐activated K+ current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier‐like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage‐dependent manner similar to pilocarpine. Single‐channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K+ current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage‐ and ligand‐specific and reinforce the importance of these properties in understanding cardiac physiology.

  June 24, 2013   doi: 10.1113/jphysiol.2013.255166   open full text
• Neuronal transporter and astrocytic ATP exocytosis underlie activity‐dependent adenosine release in the hippocampus.
  Mark J. Wall, Nicholas Dale.
  The Journal of Physiology. June 21, 2013
  •  Using microelectrode biosensors we have directly measured the adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. •  Approximately 40% of stimulated‐adenosine release occurred by translocation of adenosine from neurons via equilibrative nucleoside transporters (ENTs). •  The remaining adenosine release arises from the extracellular metabolism of ATP released from astrocytes by exocytosis. •  Isolation of the individual components of adenosine release revealed their different kinetics with adenosine release via ENTs markedly faster than the adenosine release that arises from ATP exocytosis. •  These data illustrate the complexity of activity‐dependent adenosine release: in the hippocampus, adenosine release occurs by at least two distinct mechanisms with different cellular sources and kinetics. Abstract  The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated‐adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn‐SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT‐mediated release of adenosine. These data suggest that activity‐dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources.

  June 21, 2013   doi: 10.1113/jphysiol.2013.253450   open full text
• Independent control of reciprocal and lateral inhibition at the axon terminal of retinal bipolar cells.
  Masashi Tanaka, Masao Tachibana.
  The Journal of Physiology. June 21, 2013
  •  Two different forms of feedback inhibition, reciprocal and lateral inhibition, are ubiquitously observed throughout the nervous system. •  In the retina, the axon terminal of bipolar cells receives reciprocal and lateral GABAergic inhibitory inputs from amacrine cells, but how a variety of visual inputs activate each inhibition remains largely unexplored. •  Here we show that each inhibition is independently controlled by different types of bipolar cell outputs; reciprocal inhibition is driven by strong output from each bipolar cell, whereas lateral inhibition is driven by outputs from multiple bipolar cells even when each output is weak. •  Composition of transmitter receptors and localization of Na+ channels were different between two inhibitory pathways, suggesting that different amacrine cells may mediate each inhibition. •  The dual feedback inhibition can cooperatively reduce bipolar cell outputs in response to various visual inputs without deteriorating the quality of visual signals, thereby contributing to efficient signal transmission in the visual pathway. Abstract  Bipolar cells (BCs), the second order neurons in the vertebrate retina, receive two types of GABAergic feedback inhibition at their axon terminal: reciprocal and lateral inhibition. It has been suggested that two types of inhibition may be mediated by different pathways. However, how each inhibition is controlled by excitatory BC output remains to be clarified. Here, we applied single/dual whole cell recording techniques to the axon terminal of electrically coupled BCs in slice preparation of the goldfish retina, and found that each inhibition was regulated independently. Activation voltage of each inhibition was different: strong output from a single BC activated reciprocal inhibition, but could not activate lateral inhibition. Outputs from multiple BCs were essential for activation of lateral inhibition. Pharmacological examinations revealed that composition of transmitter receptors and localization of Na+ channels were different between two inhibitory pathways, suggesting that different amacrine cells may mediate each inhibition. Depending on visual inputs, each inhibition could be driven independently. Model simulation showed that reciprocal and lateral inhibition cooperatively reduced BC outputs as well as background noise, thereby preserving high signal‐to‐noise ratio. Therefore, we conclude that excitatory BC output is efficiently regulated by the dual operating mechanisms of feedback inhibition without deteriorating the quality of visual signals.

  June 21, 2013   doi: 10.1113/jphysiol.2013.253179   open full text
• Nitric oxide‐dependent long‐term depression but not endocannabinoid‐mediated long‐term potentiation is crucial for visual recognition memory.
  Francesco Tamagnini, Gareth Barker, E. Clea Warburton, Costanza Burattini, Giorgio Aicardi, Zafar I. Bashir.
  The Journal of Physiology. June 20, 2013
  •  Perirhinal cortex (Prh) is critically involved in visual recognition memory and synaptic plasticity. •  Nitric oxide and endocannabinoids (eCBs) have been shown to act as retrograde messengers in synaptic plasticity in several brain areas, but no study has yet investigated their role in synaptic plasticity in Prh. •  Evidence is still lacking of a retrograde messenger involved in synaptic plasticity in Prh. •  In this study, we show that NO is involved in long‐term depression (LTD) but not in long‐term potentiation (LTP). Conversely, eCBs are involved in LTP but not in LTD. Crucially, inhibiition of NO signalling prevents visual recognition memory acquisition, whilst inhibition of eCB signalling does not affect recognition memory. •  These results suggest that LTD but not LTP is a neuronal correlate of visual recognition memory. Abstract  Synaptic plasticity in perirhinal cortex is essential for recognition memory. Nitric oxide and endocannabinoids (eCBs), which are produced in the postsynaptic cell and act on the presynaptic terminal, are implicated in mechanisms of long‐term potentiation (LTP) and long‐term depression (LTD) in other brain regions. In this study, we examine these two retrograde signalling cascades in perirhinal cortex synaptic plasticity and in visual recognition memory in the rat. We show that inhibition of NO‐dependent signalling prevented both carbachol‐ and activity (5 Hz)‐dependent LTD but not activity (100 Hz theta burst)‐dependent LTP in the rat perirhinal cortex in vitro. In contrast, inhibition of the eCB‐dependent signalling prevented LTP but not the two forms of LTD in vitro. Local administration into perirhinal cortex of the nitric oxide synthase inhibitor NPA (2 μm) disrupted acquisition of long‐term visual recognition memory. In contrast, AM251 (10 μm), a cannabinoid receptor 1 antagonist, did not impair visual recognition memory. The results of this study demonstrate dissociation between putative retrograde signalling mechanisms in LTD and LTP in perirhinal cortex. Thus, LTP relies on cannabinoid but not NO signalling, whilst LTD relies on NO‐ but not eCB‐dependent signalling. Critically, these results also establish, for the first time, that NO‐ but not eCB‐dependent signalling is important in perirhinal cortex‐dependent visual recognition memory.

  June 20, 2013   doi: 10.1113/jphysiol.2013.254862   open full text
• Dynamic regulation of glycine–GABA co‐transmission at spinal inhibitory synapses by neuronal glutamate transporter.
  Hitoshi Ishibashi, Junya Yamaguchi, Yoshihisa Nakahata, Junichi Nabekura.
  The Journal of Physiology. June 17, 2013
  •  Inhibition mediated by GABA and glycine is essential for controlling a balance of excitation and inhibition in the spinal cord. •  Although these transmitters are known to be co‐released from the same synaptic vesicles, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles have not been fully characterized. •  In this study, using paired whole‐cell recording, we found that raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs by enhancing glutamate uptake but reduced glycine release. •  High‐frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs at GABA/glycine mixed synapses, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. •  The present results suggest that the use of GABA as a transmitter at GABA/glycine mixed synapses may afford protection against pathophysiological hyperexcitability associated with increased extracellular glutamate concentration. Abstract  Fast inhibitory neurotransmission in the central nervous system is mediated by γ‐aminobutyric acid (GABA) and glycine, which are accumulated into synaptic vesicles by a common vesicular inhibitory amino acid transporter (VIAAT) and are then co‐released. However, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles are not fully understood. In this study, we demonstrate the dynamic control of the GABA–glycine co‐transmission by the neuronal glutamate transporter, using paired whole‐cell patch recording from monosynaptically coupled cultured spinal cord neurons derived from VIAAT‐Venus transgenic rats. Short step depolarization of presynaptic neurons evoked unitary (cell‐to‐cell) inhibitory postsynaptic currents (IPSCs). Under normal conditions, the fractional contribution of postsynaptic GABA or glycine receptors to the unitary IPSCs did not change during a 1 h recording. Intracellular loading of GABA or glycine via a patch pipette enhanced the respective components of inhibitory transmission, indicating the importance of the cytoplasmic concentration of inhibitory transmitters. Raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs but reduced glycine release by enhancing glutamate uptake. Similar effects were observed when presynaptic neurons were intracellularly perfused with glutamate. Interestingly, high‐frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. The present results suggest that the enhancement of GABA release by glutamate uptake may be advantageous for rapid vesicular refilling of the inhibitory transmitter at mixed GABA/glycinergic synapses and thus may help prevent hyperexcitability.

  June 17, 2013   doi: 10.1113/jphysiol.2012.250647   open full text
• Quantifying the origins of population variability in cardiac electrical activity through sensitivity analysis of the electrocardiogram.
  Arash Sadrieh, Stefan A. Mann, Rajesh N. Subbiah, Luke Domanski, John A. Taylor, Jamie I. Vandenberg, Adam P. Hill.
  The Journal of Physiology. May 17, 2013
  •  We used a novel high performance computing approach to conduct a sensitivity analysis of emergent properties of simulated ECGs from a transmural cable of cells. •  The rapid delayed rectifier and inward rectifying potassium currents are the primary determinants of the height of the T wave in this system. •  Theight is correlated with the temporal dispersion of repolarisation in the transmural cable while Tpeak– Tend is correlated with the interval from the time of maximum total rate of repolarisation to the end of repolarisation in the cable of cells. •  This study advances our understanding of the molecular basis of T wave morphology and the role of epistatis in the modification of cardiac electrical phenotypes. Abstract  Altered function of ion channels in the heart can increase the risk of sudden arrhythmic death. Hundreds of genetic variants exist in these cardiac ion channel genes. The challenge is how to interpret the effects of multiple conductance perturbations on the complex multi‐variable cardiac electrical system? In theory, sensitivity analysis can address this question. However, to date this approach has been restricted by computational overheads to analysis of isolated cells, which has limited extrapolation to physiologically relevant scales. The goal of this study was to extend existing sensitivity analyses to electrocardiogram (ECG) signals derived from multicellular systems and quantify the contribution of ionic conductances to emergent properties of the ECG. To achieve this, we have developed a highly parallelised simulation environment using unconventional high performance computing architectures to analyse the emergent electrical properties of a multicellular system. This has permitted the first systematic analysis of the molecular basis of the T wave amplitude, revealing important but distinct roles for delayed rectifier and inward rectifier K+ currents. In addition to quantifying how interactions between multiple ion channels influence ECG parameters we show that these sensitivities are dynamic functions of heart rate. This study provides a significant advance in our understanding both of how individual ion conductances define ECG signals and of epistatic modification of cardiac electrical phenotypes. The parallelised simulation environment we have developed removes the computational roadblock that has limited this approach and so provides the framework for future analysis of more complex tissue and whole organ systems.

  May 17, 2013   doi: 10.1113/jphysiol.2013.251710   open full text
• Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, achieving biological cardiac resynchronization post‐infarction.
  Satsuki Yamada, Timothy J. Nelson, Garvan C. Kane, Almudena Martinez‐Fernandez, Ruben J. Crespo‐Diaz, Yasuhiro Ikeda, Carmen Perez‐Terzic, Andre Terzic.
  The Journal of Physiology. May 09, 2013
  •  The pumping function of the heart depends on ordered initiation and propagation of myocardial excitation. Cardiac output is compromised by inconsistent timing and direction of wall motion, leading to dyssynchrony and organ failure. •  Myocardial infarction induces irreversible heart damage. Extensive damage hampers effective pacemaker‐based cardiac resynchronization therapy, the current standard‐of‐care. Establishment of alternative approaches is thus warranted. •  High‐resolution imaging was here utilized to non‐invasively map suitable therapeutic targets within a dyssynchronous heart. Speckle‐tracking echocardiography unmasked the source of progressive cardiac dyssynchrony within the primary infarcted region. •  Bioengineered stem cells with a capacity to induce a regenerative response were implanted into infarcted areas. Speckle‐tracking echocardiography and histology assessment revealed that cell therapy achieved cardiac resynchronization and long‐term repair. •  This proof‐of‐concept study thus introduces a stem cell‐based regenerative solution to address cardiac dyssynchrony post‐infarction. Abstract  Dyssynchronous myocardial motion aggravates cardiac pump function. Cardiac resynchronization using pacing devices is a standard‐of‐care in the management of heart failure. Post‐infarction, however, scar tissue formation impedes the efficacy of device‐based therapy. The present study tests a regenerative approach aimed at targeting the origin of abnormal motion to prevent dyssynchronous organ failure. Induced pluripotent stem (iPS) cells harbour a reparative potential, and were here bioengineered from somatic fibroblasts reprogrammed with the stemness factors OCT3/4, SOX2, KLF4, and c‐MYC. In a murine infarction model, within 30 min of coronary ligation, iPS cells were delivered to mapped infarcted areas. Focal deformation and dysfunction underlying progressive heart failure was resolved prospectively using speckle‐tracking imaging. Tracked at high temporal and spatial resolution, regional iPS cell transplantation restored, within 10 days post‐infarction, the contractility of targeted infarcted foci and nullified conduction delay in adjacent non‐infarcted regions. Local iPS cell therapy, but not delivery of parental fibroblasts or vehicle, prevented or normalized abnormal strain patterns correcting the decrease in peak strain, disparity of time‐to‐peak strain, and pathological systolic stretch. Focal benefit of iPS cell intervention translated into improved left ventricular conduction and contractility, reduced scar, and reversal of structural remodelling, protecting from organ decompensation. Thus, in ischaemic cardiomyopathy, targeted iPS cell transplantation synchronized failing ventricles, offering a regenerative strategy to achieve biological resynchronization.

  May 09, 2013   doi: 10.1113/jphysiol.2013.252288   open full text
• Preserved fertility despite erectile dysfunction in mice lacking the NO receptor.
  Dieter Groneberg, Barbara Lies, Peter Koenig, Ronald Jäger, Andreas Friebe.
  The Journal of Physiology. November 06, 2012
  Abstract  NO and cGMP have been shown to be important mediators of penile erection. Erectile dysfunction may result from reduced or non‐functional signal transduction within this cascade. There is, however, some inconsistency in the available data since mice lacking NO synthases (eNOS, nNOS or both) appear to be fertile whereas mice deficient in cGMP‐dependent protein kinase I (PKGI) suffer from erectile dysfunction. To clarify this discrepancy we performed studies on mice lacking the NO receptor, NO‐sensitive guanylyl cyclase (NO‐GC). In addition, we generated cell‐specific NO‐GC knock out lines in order to be able to investigate the function of NO in individual cell types. NO‐GC was specifically deleted in smooth muscle or endothelial cells (SM‐GCKO and EC‐GCKO, respectively) and these KO lines were compared with total knockouts (GCKO) and WT animals. We investigated expression of NO‐GC, NO‐induced relaxation of corpus cavernosum smooth muscle and their ability to generate offspring. NO‐GC‐positive immunostaining was detected in smooth muscle and endothelial cells of murine corpus cavernosum but not in interstitial cells of Cajal. NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from GCKO mice in organ bath experiments. Similar results were obtained in corpus cavernosum from SM‐GCKO mice whereas the deletion of NO‐GC in endothelial cells did not affect relaxation. The lack of NO‐induced relaxation in GCKO animals was not compensated by cAMP signalling. To our surprise, GCKO males were fertile although their ability to produce offspring was decreased. Our data show that deletion of NO‐GC specifically in smooth muscle cells abolishes NO‐induced corpus cavernosum relaxation but does not lead to infertility.

  November 06, 2012   doi: 10.1113/jphysiol.2012.24555   open full text
• Progressive limb ataxia following inferior olive lesions.
  K. M. Horn, A. Deep, A. R. Gibson.
  The Journal of Physiology. October 29, 2012
  •  The inferior olive (IO) provides climbing fibre input to the cerebellum. Kainic acid lesions of rostral IO of the cat produce several distinct movement deficits. •  Vestibular disturbances are apparent the first few days after injection but rapidly recover. •  Disturbances of grasping occur immediately and do not recover over months of testing. •  After a brief delay, limb movements during reaching and locomotion show a progressive development of ataxia that becomes severe over months of testing. •  The decomposition of movement during reaching and alterations in reach trajectories are similar to those seen in humans with cerebellar ataxias – degeneration of the IO may contribute to the progressive nature of these diseases. Abstract  Cerebellar climbing fibres originate in the inferior olive (IO). Temporary IO inactivation produces movement deficits. Does permanent inactivation produce similar deficits and, if so, do they recover? The excitotoxin, kainic acid, was injected into the rostral IO of three cats. Behaviour was measured during reaching and locomotion. Two cats were injected during the reaching task. Within minutes, grasping became difficult and the trajectories of the reaches showed higher arcing than normally seen. During locomotion, both cats showed head and trunk deviation to the injected side, walking paths curved to the injected side, and the paws were lifted higher than normal. Limbs contralateral to the injections became rigid. Within 1 day, posture had normalized, locomotion was unsteady and high lifting of the paws had reversed to a tendency to drag the dorsum of the paws. Passive body movement produced vestibular signs. Over a few days, locomotion normalized and vestibular signs disappeared. Reach trajectories were normal but grasping deficits persisted. Over the first week, the amplitude of limb lift during reaching and locomotion began to increase. The increase continued over time and, after several months, limb movements became severely ataxic. The effects followed the somatotopy of the rostral IO: a loss of cells in medial rostral IO only affected the forelimb, whereas a loss of cells in medial and lateral IO affected both forelimb and hindlimb. Deficits produced by IO lesions involve multiple mechanisms; some recover rapidly, some appear stable, and some worsen over time. The nature of the progressive deficit suggests a gradual loss of Purkinje cell inhibition on cerebellar nuclear cells.

  October 29, 2012   doi: 10.1113/jphysiol.2012.234898   open full text
• Voltage‐gated Potassium Channels and the Diversity of Electrical Signaling.
  Lily Yeh Jan, Yuh Nung Jan.
  The Journal of Physiology. March 23, 2012
  Abstract  Since Hodgkin and Huxley discovered the potassium current that underlies the falling phase of action potentials in the squid giant axon, the diversity of voltage‐gated potassium (Kv) channels has been manifested in multiple ways: The large and extended potassium channel family is evolutionarily conserved molecularly and functionally. Alternative splicing and RNA editing of Kv channel genes diversify the channel property and expression level. The mix‐and‐match of subunits in a Kv channel that contains four similar or identical pore‐forming subunits and additional auxiliary subunits further diversify Kv channels. Moreover, targeting of different Kv channels to specific subcellular compartments and local translation of Kv channel mRNA in neuronal processes diversify axonal and dendritic action potentials and influence how synaptic plasticity may be modulated. As one indication of the evolutionary conservation of Kv1 channel functions, mutations of the Shaker potassium channel gene in Drosophila and the KCNA1 gene for its mammalian ortholog Kv1.1 cause hyperexcitability near axon branch points and nerve terminals, thereby leading to uncontrolled movements and recapitulating the Episodic Ataxia‐1 (EA1) symptoms in human patients.

  March 23, 2012   doi: 10.1113/jphysiol.2011.244212   open full text
• Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice.
  Yueh‐Chen Lin, Roger H. Adamson, Joyce F. Clark, Rolf K. Reed, Fitz‐Roy E. Curry.
  The Journal of Physiology. November 16, 2011
  Non‐technical summary  When vascular volume is expanded, atrial natriuretic peptide (ANP) released from the heart acts to restore plasma volume by increasing renal water excretion, causing vasodilation and increasing vascular permeability to water and macromolecules. Previous experiments using mice with selective deletion of ANP receptors in vascular endothelial cells (Schreier et al., 2008) emphasized the importance of vascular permeability regulation by ANP in plasma volume restoration because this genetic manipulation of ANP action on vascular permeability limited the restoration of vascular volume after an acute increase in plasma volume. Here we demonstrate retention of intravenously infused fluid in wild‐type mice in which the response to endogenous ANP was attenuated by the pharmacological agent rolipram that stabilized the endothelial barrier by tightening adhesion between adjacent endothelial cells (Curry et al, 2009). The strategy may provide novel approaches to the clinical problem of maintenance of vascular volume after acute intravenous fluid infusion.Abstract  We tested the hypothesis that inhibition of phosphodiesterase 4 (PDE4) with rolipram to increase vascular endothelial cAMP and stabilize the endothelial barrier would attenuate the action of endogenous atrial natriuretic peptide (ANP) to increase vascular permeability to the plasma protein albumin after an acute plasma volume expansion. After rolipram pretreatment (8 mg (kg BW)−1, intraperitoneal, 30 min) more than 95% of the peak increase in plasma volume after volume expansion (4.5% bovine serum albumin, 114 μl (g BW)−1 hr−1, 15 min) remained in the vascular space 75 min after the end of infusion, whereas only 67% of the fluid was retained in volume expanded animals with no rolipram pretreatment. Rolipram significantly decreased 30 min fluorescently labeled albumin clearance (μl (g dry wt)−1) relative to untreated volume expanded controls in skin (e.g., back, 10.4 ± 1.6 vs 19.5 ± 3.6, P= 0.04), muscle (e.g., hamstring, 15.0 ± 1.9 vs 20.8 ± 1.4, P= 0.04) and in colon, cecum, and rectum (average reduction close to 50%). The mass of muscle and skin tissue accounted for 70% of volume expansion dependent albumin shifts from plasma to interstitium. The results are consistent with observations that the PDE4 inhibitor rolipram attenuates ANP induced increases in vascular permeability after infusion of exogenous ANP (Lin et al., 2011) and observations of elevated central venous pressure after a similar volume expansion in mice with selective deletion of the endothelial ANP receptor (Schreier et al., 2008). These observations may form the basis for new strategies to retain intravenous fluid containing macromolecules.

  November 16, 2011   doi: 10.1113/jphysiol.2011.4866   open full text