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The Journal of Physiology

Impact factor: 4.38 5-Year impact factor: 4.834 Print ISSN: 0022-3751 Online ISSN: 1469-7793 Publisher: Wiley Blackwell (Blackwell Publishing -The Physiological Society)

Subject: Psychology

Most recent papers:

  • 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. May 31, 2017
    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 pre‐determining 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. This article is protected by copyright. All rights reserved
    May 31, 2017   doi: 10.1113/JP274309   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. May 31, 2017
    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 BMI, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic‐euglycaemic clamp. Sequential muscle biopsies (0 h, 2 h, 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. In contrast, in sedentary individuals lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute FFA 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 which enables fatty acids to be stored in IMTG. This article is protected by copyright. All rights reserved
    May 31, 2017   doi: 10.1113/JP274374   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. May 31, 2017
    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 current study tested 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 ecto‐5′‐nucleotidase 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 (ΔV˙E control 74 ± 6%, ΔV˙E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as evidenced by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify ecto‐5′‐nucleotidase as a novel regulator of CB sensory activity. Future investigations are warranted to evaluate whether inhibition of ecto‐5′‐nucleotidase can effectively reduce CB activity in CB‐mediated cardiovascular pathology. This article is protected by copyright. All rights reserved
    May 31, 2017   doi: 10.1113/JP274498   open full text
  • Altered postcapillary 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. May 30, 2017
    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 leans (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 remodeling of the venular wall. Venular tone at ∼25 μm (post‐capillary) and ∼75 μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilation 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 hyperemic 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 trans‐capillary exchange of solutes and water under conditions of metabolic syndrome. This article is protected by copyright. All rights reserved
    May 30, 2017   doi: 10.1113/JP274291   open full text
  • Heterotypic endosomal fusion as an initial trigger for insulin‐induced GLUT4 translocation in skeletal muscle.
    Hiroyasu Hatakeyama, Makoto Kanzaki.
    The Journal of Physiology. May 30, 2017
    Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter GLUT4 via distinct signalling pathways, and exercise enhances insulin sensitivity. However, trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood due to technical limitations. Herein, employing various imaging techniques on isolated skeletal myofibers, 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 evidenced by these acute endomembranous activities. We also found that AS160 exhibited stripe‐like localization nearby sarcomeric α‐actinin and that insulin induced a reduction of the stripe‐like localization accompanying with changes in its detergent solubility. Our results 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 exercise benefits. This article is protected by copyright. All rights reserved
    May 30, 2017   doi: 10.1113/JP273985   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. May 29, 2017
    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 drastically increases. This paradox raises an interesting question whether increased activity recruits ATP‐independent mechanism(s) to accelerate endocytosis while preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at the 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 8–15 days, suggesting its existence before and after hearing onset. This endocytosis was not affected by reduction of exocytosis using the light chain of botulinum toxin C, or by block of clathrin‐coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre‐existing at surface, and 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. Our results 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. This article is protected by copyright. All rights reserved
    May 29, 2017   doi: 10.1113/JP274275   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. May 27, 2017
    Mutations in CLCNKB gene encoding human voltage‐gated chloride ClC‐Kb (hClC‐Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests highly variable phenotypes. The functional severity of 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 haven't been thoroughly examined, and the genotype‐phenotype association hasn't 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 eighteen 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 the protein expression. The functional severity of CLCNKB mutation is an important determinant of the phenotype in classic BS. This article is protected by copyright. All rights reserved
    May 27, 2017   doi: 10.1113/JP274344   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. May 26, 2017
    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. This article is protected by copyright. All rights reserved
    May 26, 2017   doi: 10.1113/JP274212   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. May 25, 2017
    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 (MIC) 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 5,983 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 MAPK's and CAMK's. 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 (SPEG) and obscurin) was dramatically altered by MIC, and we propose how these kinases could play a fundamental role in skeletal muscle mechanotransduction. This article is protected by copyright. All rights reserved
    May 25, 2017   doi: 10.1113/JP273904   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
  • Chronic EMGs in treadmill running SOD1 mice reveal early changes in muscle activation.
    K. A. Quinlan, E. Kajtaz, J. D. Ciolino, R. D. Imhoff‐Manuel, M. C. Tresch, C. J. Heckman, V. M. Tysseling.
    The Journal of Physiology. May 24, 2017
    To improve our understanding of early disease mechanisms and find reliable biomarkers of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, we measured electromyogram (EMG) activity in hind limb muscles of SOD1G93A mice. In contrast to clinical diagnostic measures using EMGs, which are performed on quiescent patients, we monitored activity during treadmill running in order to detect presymptomatic changes in motor patterning. Chronic EMG electrodes were implanted into vastus lateralis (VL), biceps femoris posterior (BFP), lateral gastrocnemius (LG), and tibialis anterior (TA) in mice from postnatal day (P) 55–100, and results 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) while 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 1) locomotor EMG activity might be an early measure of disease onset 2) alterations in locomotor patterning may reflect changes in neuronal drive and compensation at the network level including altered activity of spinal interneurons and 3) the increased power output necessary on an inclined treadmill was important in revealing altered activity in SOD1G93A mice. This article is protected by copyright. All rights reserved
    May 24, 2017   doi: 10.1113/JP274170   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. May 24, 2017
    Strong inward rectifier K+ (Kir2) channels primarily maintain normal resting membrane potential of cardiomyocytes. In sub‐physiological 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. Here we show that Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced 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 resting membrane potential 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 two levels of resting membrane potential, demonstrate a previously unknown mechanism of 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. This article is protected by copyright. All rights reserved
    May 24, 2017   doi: 10.1113/JP274268   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
  • 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. May 19, 2017
    Reducing the work of breathing or eliminating exercise‐induced arterial hypoxemia (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 (8M, 8F) completed three constant load exercise tests over four 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. This article is protected by copyright. All rights reserved
    May 19, 2017   doi: 10.1113/JP274068   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. May 19, 2017
    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, 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, analyzing 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 nucleus accumbens 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. This article is protected by copyright. All rights reserved
    May 19, 2017   doi: 10.1113/JP273767   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. May 18, 2017
    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 estrogen action enhancing torsadogenic effects: Models suggest estradiol interaction with hERG mutations in the pore loop containing G604, or by interaction 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. This article is protected by copyright. All rights reserved
    May 18, 2017   doi: 10.1113/JP273142   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. May 18, 2017
    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 that 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. This article is protected by copyright. All rights reserved
    May 18, 2017   doi: 10.1113/JP274252   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
  • Cortical contributions to sensory gating in the ipsilateral somatosensory cortex during voluntary activity.
    Yuming Lei, Monica A. Perez.
    The Journal of Physiology. May 17, 2017
    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 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 (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, therefore, 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, likely from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans. This article is protected by copyright. All rights reserved
    May 17, 2017   doi: 10.1113/JP274504   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. May 17, 2017
    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 favor 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 (AMPAR, ∼1 nS, ∼1 ms; NMDAR ∼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. This article is protected by copyright. All rights reserved
    May 17, 2017   doi: 10.1113/JP274321   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. May 15, 2017
    Background & aims Low aerobic capacity increases risk for NAFLD 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 runners (HCR)) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16 week ‘western diet’ high in fat (45% kcal), cholesterol (1% w w−1), and sucrose (15% kcal). Results 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, while producing greater cholesterol ester accumulation in LCR compared to HCR. Importantly, WD‐fed low‐fit LCR rats displayed greater inflammatory cell infiltration, serum ALT, expression of hepatic inflammatory markers (F4/80, MCP‐1, TLR4, TLR2, and IL‐1b), and effector caspase (caspase‐3 & ‐7) activation compared to HCR. Further, LCR rats had greater WD‐induced decreases in complete FAO and mitochondrial respiratory capacity. Conclusions 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. This article is protected by copyright. All rights reserved
    May 15, 2017   doi: 10.1113/JP274281   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
  • Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with posttraumatic stress disorder (PTSD).
    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. May 14, 2017
    Posttraumatic Stress Disorder (PTSD) is associated with increased cardiovascular (CV) risk. We tested the hypothesis that PTSD patients have augmented sympathetic nervous system (SNS) and hemodynamic reactivity during mental stress, and impaired arterial baroreflex sensitivity (BRS). 14 otherwise healthy Veterans with combat‐related PTSD were compared to 14 matched Controls without PTSD.  Muscle sympathetic nerve activity (MSNA), continuous blood pressure (BP), and electrocardiography were measured at baseline, and during two types of mental stress:  combat‐related mental stress using virtual reality combat exposure (VRCE); and noncombat related stress using mental arithmetic (MA). Cold pressor test (CPT) was administered for comparison. BRS was tested using pharmacologic manipulation of BP via the Modified Oxford technique at rest and during VRCE. Blood samples were analysed for inflammatory biomarkers. Baseline characteristics, MSNA and hemodynamics were similar between the groups. In PTSD versus Controls, MSNA (+8.2 ± 1.0 vs +1.2 ± 1.3 bursts/min P < 0.001) and heart rate (HR) responses (+3.2 ± 1.1 vs −2.3 ± 1.0 beats/min, P = 0.003) were significantly augmented during VRCE.  Similarly, in PTSD versus Controls, MSNA (+21.0 ± 2.6 vs +6.7 ± 1.5 bursts/min, 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 = NS). In the PTSD group, sympathetic BRS (‐1.2 ± 0.2 vs ‐2.0 ± 0.3 BI 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 hs‐CRP levels compared to Controls (2.1 ± 0.4 vs 1.0 ± 0.3 mg L−1, P = 0.047). Augmented SNS and hemodynamic responses to mental stress, blunted BRS, and inflammation may contribute to increased CV risk in PTSD. This article is protected by copyright. All rights reserved
    May 14, 2017   doi: 10.1113/JP274269   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
  • 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. May 10, 2017
    The nucleus tractus solitarius (NTS) neurons have been thought to function as central respiratory chemoreceptors. However, it remains unknown regarding the common molecular marker defined for these neurons. Here we ask 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, 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 favor 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. This article is protected by copyright. All rights reserved
    May 10, 2017   doi: 10.1113/JP274437   open full text
  • Light adaptation and the evolution of vertebrate photoreceptors.
    Ala Morshedian, Gordon L. Fain.
    The Journal of Physiology. May 09, 2017
    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. This article is protected by copyright. All rights reserved
    May 09, 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. May 09, 2017
    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 3 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. This article is protected by copyright. All rights reserved
    May 09, 2017   doi: 10.1113/JP274458   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
  • Blood‐flow restricted training leads to myocelullar macrophage infiltration and upregulation of heat‐shock proteins, but no apparent muscle damage.
    Jakob L. Nielsen, Per Aagaard, Tatyana A. Prokhorova, Tobias Nygaard, Rune Dueholm Bech, Charlotte Suetta, Ulrik Frandsen.
    The Journal of Physiology. May 08, 2017
    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 (3 wk/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 (CK), oxidative stress (TAC,GSH) and inflammation (MCP1,IL‐6,TNFa). 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. This article is protected by copyright. All rights reserved
    May 08, 2017   doi: 10.1113/JP273907   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. May 08, 2017
    In the present study we investigated the role of hydrocortisone (HC) on 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 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. This article is protected by copyright. All rights reserved
    May 08, 2017   doi: 10.1113/JP274200   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
  • 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. May 05, 2017
    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 SMC 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 AT1Raknockoutanimals 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 SMC 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. This article is protected by copyright. All rights reserved
    May 05, 2017   doi: 10.1113/JP274310   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
  • mTORC2 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. May 02, 2017
    Exercise increases glucose uptake into insulin‐resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. mTORC2, a regulator of insulin‐controlled glucose uptake, has been reported to interact with 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 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. Running increased muscle p‐AMPK similarly in Ric WT and Ric mKO mice and GLUT4 and HKII protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, p‐PKCα, and the mTORC2 activity readout, p‐NDRG1, 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. This article is protected by copyright. All rights reserved
    May 02, 2017   doi: 10.1113/JP274203   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. April 28, 2017
    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 to estimate 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 cross‐bridge cycling activity. To eliminate this concern, the putative thermal contribution from cross‐bridge cycling activity must be abolished, at least at minimal muscle length. We achieve 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 heat‐stress relation in its absence. We thus reach two conclusions. First, activation heat is independent of muscle length. Second, residual cross‐bridge 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. This article is protected by copyright. All rights reserved
    April 28, 2017   doi: 10.1113/JP274174   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
  • Long term plasticity of corticostriatal synapses is modulated by pathway‐specific co‐release of opioids through kappa‐opioid receptors.
    Sarah L. Hawes, Armando G. Salinas, David M. Lovinger, Kim T. Blackwell.
    The Journal of Physiology. April 27, 2017
    Synaptic plasticity in 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 kappa 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. This article is protected by copyright. All rights reserved
    April 27, 2017   doi: 10.1113/JP274190   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. April 27, 2017
    Contradictory results have been reported on the impact of ageing on structure and functions of skeletal muscle fibres likely due to 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 aging 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 torque (MVC), and patellar tendon force (Ft) were significantly lower; (ii) muscle fibres went through significant atrophy and impairment of specific force (Po/CSA) and unloaded shortening velocity (Vo); (iii) myosin/actin ratio and myosin content in individual muscle fibres were not altered; (iv) muscle proteome went through quantitative adaptations, namely an up‐regulation of the content of several groups of proteins among which myofibrillar proteins and antioxidant defence systems; (v) 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 fibres structure and function is a major feature of ageing per se and that qualitative adaptations of muscle proteome are likely more involved than quantitative adaptations in determining such phenomenon. This article is protected by copyright. All rights reserved
    April 27, 2017   doi: 10.1113/JP274148   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
  • Genotype‐specific pathogenic effects in human dilated cardiomyopathy.
    Ilse AE Bollen, Maike Schuldt, Magdalena Harakalova, Aryan Vink, Folkert W Asselbergs, Jose R Pinto, Martina Krüger, Diederik WD Kuster, Jolanda der Velden.
    The Journal of Physiology. April 24, 2017
    Background 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 K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). Methods 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. Results 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 3 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 wildtype 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. This article is protected by copyright. All rights reserved
    April 24, 2017   doi: 10.1113/JP274145   open full text
  • Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.
    Sergej Pirkmajer, Henriette Kirchner, Leonidas Lundell, Pavel V. Zelenin, Juleen R. Zierath, Kira S. Makarova, Yuri I. Wolf, Alexander V. Chibalin.
    The Journal of Physiology. April 24, 2017
    Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these proteins 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 (Chondricthyes) 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. This article is protected by copyright. All rights reserved
    April 24, 2017   doi: 10.1113/JP274254   open full text
  • Convergent ERK1/2, p38, and JNK MAPK 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. April 24, 2017
    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 independent 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. This article is protected by copyright. All rights reserved
    April 24, 2017   doi: 10.1113/JP274119   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
  • 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. April 19, 2017
    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. This article is protected by copyright. All rights reserved
    April 19, 2017   doi: 10.1113/JP273872   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. April 19, 2017
    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 if the released ATP is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In this 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 (oATP), an antagonist used to block P2 × 7 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 that airway epithelium ATP release is the molecular autocrine mechanism that regulates basal ciliary activity and the mediator of the ciliary response to chemical stimulation. This article is protected by copyright. All rights reserved
    April 19, 2017   doi: 10.1113/JP273996   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. April 18, 2017
    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 4,300 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 likely provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes. This article is protected by copyright. All rights reserved
    April 18, 2017   doi: 10.1113/JP274130   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.