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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:

  • Why don't mice lacking the mitochondrial Ca2+ uniporter experience an energy crisis?
    Pei Wang, Celia Fernandez‐Sanz, Wang Wang, Shey‐Shing Sheu.
    The Journal of Physiology. 6 days ago
    --- - |2 Abstract Current dogma holds that the heart balances energy demand and supply effectively and sustainably by sequestering enough Ca2+ into mitochondria during heartbeats to stimulate metabolic enzymes in the tricarboxylic acid (TCA) cycle and electron transport chain (ETC). This process is called excitation‐contraction‐bioenergetics (ECB) coupling. Recent breakthroughs in identifying the mitochondrial Ca2+ uniporter (MCU) and its associated proteins have opened up new windows for interrogating the molecular mechanisms of mitochondrial Ca2+ homeostasis regulation and its role in ECB coupling. Despite remarkable progress made in the past 7 years, it has been surprising, almost disappointing, that germline MCU deficiency in mice with certain genetic background yields viable pups, and knockout of the MCU in adult heart does not cause lethality. Moreover, MCU deficiency results in few adverse phenotypes, normal performance, and preserved bioenergetics in the heart at baseline. In this review, we briefly assess the existing literature on mitochondrial Ca2+ homeostasis regulation and then we consider possible explanations for why MCU‐deficient mice are spared from energy crises under physiological conditions. We propose that MCU and/or mitochondrial Ca2+ may have limited ability to set ECB coupling, that other mitochondrial Ca2+ handling mechanisms may play a role, and that extra‐mitochondrial Ca2+ may regulate ECB coupling. Since the heart needs to regenerate a significant amount of ATP to assure the perpetuation of heartbeats, multiple mechanisms are likely to work in concert to match energy supply with demand. - The Journal of Physiology, EarlyView.
    October 12, 2018   doi: 10.1113/JP276636   open full text
  • Neurocardiac regulation: From cardiac mechanisms to novel therapeutic approaches.
    E. N. Bardsley, D. J. Paterson.
    The Journal of Physiology. 7 days ago
    --- - |2+ Cardiac sympathetic over‐activity is a well‐established contributor to the progression of neurogenic hypertension and heart failure, yet the underlying pathophysiology remains unclear. Recent studies have highlighted the importance of acutely regulated cyclic nucleotides and their effectors in the control of intracellular calcium and exocytosis. Emerging evidence now suggests that a significant component of sympathetic over‐activity and enhanced transmission may arise from impaired cyclic nucleotide signalling, resulting from compromised phosphodiesterase activity, as well as alterations in receptor‐coupled G‐protein activation. In this review, we address some of the key cellular and molecular pathways that contribute to sympathetic over‐activity in hypertension and discuss their potential for therapeutic targeting. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 11, 2018   doi: 10.1113/JP276962   open full text
  • Skeletal muscle ceramides and relationship with insulin sensitivity after 2 weeks of simulated sedentary behaviour and recovery in healthy older adults.
    Paul T. Reidy, Alec I. McKenzie, Ziad Mahmassani, Vincent R. Morrow, Nikol M. Yonemura, Paul N. Hopkins, Robin L. Marcus, Matthew T. Rondina, Yu Kuei Lin, Micah J. Drummond.
    The Journal of Physiology. 9 days ago
    --- - |2+ Key points Insulin sensitivity (as determined by a hyperinsulinaemic‐euglyceamic clamp) decreased 15% after reduced activity. Despite not fully returning to baseline physical activity levels, insulin sensitivity unexpectedly, rebounded above that recorded before 2 weeks of reduced physical activity by 14% after the recovery period. Changes in insulin sensitivity in response to reduced activity were primarily driven by men but, not women. There were modest changes in ceramides (nuclear/myofibrillar fraction and serum) following reduced activity and recovery but, in the absence of major changes to body composition (i.e. fat mass), ceramides were not related to changes in inactivity‐induced insulin sensitivity in healthy older adults. Abstract Older adults are at risk of physical inactivity as they encounter debilitating life events. It is not known how insulin sensitivity is affected by modest short‐term physical inactivity and recovery in healthy older adults, nor how insulin sensitivity is related to changes in serum and muscle ceramide content. Healthy older adults (aged 64–82 years, five females, seven males) were assessed before (PRE), after 2 weeks of reduced physical activity (RA) and following 2 weeks of recovery (REC). Insulin sensitivity (hyperinsulinaemic‐euglyceamic clamp), lean mass, muscle function, skeletal muscle subfraction, fibre‐specific, and serum ceramide content and indices of skeletal muscle inflammation were assessed. Insulin sensitivity decreased by 15 ± 6% at RA (driven by men) but rebounded above PRE by 14 ± 5% at REC. Mid‐plantar flexor muscle area and leg strength decreased with RA, although only muscle size returned to baseline levels following REC. Body fat did not change and only minimal changes in muscle inflammation were noted across the intervention. Serum and intramuscular ceramides (nuclear/myofibrillar fraction) were modestly increased at RA and REC. However, ceramides were not related to changes in inactivity‐induced insulin sensitivity in healthy older adults. Short‐term inactivity induced insulin resistance in older adults in the absence of significant changes in body composition (i.e. fat mass) are not related to changes in ceramides. - The Journal of Physiology, EarlyView.
    October 09, 2018   doi: 10.1113/JP276798   open full text
  • Increased human stretch reflex dynamic sensitivity with height‐induced postural threat.
    Brian C. Horslen, Martin Zaback, J. Timothy Inglis, Jean‐Sébastien Blouin, Mark G. Carpenter.
    The Journal of Physiology. 9 days ago
    --- - |2+ Key points Threats to standing balance (postural threat) are known to facilitate soleus tendon‐tap reflexes, yet the mechanisms driving reflex changes are unknown. Scaling of ramp‐and‐hold dorsiflexion stretch reflexes to stretch velocity and amplitude were examined as indirect measures of changes to muscle spindle dynamic and static function with height‐induced postural threat. Overall, stretch reflexes were larger with threat. Furthermore, the slope (gain) of the stretch‐velocity vs. short‐latency reflex amplitude relationship was increased with threat. These findings are interpreted as indirect evidence for increased muscle spindle dynamic sensitivity, independent of changes in background muscle activity levels, with a threat to standing balance. We argue that context‐dependent scaling of stretch reflexes forms part of a multisensory tuning process where acquisition and/or processing of balance‐relevant sensory information is continuously primed to facilitate feedback control of standing balance in challenging balance scenarios. Abstract Postural threat increases soleus tendon‐tap (t‐) reflexes. However, it is not known whether t‐reflex changes are a result of central modulation, altered muscle spindle dynamic sensitivity or combined spindle static and dynamic sensitization. Ramp‐and‐hold dorsiflexion stretches of varying velocities and amplitudes were used to examine velocity‐ and amplitude‐dependent scaling of short‐ (SLR) and medium‐latency (MLR) stretch reflexes as an indirect indicator of spindle sensitivity. t‐reflexes were also performed to replicate previous work. In the present study, we examined the effects of postural threat on SLR, MLR and t‐reflex amplitude, as well as SLR‐stretch velocity scaling. Forty young‐healthy adults stood with one foot on a servo‐controlled tilting platform and the other on a stable surface. The platform was positioned on a hydraulic lift. Threat was manipulated by having participants stand in low (height 1.1 m; away from edge) then high (height 3.5 m; at the edge) threat conditions. Soleus stretch reflexes were recorded with surface electromyography and SLRs and MLRs were probed with fixed‐amplitude variable‐velocity stretches. t‐reflexes were evoked with Achilles tendon taps using a linear motor. SLR, MLR and t‐reflexes were 11%, 9.5% and 16.9% larger, respectively, in the high compared to low threat condition. In 22 out of 40 participants, SLR amplitude was correlated to stretch velocity at both threat levels. In these participants, the gain of the SLR–velocity relationship was increased by 36.1% with high postural threat. These findings provide new supportive evidence for increased muscle spindle dynamic sensitivity with postural threat and provide further support for the context‐dependent modulation of human somatosensory pathways. - The Journal of Physiology, EarlyView.
    October 09, 2018   doi: 10.1113/JP276459   open full text
  • The dynamics of cortical GABA in human motor learning.
    James Kolasinski, Emily L. Hinson, Amir P. Divanbeighi Zand, Assen Rizov, Uzay E. Emir, Charlotte J. Stagg.
    The Journal of Physiology. 9 days ago
    --- - |2+ Key points The ability to learn new motor skills is supported by plasticity in the structural and functional organisation of the primary motor cortex in the human brain. Changes inhibitory signalling by gamma‐aminobutyric acid (GABA) are thought to be crucial in inducing motor cortex plasticity. This study used magnetic resonance spectroscopy (MRS) to quantify the concentration of GABA in human motor cortex during a period of motor learning, as well as during a period of movement, and a period at rest. We report evidence for a reduction in the MRS‐measured concentration of GABA specific to learning. Further, the GABA concentration early in the learning task was strongly correlated with the magnitude of subsequent learning: higher GABA concentrations were associated with poorer learning. The results provide an initial insight into the neurochemical correlates of cortical plasticity associated with motor learning, specifically relevant in therapeutic efforts to induce cortical plasticity during stroke recovery. The ability to learn novel motor skills is a central part of our daily lives and can provide a model for rehabilitation after a stroke. However, there are still fundamental gaps in our understanding of the physiological mechanisms that underpin human motor plasticity. The acquisition of new motor skills is dependent on changes in local circuitry within the primary motor cortex (M1). This reorganisation has been hypothesised to be facilitated by a decrease in local inhibition via modulation of the neurotransmitter GABA, but this link has not been conclusively demonstrated in humans. Here, we used 7T MR Spectroscopy to investigate the dynamics of GABA concentrations in human M1 during the learning of an explicit, serial reaction time task. We observed a significant reduction in GABA concentration during motor learning that was not seen in an equivalent motor task lacking a learnable sequence, nor during a passive resting task of the same duration. No change in glutamate was observed in any group. Furthermore, M1 GABA measured early in task performance was strongly correlated with the degree of subsequent learning, such that greater inhibition was associated with poorer subsequent learning. This result suggests that higher levels of cortical inhibition may present a barrier that must be surmounted in order achieve an increase in M1 excitability, and hence encoding of a new motor skill. These results provide strong support for the mechanistic role of GABAergic inhibition in motor plasticity, raising questions regarding the link between population variability in motor learning and GABA metabolism in the brain. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 09, 2018   doi: 10.1113/JP276626   open full text
  • Shining light on the paraventricular nucleus: The role of glutamatergic PVN neurons in blood pressure control.
    Bryan K. Becker.
    The Journal of Physiology. 9 days ago
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 09, 2018   doi: 10.1113/JP277043   open full text
  • Delivering baking soda to the brain.
    Mark O. Bevensee.
    The Journal of Physiology. 9 days ago
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 09, 2018   doi: 10.1113/JP277119   open full text
  • Fat or Thin, exercise wins: Endurance exercise training reduces inflammatory circulating progenitor cells in lean and obese adults.
    Paul M. Ryan, Ryan T. Sless, Nathaniel E. Hayward.
    The Journal of Physiology. 9 days ago
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 09, 2018   doi: 10.1113/JP277229   open full text
  • Complex and spatially segregated auditory inputs of the mouse superior colliculus.
    Veronika Bednárová, Benedikt Grothe, Michael H. Myoga.
    The Journal of Physiology. 10 days ago
    --- - |2+ Key points Although the visual circuits in the superior colliculus (SC) have been thoroughly examined, the auditory circuits lack equivalent scrutiny. SC neurons receiving auditory inputs in mice were characterized and three distinguishable types of neurons were found. The auditory pathways from external nuclei of the inferior colliculus (IC) were characterized, and a novel direct inhibitory connection and an excitation that drives feed‐forward inhibitory circuits within the SC were found. The direct excitatory and inhibitory inputs exhibited distinct arbourization patterns in the SC. These findings suggest functional differences between excitatory and inhibitory sensory information that targets the auditory SC. Abstract The superior colliculus (SC) is a midbrain structure that integrates auditory, somatosensory and visual inputs to drive orientation movements. While much is known about how visual information is processed in the superficial layers of the SC, little is known about the SC circuits in the deep layers that process auditory inputs. We therefore characterized intrinsic neuronal properties in the auditory‐recipient layer of the SC (stratum griseum profundum; SGP) and confirmed three electrophysiologically defined clusters of neurons, consistent with literature from other SC layers. To determine the types of inputs to the SGP, we expressed Channelrhodopsin‐2 in the nucleus of the brachium of the inferior colliculus (nBIC) and external cortex of the inferior colliculus (ECIC) and optically stimulated these pathways while recording from SGP neurons. Probing the connections in this manner, we described a monosynaptic excitation that additionally drives feed‐forward inhibition via circuits intrinsic to the SC. Moreover, we found a profound long‐range monosynaptic inhibition in 100% of recorded SGP neurons, a surprising finding considering that only about 15% of SGP‐projecting neurons in the nBIC/ECIC are inhibitory. Furthermore, we found spatial differences in the cell body locations as well as axon trajectories between the monosynaptic excitatory and inhibitory inputs, suggesting that these inputs may be functionally distinct. Taking this together with recent anatomical evidence suggesting an auditory excitation from the nBIC and a GABAergic multimodal inhibition from the ECIC, we propose that sensory integration in the SGP is more multifaceted than previously thought. - The Journal of Physiology, EarlyView.
    October 08, 2018   doi: 10.1113/JP276370   open full text
  • And the beat goes on.
    Jack R. T. Darby, Janna L. Morrison.
    The Journal of Physiology. 10 days ago
    --- - - The Journal of Physiology, EarlyView.
    October 08, 2018   doi: 10.1113/JP277026   open full text
  • Metaboreceptor polymorphisms: do genes determine your blood pressure response to exercise?
    Jasdeep Kaur, Thales C. Barbosa, Paul J. Fadel.
    The Journal of Physiology. 10 days ago
    --- - - The Journal of Physiology, EarlyView.
    October 08, 2018   doi: 10.1113/JP276971   open full text
  • Timing is everything: maternal circadian rhythms and the developmental origins of health and disease.
    Tamara J. Varcoe.
    The Journal of Physiology. 10 days ago
    --- - - The Journal of Physiology, EarlyView.
    October 08, 2018   doi: 10.1113/JP276992   open full text
  • The postnatal development of ultrasonic vocalization‐associated breathing is altered in glycine transporter 2‐deficient mice.
    Swen Hülsmann, Yoshihiko Oke, Guillaume Mesuret, A. Tobias Latal, Michal G. Fortuna, Marcus Niebert, Johannes Hirrlinger, Julia Fischer, Kurt Hammerschmidt.
    The Journal of Physiology. 10 days ago
    --- - |2+ Key Points Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is responsible for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and die in the second postnatal week. Here we show that GlyT2‐knock out mice do not acquire adult ultrasonic vocalization‐associated breathing patterns. Despite the strong impairment of glycinergic inhibition they can produce sufficient expiratory airflow to produce ultrasonic vocalization. As mouse ultrasonic vocalization is a valuable read‐out in translational research, these data are highly relevant for a broad range of research fields. Abstract Mouse models are instrumental in elucidating the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post‐inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2‐KO mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2‐KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. In contrast, WT mice acquire different USV‐associated breathing patterns that depend on post‐inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2‐KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 08, 2018   doi: 10.1113/JP276976   open full text
  • Slow periodic activity in the longitudinal hippocampal slice can self‐propagate non‐synaptically by a mechanism consistent with ephaptic coupling.
    Chia‐Chu Chiang, Rajat S. Shivacharan, Xile Wei, Luis E. Gonzalez‐Reyes, Dominique M. Durand.
    The Journal of Physiology. 10 days ago
    --- - |2+ Key points Slow periodic activity can propagate with speeds around 0.1 m/s and be modulated by weak electric fields. Slow periodic activity in the longitudinal hippocampal slice can propagate without chemical synaptic transmission or gap junctions, but can generate electric fields which in turn activate neighboring cells. Applying local extracellular electric fields with amplitude in the range of endogenous fields is sufficient to modulate or block the propagation of this activity both in the in‐silico and in‐vitro models. Results support the hypothesis that endogenous electric fields, previously thought to be too small to trigger neural activity, play a significant role in the self‐propagation of slow periodic activity in the hippocampus. Experiments indicate that a neural network can give rise to sustained self‐propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions. Abstract Slow oscillations are a standard feature observed in the cortex and the hippocampus during slow wave sleep. Slow oscillations are characterized by low‐frequency periodic activity (<1 Hz) and are thought to be related to memory consolidation. These waves are assumed to be a reflection of the underlying neural activity, but it is not known if they can, by themselves, be self‐sustained and propagate. Previous studies have shown that slow periodic activity can be reproduced in the in‐vitro preparation to mimic in‐vivo slow oscillations. Slow periodic activity can propagate with speeds around 0.1 m/s and be modulated by weak electric fields. In the present study, we show that slow periodic activity in the longitudinal hippocampal slice is a self‐regenerating wave which can propagate with and without chemical or electrical synaptic transmission at the same speeds. We also show that applying local extracellular electric fields can modulate or even block the propagation of this wave both in in‐silico and in‐vitro models. Our results support the notion that ephaptic coupling plays a significant role in the propagation of the slow hippocampal periodic activity. Moreover, these results indicate that a neural network can give rise to sustained self‐propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 08, 2018   doi: 10.1113/JP276904   open full text
  • Guardian of mitochondrial function: an expanded role of Parkin in skeletal muscle.
    J. Botella, N. Saner, C. Granata.
    The Journal of Physiology. 10 days ago
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 08, 2018   doi: 10.1113/JP276841   open full text
  • An ex vivo bladder model with detrusor smooth muscle removed to analyze biologically active mediators released from the suburothelium.
    Leonie Durnin, Benjamin Kwok, Priya Kukadia, Roisin McAvera, Robert D. Corrigan, Sean M. Ward, Ying Zhang, Qi Chen, Sang Don Koh, Kenton M. Sanders, Violeta N. Mutafova‐Yambolieva.
    The Journal of Physiology. 13 days ago
    --- - |2+ A Key Point List Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure‐volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation, and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop the decentralized (ex vivo) detrusor smooth muscle (DSM)‐denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium‐derived mediators at the luminal and anti‐luminal aspects of the urothelium during filling. Urinary bladders were excised from C57Bl6/J mice and the DSM was removed by fine scissors dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure‐volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate‐ and lipopolysaccharide‐treated denuded bladders were characterized. The preparation wall contains intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacks the DSM and the serosa. Utility of the model for physiology research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g., ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM‐denuded bladder of Cynomolgus monkeys (Macaca fascicularis). The novel model is superior to current models utilized to study properties of the urothelium (e.g., cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enables direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium‐DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 05, 2018   doi: 10.1113/JP276924   open full text
  • Lower body negative pressure to safely reduce intracranial pressure.
    Lonnie G Petersen, Justin S Lawley, Alexander Lilja‐Cyron, Johan CG Petersen, Erin J Howden, Satyam Sarma, William K Cornwell, Rong Zhang, Louis A Whitworth, Michael A. Williams, Marianne Juhler, Benjamin D Levine.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key Points During long‐term missions, some astronauts experience structural and functional changes of the eyes and brain which resemble signs/symptoms experienced by patients with intracranial hypertension. Weightlessness prevents the normal cerebral volume and pressure “unloading” associated with upright postures on Earth, which may be part of the cerebral and ocular pathophysiology. By placing the lower body in a negative pressure device (LBNP) that pulls fluid away from cranial compartments, we simulated effects of gravity and significantly lowered pressure within the brain parenchyma and ventricle compartments. Application of incremental LBNP demonstrated a non‐linear dose‐response curve suggesting 20 mmHg LBNP as the optimal level for reducing pressure in brain without impairing cerebral perfusion pressure. This non‐invasive method of reducing pressure in the brain holds potential as a countermeasure in space as well as treatment potential for patients on Earth with traumatic brain injury or other pathology leading to intracranial hypertension. Abstract Patients with elevated intracranial pressure (ICP) exhibit neuro‐ocular symptoms including headache, papilledema, and loss of vision. Some of these symptoms are also present in astronauts during and after prolonged space‐flight where lack of gravitational stress prevents daily lowering of ICP associated with upright posture. Lower body negative pressure (LBNP) simulates the effects of gravity by displacing fluid caudally and we hypothesized that LBNP would lower ICP without compromising cerebral perfusion. Ten cerebrally intact volunteers were included: 6 ambulatory neurosurgical patients with parenchymal ICP‐sensors and 4 former cancer patients with Ommaya‐reservoirs to the frontal horn of a lateral ventricle. We applied LBNP while recording ICP and blood pressure while supine, and during simulated intracranial hypertension by 15° head‐down tilt. LBNP from 0–50 mm Hg at increments of 10 mmHg lowered ICP in a non‐linear dose‐dependent fashion; when supine (N = 10), ICP was decreased from 15 ± 2 mmHg to 14 ± 4, 12 ± 5, 11 ± 4, 10 ± 3, 9 ± 4, respectively (P < 0.0001). Cerebral perfusion pressure (CPP), calculated as mean arterial blood pressure at midbrain‐level minus ICP, was unchanged (from 70 ± 12 mmHg to 67 ± 9, 69 ± 10, 70 ± 12, 72 ± 13, 74 ± 15; P = 0.02). 15° head‐down tilt (N = 6) increased ICP to 26 ± 4 mmHg, while application of LBNP lowered ICP (to 21 ± 4, 20 ± 4, 18 ± 4, 17 ± 4, 17 ± 4; P < 0.0001) and increased CPP (P < 0.01). Twenty mmHg LBNP may be the optimal level to lower ICP without impairing CPP to counteract spaceflight associated neuro‐ocular syndrome in astronauts. Furthermore, LBNP holds clinical potential as a safe, non‐invasive method for lowering ICP and improving CPP for patients with pathologically elevated ICP on Earth. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 04, 2018   doi: 10.1113/JP276557   open full text
  • Presynaptic loss of dynamin related protein 1 impairs synaptic vesicle release and recycling at the mouse calyx of held.
    Mahendra Singh, Henry Denny, Christina Smith, Jorge Granados, Robert Renden.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key Points This study characterizes the mechanisms underlying defects in synaptic transmission when DRP1 is genetically eliminated. Viral‐mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of synaptic vesicle recycling pool, and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing intracellular calcium buffering capacity with EGTA‐AM, implying close coupling of Ca2+‐channels to SVs was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not revert synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Abstract Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin‐related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of CRE in ventral cochlear neurons of floxed‐DRP1 mice generated a presynaptic‐specific DRP1‐KO (DRP1‐preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggests SV clusters and mitochondrial content are disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage‐clamp recordings, we find that DRP1‐preKO synapses had larger evoked responses at low frequency stimulation. DRP1‐preKO synapses also had profoundly altered short‐term plasticity, due to defects in SV recycling. Readily‐releasable pool (RRP) size, estimated with high‐frequency trains, was dramatically reduced in DRP1‐preKO, suggesting an important role for DRP1 in maintenance of release competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1‐preKO. Synaptic transmission defects could be partially rescued with EGTA‐AM, indicating close coupling of Ca channel to SV distance normally found in mature terminals may be compromised by DRP1‐preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 04, 2018   doi: 10.1113/JP276424   open full text
  • Leptin acts in the carotid bodies to increase minute ventilation during wakefulness and sleep and augment the hypoxic ventilatory response.
    Candela Caballero‐Eraso, Mi‐Kyung Shin, Huy Pho, Lenise J Kim, Luis E Pichard, ZhiJuan Wu, Chenjuan Gu, Slava Berger, Luu Pham, Ho‐Yee (Bonnie) Yeung, Machiko Shirahata, Alan R Schwartz, Wan‐Yee (Winnie) Tang, James S. K. Sham, Vsevolod Y. Polotsky.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key points Leptin is a potent respiratory stimulant A long functional isoform of leptin receptor, LepRb, was detected in the carotid body (CB), a key peripheral hypoxia sensor. However, the effect of leptin on minute ventilation (VE) and the hypoxic ventilatory response (HVR) was not sufficiently studied. We report that LepRb is present in approximately 74% of the CB glomus cells Leptin increased carotid sinus nerve activity at baseline and response to hypoxia in vivo Subcutaneous infusion of leptin increased VE and the HVR in C57BL/6J mice and this effect was abolished by CB denervation Expression of LepRb in the carotid bodies of LepRb deficient obese db/db mice increased VE during wakefulness and sleep and augmented the HVR. We conclude that leptin acts on LepRb in the carotid bodies to stimulate breathing and the HVR, which may protect against sleep disordered breathing in obesity. Abstract Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb, but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and the HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 ml min‐1 g‐1 during normoxia (p < 0.01) and from 3.6 to 4.7 ml min‐1 g‐1 during hypoxia (p < 0.001), augmenting the HVR from 0.23 ml min‐1 g‐1/ΔFiO2 to 0.31 ml min‐1 g‐1/ΔFiO2 (p < 0.001). The effects of leptin on VE and the HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 ml min‐1 g‐1 during normoxia (p < 0.05,) and from 2.8 to 3.2 ml min‐1 g‐1 during hypoxia (p < 0.02), increasing the HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout NREM (20.1 vs.–27.7 ml/min respectively, p < 0.05) and REM sleep (16.5 vs 23.4 ml/min, p < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 04, 2018   doi: 10.1113/JP276900   open full text
  • Sex differences in the regulation of hepatic mitochondrial turnover following physical activity: do males need more quality control than females?
    Catherine Bellissimo, Christopher G.R. Perry.
    The Journal of Physiology. October 04, 2018
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    October 04, 2018   doi: 10.1113/JP276896   open full text
  • Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: A preclinical animal study.
    Kerstin Hoppe, Sunisa Chaiklieng, Frank Lehmann–Horn, Karin Jurkat–Rott, Scott Wearing, Werner Klingler.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key points During myotonia congenita reduced chloride (Cl−) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called “warm up”. Pharmacological inhibition of low Cl− channels by Anthracene‐9‐Carboxylic Acid from ADR muscle from mice showed a relaxation deficit at physiological conditions compared to wild‐type muscle. At increased osmolarity up to 400 mOsm the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti‐myotonic effects of hyper‐tonicity were, at least in part, antagonized by application of bumetanide. Abstract Introduction: Low chloride‐conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl−) channel gene type 1 (CLCN1). Reduced Cl− conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called “warm up”. To gain further insight into the patho‐mechanism of impaired muscle stiffness and the warm‐up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Methods: Functional force and membrane potential measurements were performed on muscle specimens of ADR mice (an animal model for low gCl‐ conductance myotonia) and pharmacologically‐induced myotonia. Specimens were exposed to solutions of increasing osmolarity, while force and membrane potentials were monitored. In the second set of experiments ADR muscle and pharmacologically‐induced myotonic muscle were exposed to an antagonist of NKCC1. Measurements and Main Results: Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control WT muscle. High osmolarity diminished myotonia and facilitated the warm‐up phenomenon as depicted by a faster muscle relaxation time (T90/10). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the antimyotonic effect of high osmolarity. Conclusions: Increased osmolarity decreased signs of myotonia and facilitated the warm‐up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm‐up and reduces the number of contractions required to achieve normal relaxation kinetics. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 04, 2018   doi: 10.1113/JP276528   open full text
  • Effects of lorazepam and baclofen on short‐ and long‐latency afferent inhibition.
    Claudia V. Turco, Jenin El‐Sayes, Mitchell B. Locke, Robert Chen, Steven Baker, Aimee J. Nelson.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key points Short‐latency afferent inhibition (SAI) is modulated by GABAA receptor activity, whereas the pharmacological origin of long‐latency afferent inhibition remains unknown. This is the first study to report that long‐latency afferent inhibition (LAI) is reduced by the GABAA positive allosteric modulator lorazepam, and that both SAI and LAI are not modulated by the GABAB agonist baclofen. These findings advance our understanding of the neural mechanisms underlying afferent inhibition. Abstract The afferent volley evoked by peripheral nerve stimulation has an inhibitory influence on transcranial magnetic stimulation induced motor evoked potentials. This phenomenon, known as afferent inhibition, occurs in two phases: short‐latency afferent inhibition (SAI) and long‐latency afferent inhibition (LAI). SAI exerts its inhibitory influence via cholinergic and GABAergic activity. The neurotransmitter receptors that mediate LAI remain unclear. The present study aimed to determine whether LAI is contributed by GABAA and/or GABAB receptor activity. In a double‐blinded, placebo‐controlled study, 2.5 mg of lorazepam (GABAA agonist), 20 mg of baclofen (GABAB agonist) and placebo were administered to 14 males (mean age 22.7 ± 1.9 years) in three separate sessions. SAI and LAI, evoked by stimulation of the median nerve and recorded from the first dorsal interosseous muscle, were quantified before and at the peak plasma concentration following drug ingestion. Results indicate that lorazepam reduced LAI by ∼40% and, in support of previous work, reduced SAI by ∼19%. However, neither SAI, nor LAI were altered by baclofen. In a follow‐up double‐blinded, placebo‐controlled study, 10 returning participants received placebo or 40 mg of baclofen (double the dosage used in Experiment 1). The results obtained indicate that SAI and LAI were unchanged by baclofen. This is the first study to show that LAI is modulated by GABAA receptor activity, similar to SAI, and that afferent inhibition does not appear to be a GABAB mediated process. - The Journal of Physiology, EarlyView.
    October 04, 2018   doi: 10.1113/JP276710   open full text
  • Acute and chronic exercise in patients with heart failure with reduced ejection fraction: evidence of structural and functional plasticity and intact angiogenic signalling in skeletal muscle.
    Fabio Esposito, Odile Mathieu‐Costello, Peter D. Wagner, Russell S. Richardson.
    The Journal of Physiology. October 04, 2018
    --- - |2+ Key points The vascular endothelial growth factor (VEGF) responses to acute submaximal exercise and training effects in patients with heart failure with reduced ejection fraction (HFrEF) were investigated. Six patients and six healthy matched controls performed knee‐extensor exercise (KE) at 50% of maximum work rate before and after (only patients) KE training. Muscle biopsies were taken to assess skeletal muscle structure and the angiogenic response. Before training, during this submaximal KE exercise, patients with HFrEF exhibited higher leg vascular resistance and greater noradrenaline spillover. Skeletal muscle structure and VEGF response were generally not different between groups. Following training, resistance was no longer elevated and noradrenaline spillover was curtailed in the patients. Although, in the trained state, VEGF did not respond to acute exercise, capillarity was augmented. Muscle fibre cross‐sectional area and percentage area of type I fibres increased and mitochondrial volume density exceeded that of controls. Structural/functional plasticity and appropriate angiogenic signalling were observed in skeletal muscle of patients with HFrEF. Abstract This study examined the response to acute submaximal exercise and the effect of training in patients with heart failure with reduced ejection fraction (HFrEF). The acute angiogenic response to submaximal exercise in HFrEF after small muscle mass training is debated. The direct Fick method, with vascular pressures, was performed across the leg during knee‐extensor exercise (KE) at 50% of maximum work rate (WRmax) in patients (n = 6) and controls (n = 6) and then after KE training in patients. Muscle biopsies facilitated the assessment of skeletal muscle structure and vascular endothelial growth factor (VEGF) mRNA levels. Prior to training, HFrEF exhibited significantly higher leg vascular resistance (LVR) (≈15%) and significantly greater noradrenaline spillover (≈385%). Apart from mitochondrial volume density, which was significantly lower (≈22%) in HFrEF, initial skeletal muscle structure, including capillarity, was not different between groups. Resting VEGF mRNA levels, and the increase with exercise, was not different between patients and controls. Following training, LVR was no longer elevated and noradrenaline spillover was curtailed. Skeletal muscle capillarity increased with training, as assessed by capillary‐to‐fibre ratio (≈13%) and number of capillaries around a fibre (NCAF) (≈19%). VEGF mRNA was now not significantly increased by acute exercise. Muscle fibre cross‐sectional area and percentage area of type I fibres both increased significantly with training (≈18% and ≈21%, respectively), while the percentage area of type II fibres fell significantly (≈11%), and mitochondrial volume density now exceeded that of controls. These data reveal structural and functional plasticity and appropriate angiogenic signalling in skeletal muscle of HFrEF patients. - The Journal of Physiology, EarlyView.
    October 04, 2018   doi: 10.1113/JP276678   open full text
  • What short‐term potentiation is and why it may be relevant to obstructive sleep apnoea.
    Magdy Younes.
    The Journal of Physiology. October 04, 2018
    --- - - The Journal of Physiology, EarlyView.
    October 04, 2018   doi: 10.1113/JP276848   open full text
  • When gain is greater than loss: effects of physical activity on insulin sensitivity after short‐term inactivity in older subjects.
    Jaume Padilla, Nathan C. Winn, Lauren K. Walsh.
    The Journal of Physiology. October 04, 2018
    --- - - The Journal of Physiology, EarlyView.
    October 04, 2018   doi: 10.1113/JP277110   open full text
  • The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain.
    Jun‐Nan Li, Patrick L. Sheets.
    The Journal of Physiology. October 03, 2018
    --- - |2+ Key points The central nucleus of the amygdala (CeA) encompasses the main output pathways of the amygdala, a temporal lobe structure essential in affective and cognitive dimensions of pain. A major population of neurons in the CeA send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. CeA‐PAG neurons are topographically organized based on their targeted subregion within the PAG. PAG‐projecting neurons in the central medial (CeM) and central lateral (CeL) regions of CeA are intrinsically distinct. CeL‐PAG neurons are a homogeneous population of intrinsically distinct neurons while CeM‐PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM‐PAG subtypes are altered in the Complete Freund's Adjuvant (CFA) model of inflammatory pain. Abstract Background A major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA‐PAG pathway has proved to be a component of descending antinociceptive circuitry, the functional organization of CeA‐PAG neurons remains unclear. Study design We identified CeA‐PAG neurons in C57BL/6 mice of both sexes using intracranial injection of fluorescent retrograde tracer into the PAG. In acute brain slice, we investigated the topographical and intrinsic characteristics of retrogradely‐labeled CeA‐PAG neurons using epifluorescence and whole‐cell electrophysiology. We also measured changes to CeA‐PAG neurons in the Complete Freund's Adjuvant (CFA) model of inflammatory pain. Results Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL‐PAG neurons are comprised of a relatively homogeneous population of intrinsically distinct neurons while CeM‐PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM‐PAG subtypes are altered one day following induction of CFA inflammatory pain model. Conclusion Collectively, our results provide insight into pain‐induced changes to a specific population of CeA neurons that likely play a key role in the integration of noxious input with endogenous analgesia and behavioral coping response. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 03, 2018   doi: 10.1113/JP276935   open full text
  • Visual response properties of neurons in the superficial layers of superior colliculus of awake mouse.
    Gioia Franceschi, Samuel G. Solomon.
    The Journal of Physiology. October 03, 2018
    --- - |2+ Key points In rodents including mice, the superior colliculus is the major target of the retina, but its visual response is not well characterised We made extracellular recordings from single nerve cells in the superficial layers of superior colliculus in awake, head‐restrained mice, and measured their responses to visual stimuli We find that these neurons show brisk, highly sensitive and short latency visual responses, a preference for black over white stimuli, and diverse responses to moving patterns At least 5 broad classes can be defined by the variation in functional properties among units We show that eye movements have measurable impact on visual responses in awake animals and show how they may be mitigated in analyses. Abstract The mouse is an increasingly important animal model of visual function in health and disease. In mice, most retinal signals are routed through the superficial layers of the midbrain superior colliculus, and it is well established that much of mouse visual behaviour relies on activity in the superior colliculus. The functional organisation of visual signals in the mouse superior colliculus is, however, not well established in awake animals. We therefore made extracellular recordings from the superficial layers of superior colliculus in awake mice, while the animals viewed visual stimuli including flashed spots and drifting gratings. We find that neurons in the superficial layers of awake mouse superior colliculus generally show short latency, brisk responses. Receptive fields are usually ‘ON‐OFF’ with a preference for black stimuli, and are weakly non‐linear in response to gratings and other forms of luminance modulation. Population responses to drifting gratings are highly contrast sensitive, with robust response to spatial frequencies above 0.3 cycles/degree and temporal frequencies above 15 Hz. The receptive fields are also often speed‐tuned or direction‐selective. Analysis of response across multiple stimulus dimensions reveals at least 5 functionally distinct groups of units. We also find that eye movements affect measurements of receptive field properties in awake animals, and show how these may be mitigated in analyses. Qualitatively similar responses were obtained in urethane‐anaesthetised animals, but receptive fields in awake animals had higher contrast sensitivity, shorter visual latency, and stronger response to high temporal frequencies. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 03, 2018   doi: 10.1113/JP276964   open full text
  • Increased sensitivity of the circadian system to light in delayed sleep‐wake phase disorder.
    Lauren A. Watson, Andrew J. K. Phillips, Ihaia T. Hosken, Elise M. McGlashan, Clare Anderson, Leon C. Lack, Steven W. Lockley, Shantha M. W. Rajaratnam, Sean W. Cain.
    The Journal of Physiology. October 03, 2018
    --- - |2+ Key Points This is the first study to demonstrate an altered circadian phase shifting response in a circadian rhythm sleep disorder. Patients with Delayed Sleep‐Wake Phase Disorder (DSWPD) demonstrate greater sensitivity of the circadian system to the phase delaying effects of light. Increased circadian sensitivity to light is associated with later circadian timing within both control and DSWPD groups. DSWPD patients had a greater sustained pupil response after light exposure. Treatments for DSWPD should consider sensitivity of the circadian system to light as a potential underlying vulnerability, making patients susceptible to relapse. Abstract Patients with Delayed Sleep‐Wake Phase Disorder (DSWPD) exhibit delayed sleep‐wake behavior relative to desired bedtime, often leading to chronic sleep restriction and daytime dysfunction. The majority of DSWPD patients also display delayed circadian timing in the melatonin rhythm. Hypersensitivity of the circadian system to phase delaying light is a plausible physiological basis for DSWPD vulnerability. We compared the phase shifting response to a 6.5‐h light exposure (∼150 lux) between male patients with diagnosed DSWPD (n = 10; aged 22.4 ± 3.3 years) and male healthy controls (n = 11; aged 22.4 ± 2.4 years). Salivary dim light melatonin onset (DLMO) was measured under controlled conditions in dim light (<3 lux) before and after light exposure. Correcting for the circadian time of the light exposure, DSWPD patients exhibited 31.5% greater phase delay shifts than healthy controls. In both groups, a later initial phase of the melatonin rhythm was associated with greater magnitude of phase shifts, indicating that increased circadian sensitivity to light may be a factor that contributes to delayed phase, even in non‐clinical groups. DSWPD patients also had reduced pupil size following the light exposure, and showed a trend towards increased melatonin suppression during light exposure. These findings indicate that, for patients with DSWPD, assessment of light sensitivity may be an important factor that can inform behavioral therapy, including minimization of exposure to phase‐delaying night‐time light. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    October 03, 2018   doi: 10.1113/JP275917   open full text
  • Recovery of respiratory function in mdx mice co‐treated with neutralizing interleukin‐6 receptor antibodies and urocortin‐2.
    David P. Burns, Leonie Canavan, Jane Rowland, Robin O'Flaherty, Molly Brannock, Sarah E. Drummond, Dervla O'Malley, Deirdre Edge, Ken D. O'Halloran.
    The Journal of Physiology. October 03, 2018
    --- - |2+ Key points Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2‐week intervention in young wild‐type and mdx mice, consisting of either injection of saline or co‐administration of a neutralizing interleukin‐6 receptor antibody (xIL‐6R) and urocortin‐2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL‐6R and Ucn2 co‐treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro‐inflammatory cytokine interleukin‐1β was reduced and the concentration of the anti‐inflammatory cytokine interleukin‐10 was increased in mdx diaphragm following drug co‐treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing. Abstract The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six‐week‐old male mdx (n = 32) and wild‐type (WT; n = 32) mice received either saline (0.9% w/v) or a co‐administration of neutralizing interleukin‐6 receptor antibodies (xIL‐6R; 0.2 mg kg−1) and corticotrophin‐releasing factor receptor 2 agonist (urocortin‐2; 30 μg kg−1) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force‐ and power‐generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co‐treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co‐treatment preserved MyHC type IIx complement in mdx muscle. Drug co‐treatment increased the cross‐sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL‐1β, IL‐6, KC/GRO and TNF‐α were significantly increased in mdx diaphragm compared with WT. Drug co‐treatment significantly decreased IL‐1β and increased IL‐10 in mdx diaphragm. Drug co‐treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies. - The Journal of Physiology, EarlyView.
    October 03, 2018   doi: 10.1113/JP276954   open full text
  • Altered anabolic signalling and reduced stimulation of myofibrillar protein synthesis after feeding and resistance exercise in people with obesity.
    Joseph W. Beals, Sarah K. Skinner, Colleen F. McKenna, Elizabeth G. Poozhikunnel, Samee A. Farooqi, Stephan Vliet, Isabel G. Martinez, Alexander V. Ulanov, Zhong Li, Scott A. Paluska, Nicholas A. Burd.
    The Journal of Physiology. October 01, 2018
    --- - |2+ Key points Lifestyle modifications that include the regular performance of exercise are probably important for counteracting the negative consequences of obesity on postprandial myofibrillar protein synthetic responses to protein dense food ingestion. We show that the interactive effect of resistance exercise and feeding on the stimulation of myofibrillar protein synthesis rates is diminished with obesity compared to normal weight adults. The blunted myofibrillar protein synthetic response with resistance exercise in people with obesity may be underpinned by alterations in muscle anabolic signalling phosphorylation (p70S6K and 4E‐BP1). The results obtained in the present study suggest that further exercise prescription manipulation may be necessary to optimize post‐exercise myofibrillar protein synthesis rates in adults with obesity. Abstract We aimed to determine whether obesity alters muscle anabolic and inflammatory signalling phosphorylation and also muscle protein synthesis within the myofibrillar (MYO) and sarcoplasmic (SARC) protein fractions after resistance exercise. Nine normal weight (NW) (21 ± 1 years, body mass index 22 ± 1 kg m−2) and nine obese (OB) (22 ± 1 years, body mass index 36 ± 2 kg m−2) adults received l‐[ring‐13C6]phenylalanine infusions with blood and muscle sampling at basal and fed‐state of the exercise (EX) and non‐exercise (CON) legs. Participants performed unilateral leg extensions and consumed pork (36 g of protein) immediately after exercise. Basal muscle Toll‐like receptor 4 (TLR4) protein was similar between OB and NW groups (P > 0.05) but increased at 300 min after pork ingestion only in the OB group (P = 0.03). Resistance exercise reduced TLR4 protein in the OB group at 300 min (EX vs. CON leg in OB: P = 0.04). Pork ingestion increased p70S6K phosphorylation at 300 min in CON and EX of the OB and NW groups (P > 0.05), although the response was lower in the EX leg of OB vs. NW at 300 min (P = 0.05). Basal MYO was similar between the NW and OB groups (P > 0.05) and was stimulated by pork ingestion in the EX and CON legs in both groups (Δ from basal NW: CON 0.04 ± 0.01% h−1; EX 0.10 ± 0.02% h−1; OB: CON 0.06 ± 0.01% h−1; EX 0.06 ± 0.01% h−1; P < 0.05). MYO was more strongly stimulated in the EX vs. CON legs in NW (P = 0.02) but not OB (P = 0.26). SARC was feeding sensitive but not further potentiated by resistance exercise in both groups. Our results suggest that obesity may attenuate the effectiveness of resistance exercise to augment fed‐state MYO. - The Journal of Physiology, EarlyView.
    October 01, 2018   doi: 10.1113/JP276210   open full text
  • Parkin: one of the guardians of mitochondrial function and skeletal muscle contractility.
    Gabriel S. Arini, Ancély F. dos Santos.
    The Journal of Physiology. October 01, 2018
    --- - - The Journal of Physiology, EarlyView.
    October 01, 2018   doi: 10.1113/JP276872   open full text
  • Issue Information.

    The Journal of Physiology. October 01, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4549-4550, 1 October 2018.
    October 01, 2018   doi: 10.1113/tjp.12592   open full text
  • Feedforward‐ and motor effort‐dependent increase in prefrontal oxygenation during voluntary one‐armed cranking.
    Kei Ishii, Nan Liang, Ryota Asahara, Makoto Takahashi, Kanji Matsukawa.
    The Journal of Physiology. September 30, 2018
    --- - |2+ Key points Some cortical areas are believed to transmit a descending signal in association with motor intention and/or effort that regulates the cardiovascular system during exercise (termed central command). However, there was no evidence for the specific cortical area responding prior to arbitrary motor execution and in proportion to the motor effort. Using a multichannel near‐infrared spectroscopy system, we found that the oxygenation of the dorsolateral and ventrolateral prefrontal cortices on the right side increases in a feedforward‐ and motor effort‐dependent manner during voluntary one‐armed cranking with the right arm. This finding may suggest a role of the dorsolateral and ventrolateral prefrontal cortices in triggering off central command and may help us to understand impaired regulation of the cardiovascular system in association with lesion of the prefrontal cortex. Abstract Output from higher brain centres (termed central command) regulates the cardiovascular system during exercise in a feedforward‐ and motor effort‐dependent manner. This study aimed to determine a cortical area responding prior to arbitrarily started exercise and in proportion to the effort during exercise. The oxygenation responses in the frontal and frontoparietal areas during one‐armed cranking with the right arm were measured using multichannel near‐infrared spectroscopy, as indexes of regional blood flow responses, in 20 subjects. The intensity of voluntary exercise was 30% and 60% of the maximal voluntary effort (MVE). At the start period of both voluntary cranking tasks, the oxygenation increased (P < 0.05) only in the lateral and dorsal part of the dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex (VLPFC) and sensorimotor cortices. Then, the oxygenation increased gradually in all cortical areas during cranking at 60% MVE, while oxygenation increased only in the frontoparietal area and some of the frontal area during cranking at 30% MVE. The rating of perceived exertion to the cranking tasks correlated (P < 0.05) with the oxygenation responses on the right side of the lateral‐DLPFC (r = 0.46) and VLPFC (r = 0.48) and the frontopolar areas (r = 0.47–0.49). Motor‐driven passive one‐armed cranking decreased the oxygenation in most cortical areas, except the contralateral frontoparietal areas. Accordingly, the lateral‐DLPFC and VLPFC on the right side would respond in a feedforward‐ and motor effort‐dependent manner during voluntary exercise with the right arm. Afferent inputs from mechanosensitive afferents may decrease the cortical oxygenation. - The Journal of Physiology, EarlyView.
    September 30, 2018   doi: 10.1113/JP276956   open full text
  • Renal reactivity: Acid‐base compensation during incremental ascent to high altitude.
    Shaelynn M. Zouboules, Hailey C. Lafave, Ken D. O'Halloran, Tom D. Brutseart, Heidi E. Nysten, Cassandra E. Nysten, Craig D. Steinback, Mingma T. Sherpa, Trevor A. Day.
    The Journal of Physiology. September 29, 2018
    --- - |2+ Key points Ascent to high altitude imposes an acid‐base challenge in which renal compensation is integral to maintain pH homeostasis, facilitate acclimatization and help prevent mountain sicknesses. The time‐course and extent of plasticity of this important renal response during incremental ascent to altitude is unclear. We created a novel index that accurately quantifies renal acid‐base compensation, which may have laboratory, fieldwork and clinical applications. Using this index, we found that renal compensation increased and plateaued after five days of incremental altitude exposure, suggesting plasticity in renal acid‐base compensation mechanisms. The time‐course and extent of plasticity in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude. Abstract Ascent to high altitude, and the associated hypoxic ventilatory response, imposes an acid‐base challenge, namely chronic hypocapnia and respiratory alkalosis. The kidneys impart a relative compensatory metabolic acidosis through the elimination of bicarbonate (HCO3−) in urine. The time‐course and extent of plasticity of the renal response during incremental ascent is unclear. We developed an index of renal reactivity (RR), indexing the relative change in arterial bicarbonate concentration ([HCO3−]a; i.e., renal response) against the relative change in arterial pressure of CO2 (PaCO2; i.e. renal stimulus) during incremental ascent to altitude (Δ[HCO3−]a/ΔPaCO2). We aimed to assess if (1) RR magnitude was inversely‐correlated with relative changes in arterial pH (ΔpHa) with ascent and (2) RR increased over time and altitude exposure (i.e., plasticity). During ascent to 5160 m over 10 days in the Nepal Himalaya, arterial blood was drawn from the radial artery for measurement of blood gas/acid‐base variables in lowlanders at 1045/1400 m and following one night sleep at 3440 m (day 3), 3820 m (day 5), 4240 m (day 7) and 5160 m (day 10) during ascent. At 3820 m and higher, RR significantly increased and plateaued compared to 3440 m (P < 0.04), suggesting plasticity in renal acid‐base compensations. At all altitudes, we observed a strong negative correlation (r ≥ −0.71; P < 0.001) between RR and ΔpHa from baseline. Renal compensation plateaued after five days of altitude exposure, despite subsequent exposure to higher altitudes. The time‐course, extent of plasticity and plateau in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 29, 2018   doi: 10.1113/JP276973   open full text
  • Investigating Cerebral Blood Flow Control to Save the Newborn Brain.
    Vignesh Murali, Cecilia G. Freeman, Ronée E. Harvey, Nicole C. Baig, Jennifer L. Helmond, Noud Helmond.
    The Journal of Physiology. September 28, 2018
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 28, 2018   doi: 10.1113/JP277045   open full text
  • Sex differences in diaphragmatic fatigue: do young women have an advantage?
    Claire M. DeLucia, Daniel H. Craighead.
    The Journal of Physiology. September 28, 2018
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 28, 2018   doi: 10.1113/JP277120   open full text
  • TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.
    M. Choudhury, N. Black, A. Alghmdi, A. D'Souza, R. Wang, J. Yanni, H. Dobrzynski, P.A. Kingston, H. Zhang, M.R. Boyett, G.M Morris.
    The Journal of Physiology. September 27, 2018
    --- - |2+ Key points The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or ‘sick sinus syndrome’, can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. ‘Biopacemaking’ utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the ‘HCN’ pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+/Ca2+ exchanger ‘NCX1’, and the transcription factors ‘TBX3’ and ‘TBX18’ known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue. Gene therapy targeting TBX18 could therefore have potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. Abstract The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome (SSS), causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+/Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5+/‐13.6 bpm, SAP 144.1+/‐8.6 bpm, SAP‐TBX18 214.4+/‐14.4 bpm; p<0.001), improved heart rate stability (SDRR from 39.3+/‐7.2 ms to 6.9+/‐0.8 ms, p<0.01; RMSDD from 41.7+/‐8.2 ms to 6.1+/‐1.2 ms, p<0.01; Poincaré SD1 from 29.5+/‐5.8 ms to 7.9+/‐2.0 ms, p<0.05) and restoration of isoprenaline response (increases in rates of: SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4%, and SAP‐TBX18 103.3 ± 10.2%; p<0.001). These changes were driven by a TBX18‐induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10‐5 ± 2.2 × 10‐6 to 2.8 × 10–5 ± 4.3 × 10‐6 arbitrary units, p<0.001). Biophysically detailed computer modelling incorporating isoform specific HCN channel electrophysiology confirmed that the measured changes in HCN abundances could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 27, 2018   doi: 10.1113/JP276508   open full text
  • NHA2 promotes cyst development in an in vitro model of polycystic kidney disease.
    Hari Prasad, Donna K. Dang, Kalyan C. Kondapalli, Niranjana Natarajan, Valeriu Cebotaru, Rajini Rao.
    The Journal of Physiology. September 22, 2018
    --- - |2+ Key Points We observed significant and selective up‐regulation of the Na+/H+ exchanger NHA2 (SLC9B2) in cysts of patients with autosomal dominant polycystic kidney disease. Using the MDCK model of cystogenesis, we find NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro. Polycystin‐1 represses NHA2 expression via Ca2+/NFAT signalling whereas the dominant negative membrane‐anchored C‐terminal fragment (PC1‐MAT) increased NHA2 levels. Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression. Taken together, our findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of PKD. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin‐1 (PC1) and polycystin‐2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up‐regulation of NHA2, a member of the SLC9B family of Na+/H+ exchangers that correlated with cyst size and disease severity in ADPKD patients. Using three‐dimensional cultures of MDCK cells to model cystogenesis in vitro, we show that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store‐dependent and independent pathways, whereas reciprocal activation of Ca2+ influx by a dominant negative, membrane‐anchored C‐terminal tail fragment of PC1 elevated NHA2. We show that NHA2 is a target of Ca2+/NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observe robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up‐regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for PKD and reveal a potential new regulator of transepithelial salt and water transport in the kidney. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 22, 2018   doi: 10.1113/JP276796   open full text
  • Rapid saline infusion and/or drinking enhance skin sympathetic nerve activity components reduced by hypovolaemia and hyperosmolality in hyperthermia.
    Yoshi‐ichiro Kamijo, Kazunobu Okazaki, Shigeki Ikegawa, Yoshiyuki Okada, Hiroshi Nose.
    The Journal of Physiology. September 22, 2018
    --- - |2+ Key point summary In hyperthermia, plasma hyperosmolality suppresses both cutaneous vasodilatation and sweating responses and these suppressions are removed by oropharyngeal stimulation such as drinking. Hypovolaemia suppresses only cutaneous vasodilatation, which is enhanced by rapid infusion in hyperthermia. Our recent studies suggest that skin sympathetic nerve activity (SSNA) involved components synchronised and non‐synchronised with the cardiac cycle, which are associated with an active vasodilator and a sudomotor, respectively. In the present study, plasma hyperosmolality suppressed both components, drinking removed the hyperosmolality‐induced suppressions, simultaneously with increases in cutaneous vasodilatation and sweating with not altering plasma volume and osmolality. Furthermore, a rapid saline infusion increased synchronised component and cutaneous vasodilatation in hypovolaemic and hyperthermic humans. The results support our idea that SSNA involves active cutaneous vasodilator and sudomotor and a site where osmolality signals are projected to control thermoregulation is located more superior than the medulla where signals from baroreceptors are projected. Abstract We reported that skin sympathetic nerve activity (SSNA) involved components synchronised and non‐synchronised with the cardiac cycle, both components increased in hyperthermia and our results suggest that the components are associated with active vasodilator and sudomotor, respectively. In the present study, we examined whether the increases in the components in hyperthermia would be suppressed by plasma hyperosmolality simultaneously with suppressions of cutaneous vasodilatation and sweating and whether these suppressions were released by oropharyngeal stimulation (drinking). Also, effects of a rapid saline infusion on the both components and responses of cutaneous vasodilatation and sweating were tested in hypovolaemic and hyperthermic subjects. We found that 1) plasma hyperosmolality suppressed both components in hyperthermia, 2) the suppressions were released by drinking of 200‐mL water simultaneously with enhanced cutaneous vasodilatation and sweating responses, and 3) a rapid infusion; at 1.0 and 0.2 ml min‐1 kg−1 for the first 10 min and the following 20 min, respectively, increased the synchronised component and cutaneous vasodilatation in diuretic‐induced hypovolaemia greater than those in time control; at 0.1 ml min‐1 kg−1 for 30 min, and no greater increases in the non‐synchronised component and sweating responses were observed during rapid infusion than time control. The results support the idea that SSNA involves the components synchronised and non‐synchronised with the cardiac cycle, associated with the active cutaneous vasodilator and sudomotor, and a site of osmolality‐induced modulation for thermoregulation is located superior to the medulla where signals from baroreceptors are projected. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 22, 2018   doi: 10.1113/JP276633   open full text
  • Commissural communication allows mouse intergeniculate leaflet and ventral lateral geniculate neurons to encode interocular differences in irradiance.
    A. Pienaar, L. Walmsley, E. Hayter, M. Howarth, T. M. Brown.
    The Journal of Physiology. September 21, 2018
    --- - |2+ Key Points Unlike other visual thalamic regions, the intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/vLGN) possess extensive reciprocal commissural connections, the functions of which are unknown. Using electrophysiological approaches, we show that commissural projecting IGL/vLGN cells are primarily activated by light increments to the contralateral eye while cells receiving commissural input typically exhibit antagonistic binocular responses. Across antagonistic cells, the nature of the commissural input (excitatory or inhibitory) corresponds to the presence of ipsilateral ON or OFF visual responses and in both cases antagonistic responses disappear following inactivation of the contralateral thalamus. We go on to show that the steady state firing rates of antagonistic cells uniquely encode interocular differences in irradiance. Collectively our data reveal a pivotal role for IGL/vLGN commissural signalling in generating new sensory properties that are potentially useful the proposed roles for these nuclei in visuomotor/vestibular and circadian control. Abstract The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/vLGN) are portions of the visual thalamus implicated in circadian and visuomotor/vestibular control. A defining feature of IGL/vLGN organisation is the presence of extensive reciprocal commissural connections, the functions of which are presently unknown. Here we use a combination of multielectrode recording, electrical microstimulation, thalamic inactivation and a range of visual stimuli in mice to address this deficit. Our data indicate that, like most IGL/vLGN cells, those that project commissurally primarily convey contralateral‐ON visual signals while most IGL/vLGN neurons that receive this input exhibit antagonistic binocular responses (i.e. excitatory responses driven by one eye and inhibitory responses driven by the other), enabling them to encode interocular differences in irradiance. We also confirm that this property derives from commissural input since, following inactivation of the contralateral visual thalamus, these cells instead display monocular contralateral‐driven ON responses. Our data thereby reveal a fundamental role for commissural signalling in generating new visual response properties at the level of the visual thalamus. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 21, 2018   doi: 10.1113/JP276917   open full text
  • UBC‐Nepal expedition: peripheral fatigue recovers faster in Sherpa than Lowlanders at high‐altitude.
    Luca Ruggiero, Ryan L. Hoiland, Alexander B. Hansen, Philip N. Ainslie, Chris J. McNeil.
    The Journal of Physiology. September 21, 2018
    --- - |2+ Key points The reduced oxygen tension of high‐altitude compromises performance in Lowlanders. In this environment, Sherpa display superior performance, and little is known on this issue. Sherpa present unique genotypic and phenotypic characteristics at the muscular level, which may enhance resistance to peripheral fatigue at high‐altitude compared to Lowlanders. We studied the impact of gradual ascent and exposure to high‐altitude (5050 m) on peripheral fatigue in age‐matched Lowlanders and Sherpa, using intermittent electrically‐evoked contractions of the knee extensors. Peripheral fatigue (force loss) was lower in Sherpa during the first part of the protocol. Post‐protocol, the rate of force development and contractile impulse recovered faster in Sherpa than Lowlanders. At any time, indices of muscle oxygenation were not different between groups. Muscle contractile properties in Sherpa, independent of muscle oxygenation, were less perturbed by non‐volitional fatigue. Hence, elements within the contractile machinery contribute to the superior physical performance of Sherpa at high‐altitude. Abstract Altitude‐related acclimatisation is characterised by marked muscular adaptations. Lowlanders and Sherpa differ in their muscular genotypic and phenotypic characteristics, which may influence peripheral fatigability at altitude. After gradual ascent to 5050m, 12 Lowlanders and 10 age‐matched Sherpa (32 ± 10 vs. 31 ± 11 years, respectively) underwent 3 bouts (separated by 15s rest) of 75 intermittent electrically‐evoked contractions (12 pulses at 15 Hz, 1.6s between train onsets) of the dominant leg quadriceps, at the intensity which initially evoked 30% of maximal voluntary force. Trains were also delivered at minutes 1, 2, and 3 after the protocol to measure recovery. Tissue oxygenation index (TOI) and total haemoglobin (tHb) were quantified by a NIRS probe secured over rectus femoris. Superficial femoral artery blood flow was recorded using ultrasonography, and delivery of oxygen was estimated (eDO2). At the end of bout 1, peak force was greater in Sherpa than Lowlanders (91.5% vs. 84.5% baseline, respectively; P<0.05). Peak rate of force development (pRFD), contractile impulse (CI200), and half‐relaxation time (HRT) recovered faster in Sherpa than Lowlanders (% baseline at 1 min; pRFD: 89% vs. 74%; cIM: 91% vs. 80%; HRT: 113% vs. 123%, respectively; P<0.05). Vascular measures were pooled for Lowlanders and Sherpa as they did not differ during fatigue or recovery (P<0.05). Mid‐bout 3, TOI was decreased (90% baseline) whereas tHb was increased (109% baseline). After bout 3, eDO2 was markedly increased (1266% baseline). The skeletal muscle of Sherpa seemingly favours repeated force production at altitude for similar oxygen delivery compared to Lowlanders. Luca Ruggiero is completing his PhD in the Integrative Neuromuscular Physiology Laboratory at the Okanagan Campus of the University of British Columbia. His PhD research focuses on neuromuscular fatigue and motor control at high‐altitude as well as the neuromuscular advantages of Sherpa compared to Lowlanders in such an environment. His main research interests include neuromuscular fatigue, environmental stress, and biomechanics. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 21, 2018   doi: 10.1113/JP276599   open full text
  • Haptic exploration attenuates and alters somatosensory cortical oscillations.
    Max J. Kurz, Alex I. Wiesman, Nathan M. Coolidge, Tony W. Wilson.
    The Journal of Physiology. September 21, 2018
    --- - |2+ Key points Several behavioural studies have shown the sensory perceptions are reduced during movement; yet the neurophysiological reason for this is not clear. Participants underwent stimulation of the median nerve when either sitting quietly (i.e. passive stimulation condition) or performing haptic exploration of a ball with the left hand. Magnetoencephalographic brain imaging and advanced beamforming methods were used to identify the differences in somatosensory cortical responses. We show that the neural populations active during the passive stimulation condition were strongly gated during the haptic exploration task. These results imply that the reduced haptic perceptions might be governed by gating of certain somatosensory neural populations. Abstract Several behavioural studies have shown that children have reduced sensory perceptions during movement; however, the neurophysiological nexus for these altered perceptions remains unknown. We used magnetoencephalographic brain imaging and advanced beamforming methods to address this knowledge gap. In our experiment, a cohort of children (aged 10–18 years) underwent stimulation of the median nerve when either sitting quietly (i.e. passive stimulation condition) or performing haptic exploration of a ball with the left hand. Our results revealed two novel observations. First, there was a relationship between the child's age and the strength of the beta (18–26 Hz) response seen within the somatosensory cortices during the passive stimulation condition. This suggests that there may be an age‐dependent change in the processing of peripheral feedback by the somatosensory cortices. Second, all of the cortical regions that were active during the passive stimulation condition were almost completely gated during the haptic task. Instead, the haptic task involved neural oscillations within Brodmann area 2, which is known to convey less spatially precise tactile information but is involved in the processing of more complex somatosensations across the respective digits. These results imply that the reduced somatosensory perceptions seen during movements in healthy children may be related to the gating of certain neural generators, as well as activation of haptic‐specific neural generators within the somatosensory cortices. The utilization of such haptic‐specific circuits during development may lead to the enhanced somatosensory processing during haptic exploration seen in healthy adults. - The Journal of Physiology, EarlyView.
    September 21, 2018   doi: 10.1113/JP276263   open full text
  • Turnaround in the history of carotid chemoreflex contribution for cardiorespiratory control in COPD: what are the upcoming chapters?
    Diogo Machado Oliveira, Indyanara Cristina Ribeiro, Tamires Silva Cesar, Tiago Obeid Freitas, Liliane Cunha Aranda.
    The Journal of Physiology. September 20, 2018
    --- - - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 20, 2018   doi: 10.1113/JP276986   open full text
  • Nitric oxide‐dependent attenuation of noradrenaline‐induced vasoconstriction is impaired in the canine model of Duchenne muscular dystrophy.
    Kasun Kodippili, Chady H. Hakim, Hsiao T. Yang, Xiufang Pan, N. Nora Yang, Maurice H. Laughlin, Ronald L. Terjung, Dongsheng Duan.
    The Journal of Physiology. September 20, 2018
    --- - |2+ Key points We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS‐derived NO‐dependent and NO‐independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO‐independent sympatholysis was not affected in the canine DMD model. Abstract The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane‐associated nNOS to blood flow regulation in dystrophin‐deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO‐independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD. - The Journal of Physiology, EarlyView.
    September 20, 2018   doi: 10.1113/JP275672   open full text
  • The effect of maternal metabolic status on offspring health: a role for skeletal muscle?
    Jasmine Mikovic, Séverine Lamon.
    The Journal of Physiology. September 20, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 20, 2018   doi: 10.1113/JP276929   open full text
  • Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.
    Tyler Basting, Jiaxi Xu, Snigdha Mukerjee, Joel Epling, Robert Fuchs, Srinivas Sriramula, Eric Lazartigues.
    The Journal of Physiology. September 20, 2018
    --- - |2+ Key points Recurrent periods of over‐excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over‐activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin‐2 leads to an immediate frequency‐dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)‐salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. Abstract Neuro‐cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over‐excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin‐2 using an adeno‐associated virus (CamKII‐ChR2‐eYFP‐AAV2) in wildtype mice (n = 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency‐dependent (2, 10 and 20 Hz) increase in BP from baseline by ∼9 mmHg at 20 Hz stimulation (P < 0.001). Additionally, in vGlut2‐cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre‐dependent caspase (AAV2‐flex‐taCASP3‐TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA‐salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P < 0.05) resulted in an attenuated rise in BP following DOCA‐salt treatment (P < 0.05 at 7 day time point, n = 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA‐salt treatment in the lesioned animals (P < 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP. - The Journal of Physiology, EarlyView.
    September 20, 2018   doi: 10.1113/JP276229   open full text
  • Ageing changes in biventricular cardiac function in male and female baboons (Papio spp.).
    Anderson H. Kuo, Cun Li, Hillary F. Huber, Peter W. Nathanielsz, Geoffrey D. Clarke.
    The Journal of Physiology. September 19, 2018
    --- - |2+ Key points Life course changes in cardiovascular function in a non‐human primate have been comprehensively characterized. Age‐related declines in normalized left ventricular stroke volume and cardiac output were found with corresponding decreases in biventricular ejection fractions and filling rates. There were age‐related decreases in male and female baboon normalized left ventricular myocardial mass index, which declined at similar rates. Systolic functional declines in right ventricular function were observed with age, similar to the left ventricle. Sex differences were found in the rates and directions of right ventricular volume changes along with decreased end‐systolic right ventricular sphericity. The results validate the baboon as an appropriate model for translational studies of cardiovascular functional decline with ageing. Abstract Previous studies reported cardiac function declines with ageing. This study determined changes in biventricular cardiac function in a well‐characterized baboon model. Cardiac magnetic resonance imaging measured key biventricular parameters in 47 baboons (22 female, age 4–23 years). ANCOVA assessed sex and age changes with P < 0.05 deemed significant. Stroke volume, cardiac output and other cardiac functional parameters were normalized to body surface area. There were similar, age‐related rates of decrease in male (M) and female (F) normalized left ventricular (LV) myocardial mass index (M: −1.2 g m−2 year−1, F: −0.9 g m−2 year−1). LV ejection fraction declined at −0.96% year−1 (r = −0.43, P = 0.002) and right ventricular (RV) ejection fraction decreased at −1.2% year−1 (r = −0.58, P < 0.001). Normalized LV stroke volume fell at −1.1 ml m−2 year−1 (r = −0.47, P = 0.001), normalized LV ejection rate at −3.8 ml s−1 m−2 year−1 (r = −0.43, P < 0.005) and normalized LV filling rate at −4.1 ml s−1 m−2 year−1 (r = −0.44, P < 0.005). Also, RV wall thickening fraction decreased with age (slope = −1% year−1, P = 0.008). RV ejection rate decreased at −3.6 ml s−1 m−2 year−1 (P = 0.002) and the normalized average RV filling rate dropped at −3.7 ml s−1 m−2 year−1 (P < 0.0001). End‐systolic RV sphericity index also dropped with age (r = −0.33, P = 0.02). Many observed changes parallel previously reported data in human and animal studies. These measured biventricular functional declines in hearts with ageing from the closest experimental primate species to man underscore the utility of the baboon model for investigating mechanisms related to heart ageing. - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276338   open full text
  • A simple decision to move in response to touch reveals basic sensory memory and mechanisms for variable response times.
    Stella Koutsikou, Robert Merrison‐Hort, Edgar Buhl, Andrea Ferrario, Wen‐Chang Li, Roman Borisyuk, Stephen R. Soffe, Alan Roberts.
    The Journal of Physiology. September 19, 2018
    --- - |2+ Key points Short‐term working memory and decision‐making are usually studied in the cerebral cortex; in many models of simple decision making, sensory signals build slowly and noisily to threshold to initiate a motor response after long, variable delays. When touched, hatchling frog tadpoles decide whether to swim; we define the long and variable delays to swimming and use whole‐cell recordings to uncover the neurons and processes responsible. Firing in sensory and sensory pathway neurons is short latency, and too brief and invariant to explain these delays, while recordings from hindbrain reticulospinal neurons controlling swimming reveal a prolonged and variable build‐up of synaptic excitation which can reach firing threshold and initiate swimming. We propose this excitation provides a sensory memory of the stimulus and may be generated by small reverberatory hindbrain networks. Our results uncover fundamental network mechanisms that allow animals to remember brief sensory stimuli and delay simple motor decisions. Abstract Many motor responses to sensory input, like locomotion or eye movements, are much slower than reflexes. Can simpler animals provide fundamental answers about the cellular mechanisms for motor decisions? Can we observe the ‘accumulation’ of excitation to threshold proposed to underlie decision making elsewhere? We explore how somatosensory touch stimulation leads to the decision to swim in hatchling Xenopus tadpoles. Delays measured to swimming in behaving and immobilised tadpoles are long and variable. Activity in their extensively studied sensory and sensory pathway neurons is too short‐lived to explain these response delays. Instead, whole‐cell recordings from the hindbrain reticulospinal neurons that drive swimming show that these receive prolonged, variable synaptic excitation lasting for nearly a second following a brief stimulus. They fire and initiate swimming when this excitation reaches threshold. Analysis of the summation of excitation requires us to propose extended firing in currently undefined presynaptic hindbrain neurons. Simple models show that a small excitatory recurrent‐network inserted in the sensory pathway can mimic this process. We suggest that such a network may generate slow, variable summation of excitation to threshold. This excitation provides a simple memory of the sensory stimulus. It allows temporal and spatial integration of sensory inputs and explains the long, variable delays to swimming. The process resembles the ‘accumulation’ of excitation proposed for cortical circuits in mammals. We conclude that fundamental elements of sensory memory and decision making are present in the brainstem at a surprisingly early stage in development. - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276356   open full text
  • Somatic modulation of ectopic action potential initiation in distal axons.
    Aurélie Fékété, Dominique Debanne.
    The Journal of Physiology. September 19, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP277012   open full text
  • VEGF‐A165b to the rescue: vascular integrity and pain sensitization.
    Madhavi Jere, Ryan M. Cassidy.
    The Journal of Physiology. September 19, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276902   open full text
  • TRPV1 and BDKRB2 receptor polymorphisms can influence the exercise pressor reflex.
    Karambir Notay, Shannon L. Klingel, Jordan B. Lee, Connor J. Doherty, Jeremy D. Seed, Michal Swiatczak, David M. Mutch, Philip J. Millar.
    The Journal of Physiology. September 19, 2018
    --- - |2+ Key points The mechanisms responsible for the high inter‐individual variability in blood pressure responses to exercise remain unclear. Common genetic variants of genes related to the vascular transduction of sympathetic outflow have been investigated, but variants influencing skeletal muscle afferent feedback during exercise have not been explored. Single nucleotide polymorphisms in TRPV1 rs222747 and BDKRB2 rs1799722 receptors present in skeletal muscle were associated with differences in the magnitude of the blood pressure response to static handgrip exercise but not mental stress. The combined effects of TRPV1 rs222747 and BDKRB2 rs1799722 on blood pressure and heart rate responses during exercise were additive, and primarily found in men. Genetic differences in skeletal muscle metaboreceptors may be a risk factor for exaggerated blood pressure responses to exercise. Abstract Exercise blood pressure (BP) responses demonstrate high inter‐individual variability, which could relate to differences in metabolically sensitive afferent feedback from the exercising muscle. We hypothesized that single‐nucleotide polymorphisms (SNPs) in genes encoding metaboreceptors present in group III/IV skeletal muscle afferents can influence the exercise pressor response. Two hundred men and women underwent measurements of continuous BP and heart rate at baseline and during 2 min of static handgrip exercise (30% maximal volitional contraction), post‐exercise circulatory occlusion and mental stress (serial subtraction; internal control). Participants were genotyped for SNPs in TRPV1 (rs222747; G/C), ASIC3 (rs2288645; G/A), BDKRB2 (rs1799722; C/T), PTGER2 (rs17197; A/G) and P2RX4 (rs25644; A/G). Exercise systolic BP (19 ± 10 vs. 22 ± 10 mmHg, P = 0.03) was lower in GG versus GC/CC minor allele carriers for TRPV1 rs222747, while exercise diastolic BP (14 ± 7 vs. 17 ± 7 mmHg, P = 0.007) and heart rate (12 ± 8 vs. 15 ± 9 beats min−1, P = 0.03) were lower in CC versus CT/TT minor allele carriers for BDKRB2 rs1799722. Individuals carrying both minor alleles for TRPV1 rs222747 and BDKRB2 rs1799722 had greater systolic (22 ± 11 vs. 17 ± 10 mmHg, P = 0.04) and diastolic (18 ± 7 vs. 14 ± 7 mmHg, P = 0.01) BP responses than those with no minor alleles; these differences were larger in men. No differences in BP or heart rate responses were detected during static handgrip with ASIC3 rs2288645, PTGER2 rs17197 or P2RX4 rs25644. None of the selected SNPs were associated with differences during mental stress. These findings demonstrate that variants in TRPV1 and BDKRB2 receptors can contribute to BP differences during static exercise in an additive manner. - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276526   open full text
  • Inhibition of GluN2A NMDA receptors ameliorates synaptic plasticity deficits in the Fmr1−/y mouse model.
    Camilla J. Lundbye, Anna Karina H. Toft, Tue G. Banke.
    The Journal of Physiology. September 19, 2018
    --- - |2+ Key points Fragile X syndrome (FXS) is a genetic condition that is the most common form of inherited intellectual impairment and causes a range of neurodevelopmental complications including learning disabilities and intellectual disability and shares many characteristics with autism spectrum disorder (ASD). In the FXS mouse model, Fmr1−/y, impaired synaptic plasticity was restored by pharmacologically inhibiting GluN2A‐containing NMDA receptors but not GluN2B‐containing receptors. Similar results were obtained by crossing Fmr1−/y with GluN2A knock‐out (Grin2A−/−) mice. These results suggest that dampening the elevated levels of GluN2A‐containing NMDA receptors in Fmr1−/y mice has the potential to restore hyperexcitability of the neural circuitry to (a more) normal‐like level of brain activity. Abstract NMDA receptors (NMDARs) play important roles in synaptic plasticity at central excitatory synapses, and dysregulation of their function may lead to severe disorders such Fragile X syndrome (FXS). FXS is caused by transcriptional silencing of the FMR1 gene followed by lack of the encoding protein. Here we examined the effects of pharmacological and genetic manipulation of hippocampal NMDAR functions in long‐term potentiation (LTP) and depression (LTD). We found impaired NMDAR‐dependent LTP in the Fmr1‐deficient mice, which could be fully restored when GluN2A‐containing NMDARs was pharmacological inhibited. Interestingly, similar LTP effects were observed when the GluN2A gene (Grin2a) was deleted in Fmr1−/y mice (Fmr1−/y/Grin2a−/− double knockout). In addition, GluN2A inhibition improved elevated mGluR5‐dependent LTD to normal level in the Fmr1−/y mouse. These findings suggest that GluN2A is a promising target in FXS research that could help us better understand the disorder. - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276304   open full text
  • Synaptic entrainment of ectopic action potential generation in hippocampal pyramidal neurons.
    Christian Thome, Fabian C. Roth, Joshua Obermayer, Antonio Yanez, Andreas Draguhn, Alexei V. Egorov.
    The Journal of Physiology. September 19, 2018
    --- - |2+ Key points Ectopic action potentials (EAPs) arise at distal locations in axonal fibres and are often associated with neuronal pathologies such as epilepsy or nerve injury, but they also occur during physiological network conditions. This study investigates whether initiation of such EAPs is modulated by subthreshold synaptic activity. Somatic subthreshold potentials invade the axonal compartment to considerable distances (>350 μm), whereas spread of axonal subthreshold potentials to the soma is inefficient. Ectopic spike generation is entrained by conventional synaptic signalling mechanisms. Excitatory synaptic potentials promote EAPs, whereas inhibitory synaptic potentials block EAPs. The modulation of ectopic excitability depends on propagation of somatic voltage deflections to the axonal EAP initiation site. Synaptic modulation of EAP initiation challenges the view of the distal axon being independent of synaptic activity and may contribute to mechanisms underlying fast network oscillations and pathological network activity. Abstract While most action potentials are generated at the axon initial segment, they can also be triggered at more distal sites along the axon. Such ectopic action potentials (EAPs) occur during several neuronal pathologies such as epilepsy, nerve injuries and inflammation but have also been observed during physiological network activity. EAPs propagate antidromically towards the somato‐dendritic compartment where they modulate synaptic plasticity. Here we investigate the converse signal direction: do somato‐dendritic synaptic potentials affect the generation of ectopic spikes? We measured anti‐ and orthodromic spikes in the soma and axon of mouse hippocampal CA1 pyramidal cells. We found that synaptic potentials propagate reliably through the axon, causing significant voltage transients at distances >350 μm. At these sites, excitatory input efficiently facilitated EAP initiation in distal axons and, conversely, inhibitory input suppressed EAP initiation. Our data reveal a new mechanism by which ectopically generated spikes can be entrained by conventional synaptic signalling during normal and pathological network activity. - The Journal of Physiology, EarlyView.
    September 19, 2018   doi: 10.1113/JP276720   open full text
  • Gestational chronodisruption leads to persistent changes in the rat fetal and adult adrenal clock and function.
    E. R. Salazar, H. G. Richter, C. Spichiger, N. Mendez, D. Halabi, K. Vergara, I. P. Alonso, F. A. Corvalán, C. Azpeleta, M. Seron‐Ferre, C. Torres‐Farfan.
    The Journal of Physiology. September 18, 2018
    --- - |2+ Key points Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Here we explored the developmental impact of gestational chronodisruption (chronic photoperiod shift, CPS) on adult and fetal adrenal biorhythms and function. We found that gestational chronodisruption altered fetal and adult adrenal function, at the molecular, morphological and physiological levels. The differences between control and CPS offspring suggest desynchronization of the adrenal circadian clock and steroidogenic pathway, leading to abnormal stress responses and metabolic adaptation, potentially increasing the risk of developing chronic diseases. Abstract Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long‐term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down‐regulated and 437 up‐regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48–20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long‐term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases. - The Journal of Physiology, EarlyView.
    September 18, 2018   doi: 10.1113/JP276083   open full text
  • P2X4 receptor re‐sensitization depends on a protonation/deprotonation cycle mediated by receptor internalization and recycling.
    Giorgio Fois, Karl J. Föhr, Carolin Kling, Michael Fauler, Oliver H. Wittekindt, Paul Dietl, Manfred Frick.
    The Journal of Physiology. September 18, 2018
    --- - |2+ Key points Re‐sensitization of P2X4 receptors depends on a protonation/de‐protonation cycle Protonation and de‐protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartments Protonation and de‐protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptors Re‐sensitization is blocked in the presence of the receptor agonist ATP Abstract P2X4 receptors are members of the P2X receptor family of cation‐permeable, ligand‐gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re‐sensitization is required to return the receptor into its resting state. Desensitization and re‐sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re‐sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re‐sensitization. Re‐sensitization depends on a protonation/de‐protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re‐sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP. - The Journal of Physiology, EarlyView.
    September 18, 2018   doi: 10.1113/JP275448   open full text
  • Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue.
    Stefan Passlick, Ek Raj Thapaliya, Zuxin Chen, Matthew T. Richers, Graham C. R. Ellis‐Davies.
    The Journal of Physiology. September 17, 2018
    --- - |2+ Key points We developed a new caged nicotinic acetylcholine receptor (nAChR) agonist, ABT594, which is photolyzed by one‐ and two‐photon excitation. The caged compound is photolyzed with a quantum yield of 0.20 One‐photon uncaging of ABT594 elicited large currents and Ca2+ transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca2+‐permeable nAChRs on axons of MHb neurons. Abstract Photochemical release of neurotransmitters has been instrumental to the study of the underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. Therefore, we caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one‐ or two‐photon excitation. Brief flashes (0.5 – 3.0 ms) of 410 nm light evoked large currents and Ca2+ transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR‐mediated Ca2+ signals along the axons of MHb neurons. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 17, 2018   doi: 10.1113/JP276615   open full text
  • Astrocyte‐mediated primary afferent depolarization: a new twist to a complicated tale?
    Arlette Kolta.
    The Journal of Physiology. September 16, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 16, 2018   doi: 10.1113/JP276949   open full text
  • Differential targeting and signalling of voltage‐gated T‐type Cav3.2 and L‐type Cav1.2 channels to ryanodine receptors in mesenteric arteries.
    Gang Fan, Mario Kaßmann, Ahmed M. Hashad, Donald G. Welsh, Maik Gollasch.
    The Journal of Physiology. September 16, 2018
    --- - |2+ Key points In arterial smooth muscle, Ca2+ sparks are elementary Ca2+‐release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+‐sensitive K+ (BKCa) channels. This study elucidated the contribution of T‐type Cav3.2 channels in caveolae and their functional interaction with L‐type Cav1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L‐type Cav1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T‐type Cav3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T‐type Cav3.2 channels and RyRs to ignite Ca2+ sparks. Abstract Recent data suggest that T‐type Cav3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav3.2 channels and their functional interaction with Cav1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen‐inducible smooth muscle‐specific Cav1.2−/− (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+, Cd2+ and methyl‐β‐cyclodextrin were used to inhibit Cav3.2 channels, Cav1.2 channels and caveolae, respectively. Ni2+ (50 μmol L−1) and methyl‐β‐cyclodextrin (10 mmol L−1) decreased Ca2+ spark frequency by ∼20–30% in mesenteric VSMCs in a non‐additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 μmol L−1) suppressed Ca2+ sparks in mesenteric arteries by ∼70–80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl‐β‐cyclodextrin. Our data demonstrate that Ca2+ influx through CaV1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries. - The Journal of Physiology, EarlyView.
    September 16, 2018   doi: 10.1113/JP276923   open full text
  • Spatial receptive field shift by preceding cross‐modal stimulation in the cat superior colliculus.
    Jinghong Xu, Tingting Bi, Jing Wu, Fanzhu Meng, Kun Wang, Jiawei Hu, Xiao Han, Jiping Zhang, Xiaoming Zhou, Les Keniston, Liping Yu.
    The Journal of Physiology. September 16, 2018
    --- - |2+ Key points It has been known for some time that sensory information of one type can bias the spatial perception of another modality. However, there is a lack of evidence of this occurring in individual neurons. In the present study, we found that the spatial receptive field of superior colliculus multisensory neurons could be dynamically shifted by a preceding stimulus in a different modality. The extent to which the receptive field shifted was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. This result provides a neural mechanism that could underlie the process of cross‐modal spatial calibration. Abstract Psychophysical studies have shown that the different senses can be spatially entrained by each other. This can be observed in certain phenomena, such as ventriloquism, in which a visual stimulus can attract the perceived location of a spatially discordant sound. However, the neural mechanism underlying this cross‐modal spatial recalibration has remained unclear, as has whether it takes place dynamically. We explored these issues in multisensory neurons of the cat superior colliculus (SC), a midbrain structure that involves both cross‐modal and sensorimotor integration. Sequential cross‐modal stimulation showed that the preceding stimulus can shift the receptive field (RF) of the lagging response. This cross‐modal spatial calibration took place in both auditory and visual RFs, although auditory RFs shifted slightly more. By contrast, if a preceding stimulus was from the same modality, it failed to induce a similarly substantial RF shift. The extent of the RF shift was dependent on both temporal and spatial gaps between the preceding and following stimuli, as well as the salience of the preceding stimulus. A narrow time gap and high stimulus salience were able to induce larger RF shifts. In addition, when both visual and auditory stimuli were presented simultaneously, a substantial RF shift toward the location‐fixed stimulus was also induced. These results, taken together, reveal an online cross‐modal process and reflect the details of the organization of SC inter‐sensory spatial calibration. - The Journal of Physiology, EarlyView.
    September 16, 2018   doi: 10.1113/JP275427   open full text
  • Specialized mechanoreceptor systems in rodent glabrous skin.
    Jan Walcher, Julia Ojeda‐Alonso, Julia Haseleu, Maria K. Oosthuizen, Ashlee H. Rowe, Nigel C. Bennett, Gary R. Lewin.
    The Journal of Physiology. September 16, 2018
    --- - |2+ Key points An ex vivo preparation was developed to record from single sensory fibres innervating the glabrous skin of the mouse forepaw. The density of mechanoreceptor innervation of the forepaw glabrous skin was found to be three times higher than that of hindpaw glabrous skin. Rapidly adapting mechanoreceptors that innervate Meissner's corpuscles were severalfold more responsive to slowly moving stimuli in the forepaw compared to those innervating hindpaw skin. We found a distinct group of small hairs in the centre of the mouse hindpaw glabrous skin that were exclusively innervated by directionally sensitive D‐hair receptors. The directional sensitivity, but not the end‐organ anatomy, were the opposite to D‐hair receptors in the hairy skin. Glabrous skin hairs in the hindpaw are not ubiquitous in rodents, but occur in African and North American species that diverged more than 65 million years ago. Abstract Rodents use their forepaws to actively interact with their tactile environment. Studies on the physiology and anatomy of glabrous skin that makes up the majority of the forepaw are almost non‐existent in the mouse. Here we developed a preparation to record from single sensory fibres of the forepaw and compared anatomical and physiological receptor properties to those of the hindpaw glabrous and hairy skin. We found that the mouse forepaw skin is equipped with a very high density of mechanoreceptors; >3 times more than hindpaw glabrous skin. In addition, rapidly adapting mechanoreceptors that innervate Meissner's corpuscles of the forepaw were severalfold more sensitive to slowly moving mechanical stimuli compared to their counterparts in the hindpaw glabrous skin. All other mechanoreceptor types as well as myelinated nociceptors had physiological properties that were invariant regardless of which skin area they occupied. We discovered a novel D‐hair receptor innervating a small group of hairs in the middle of the hindpaw glabrous skin in mice. These glabrous skin D‐hair receptors were direction sensitive albeit with an orientation sensitivity opposite to that described for hairy skin D‐hair receptors. Glabrous skin hairs do not occur in all rodents, but are present in North American and African rodent species that diverged more than 65 million years ago. The function of these specialized hairs is unknown, but they are nevertheless evolutionarily very ancient. Our study reveals novel physiological specializations of mechanoreceptors in the glabrous skin that likely evolved to facilitate tactile exploration. - The Journal of Physiology, EarlyView.
    September 16, 2018   doi: 10.1113/JP276608   open full text
  • Nicotine modulates human brain plasticity via calcium‐dependent mechanisms.
    Grundey Jessica, Barlay Jerick, Batsikadze Giorgi, Kuo Min‐Fang, Paulus Walter, Nitsche Michael.
    The Journal of Physiology. September 15, 2018
    --- - |2+ Key point Nicotine modulates cognition and memory function by targeting the nicotinic acetylcholine receptor and releasing different transmitter systems postsynaptically. With both nicotine‐generated mechanisms, calcium influx and calcium permeability can be regulated, which is a key requirement for the induction of long‐term potentiation, the physiological basis of learning and memory function. We try to unmask the underlying mechanism of nicotinic effects on anodal tDCS‐induced LTP‐like plasticity based on the hypothesis of calcium‐dependency. Abolished tDCS‐induced neuroplasticity by nicotine administration is reversed by calcium channel blockade with flunarizine in a dosage‐dependent manner. It suggests dose determination of nicotine/nicotine agonists in therapeutical settings when treating cognitive dysfunction and explains partially heterogeneous results on cognition in subjects in different experimental settings. Abstract Nicotine (NIC) modulates neuroplasticity and improves cognitive performance in animals and humans mainly by increased calcium permeability and modulation of diverse transmitter systems. NIC administration impairs calcium‐dependent plasticity induced by non‐invasive brain stimulation with transcranial direct current stimulation (tDCS) in non‐smoking participants probably by intracellular calcium overflow. In order to test this hypothesis, we analysed the effect of calcium channel blockage with flunarizine (FLU) on anodal tDCS‐induced cortical excitability changes in healthy non‐smokers under NIC. We applied anodal tDCS combined with nicotine patch and FLU in three different doses (2.5; 5 and 10 mg) or placebo medication. NIC abolished anodal tDCS‐induced neuroplasticity. Under medium dosage, but not under low and high dosage of FLU, combined with NIC plasticity was re‐established. For FLU alone, the lowest dosage weakened LTP‐like plasticity, while the highest dosage again abolished tDCS‐induced plasticity. The medium dosage turned LTP‐like plasticity in long‐term depression‐like (LTD) plasticity. Our results suggest a key role of calcium influx and calcium levels in nicotinic effects on LTP‐like plasticity in humans. This knowledge might be relevant for the development of new therapeutic strategies in cognitive dysfunction. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 15, 2018   doi: 10.1113/JP276502   open full text
  • Ventilatory and integrated physiological responses to chronic hypercapnia in goats.
    Nicholas J. Burgraff, Suzanne E. Neumueller, Kirstyn Buchholz, Thomas M. Langer, Matthew R. Hodges, Lawrence Pan, Hubert V. Forster.
    The Journal of Physiology. September 13, 2018
    --- - |2+ Key points summary Chronic hypercapnia per se has distinct effects on mechanisms regulating steady state ventilation and the CO2/H+ chemoreflex. Chronic hypercapnia leads to sustained hyperpnea that exceeds predicted ventilation based upon the CO2/H+ chemoreflex. There is an integrative ventilatory, cardiovascular, and metabolic physiologic response to chronic hypercapnia. Chronic hypercapnia leads to deterioration of cognitive function. Abstract Respiratory diseases such as chronic obstructive pulmonary disease (COPD) often lead to chronic hypercapnia which may exacerbate progression of the disease, increase risk of mortality, and contribute to comorbidities such as cognitive dysfunction. Determining the contribution of hypercapnia per se to adaptations in ventilation and cognitive dysfunction within this patient population is complicated by the presence of multiple comorbidities. Herein, we sought to determine the role of chronic hypercapnia per se on the temporal pattern of ventilation and the ventilatory CO2/H+ chemoreflex by exposing healthy goats to either room air or an elevated inspired CO2 (InCO2) of 6% for 30 days. A second objective was to determine whether chronic hypercapnia per se contributes to cognitive dysfunction. During 30 days of exposure to 6% InCO2, steady state (SS) ventilation (V̇I) initially increased to 335% of control, and then within 1–5 days decreased and stabilized at ∼230% of control. There was an initial respiratory acidosis that was partially mitigated over time due to increased arterial [HCO3−]. There was a transient decrease in the ventilatory CO2/H+ chemoreflex, followed by return to pre‐exposure levels. The SS V̇I during chronic hypercapnia was greater than predicted from the acute CO2/H+ chemoreflex, suggesting separate mechanisms regulating SS V̇I and the chemoreflex. Finally, as assessed by a shape discrimination test, we found a sustained decrease in cognitive function during chronic hypercapnia. We conclude that chronic hypercapnia per se results in: a) a disconnect between SS V̇I and the CO2/H+ chemoreflex, and b) deterioration of cognitive function. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 13, 2018   doi: 10.1113/JP276666   open full text
  • Passive heat therapy protects against endothelial cell hypoxia‐reoxygenation via effects of elevations in temperature and circulating factors.
    Vienna E. Brunt, Karen Wiedenfeld‐Needham, Lindan N. Comrada, Christopher T. Minson.
    The Journal of Physiology. September 13, 2018
    --- - |2+ Key Points Accumulating evidence indicates that passive heat therapy (chronic use of hot tubs or saunas) has widespread physiological benefits, including enhanced resistance against novel stressors (‘stress resistance’). Using a cell culture model to isolate the key stimuli that are likely to underlie physiological adaptation with heat therapy, we showed that both mild elevations in temperature (to 39°C) and exposure to serum from human subjects who have undergone 8 weeks of heat therapy (i.e. altered circulating factors) independently prevented oxidative and inflammatory stress associated with hypoxia‐reoxygenation in cultured endothelial cells. Our results elucidate some of the mechanisms (i.e. direct effects of temperature vs. circulating factors) by which heat therapy seems to improve resistance against oxidative and inflammatory stress. Heat therapy may be a promising intervention for reducing cellular damage following ischaemic events, which has broad implications for patients with cardiovascular diseases and conditions characterized by ‘chronic’ ischaemia (e.g. peripheral artery disease, metabolic diseases, obesity). Abstract Repeated exposure to passive heat stress (‘heat therapy’) has widespread physiological benefits, including cellular protection against novel stressors. Increased heat shock protein (HSP) expression and upregulation of circulating factors may impart this protection. We tested the isolated abilities of mild heat pretreatment and serum from human subjects (n = 10) who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia‐reoxygenation (H/R), a model of ischaemic cardiovascular events. Cultured human umbilical vein endothelial cells were incubated for 24 h at 37°C (control), 39°C (heat pretreatment) or 37°C with 10% serum collected before and after 8 weeks of passive heat therapy (four to five times per week to increase rectal temperature to ≥ 38.5°C for 60 min). Cells were then collected before and after incubation at 1% O2 for 16 h (hypoxia; 37°C), followed by 20% O2 for 4 h (reoxygenation; 37°C) and assessed for markers of cell stress. In control cells, H/R increased nuclear NF‐κB p65 protein (i.e. activation) by 106 ± 38%, increased IL‐6 release by 37 ± 8% and increased superoxide production by 272 ± 45%. Both heat pretreatment and exposure to heat therapy serum prevented H/R‐induced NF‐κB activation and attenuated superoxide production; by contrast, only exposure to serum attenuated IL‐6 release. H/R also decreased cytoplasmic haemeoxygenase‐1 (HO‐1) protein (known to suppress NF‐κB), in control cells (−25 ± 8%), whereas HO‐1 protein increased following H/R in cells pretreated with heat or serum‐exposed, providing a possible mechanism of protection against H/R. These data indicate heat therapy is capable of imparting resistance against inflammatory and oxidative stress via direct heat and humoral factors. - The Journal of Physiology, EarlyView.
    September 13, 2018   doi: 10.1113/JP276559   open full text
  • Pulmonary vascular dysfunction in metabolic syndrome.
    Conor Willson, Makiko Watanabe, Atsumi Tsuji‐Hosokawa, Ayako Makino.
    The Journal of Physiology. September 13, 2018
    --- - |2+ Abstract Metabolic syndrome is a critically important precursor to the onset of many diseases, such as cardiovascular disease, and cardiovascular disease is the leading cause of death worldwide. The primary risk factors of metabolic syndrome include hyperglycaemia, abdominal obesity, dyslipidaemia, and high blood pressure. It has been well documented that metabolic syndrome alters vascular endothelial and smooth muscle cell functions in the heart, brain, kidney and peripheral vessels. However, there is less information available regarding how metabolic syndrome can affect pulmonary vascular function and ultimately increase an individual's risk of developing various pulmonary vascular diseases, such as pulmonary hypertension. Here, we review in detail how metabolic syndrome affects pulmonary vascular function. - The Journal of Physiology, EarlyView.
    September 13, 2018   doi: 10.1113/JP275856   open full text
  • Acute hydrocortisone administration reduces cardiovagal baroreflex sensitivity and heart rate variability in young men.
    Ahmed M. Adlan, Jet J. C. S. Veldhuijzen van Zanten, Gregory Y. H. Lip, Julian F. R. Paton, George D. Kitas, James P. Fisher.
    The Journal of Physiology. September 13, 2018
    --- - |2+ Key points A surge in cortisol during acute physiological and pathophysiological stress may precipitate ventricular arrhythmia and myocardial infarction. Reduced cardiovagal baroreflex sensitivity and heart rate variability are observed during acute stress and are associated with an increased risk of acute cardiac events. In the present study, healthy young men received either a single iv bolus of saline (placebo) or hydrocortisone, 1 week apart, in accordance with a randomized, placebo‐controlled, cross‐over study design. Hydrocortisone acutely increased heart rate and blood pressure and reduced cardiovagal baroreflex sensitivity and heart rate variability in young men. These findings suggest that, by reducing cardiovagal baroreflex sensitivity and heart rate variability, acute surges in cortisol facilitate a pro‐arrhythmic milieu and provide an important mechanistic link between stress and acute cardiac events Abstract Surges in cortisol concentration during acute stress may increase cardiovascular risk. To better understand the interactions between cortisol and the autonomic nervous system, we determined the acute effects of hydrocortisone administration on cardiovagal baroreflex sensitivity (BRS), heart rate variability (HRV) and cardiovascular reactivity. In a randomized, placebo‐controlled, single‐blinded cross‐over study, 10 healthy males received either a single iv bolus of saline (placebo) or 200 mg of hydrocortisone, 1 week apart. Heart rate (HR), blood pressure (BP) and limb blood flow were monitored 3 h later, at rest and during the sequential infusion of sodium nitroprusside and phenylephrine (modified Oxford Technique), a cold pressor test and a mental arithmetic stress task. HRV was assessed using the square root of the mean of the sum of the squares of differences between successive R‐R intervals (rMSSD). Hydrocortisone markedly increased serum cortisol 3 h following infusion and also compared to placebo. In addition, hydrocortisone elevated resting HR (+7 ± 4 beats min−1; P < 0.001) and systolic BP (+5 ± 5 mmHg; P = 0.008); lowered cardiovagal BRS [geometric mean (95% confidence interval) 15.6 (11.1–22.1) ms/mmHg vs. 26.2 (17.4––39.5) ms/mmHg, P = 0.011] and HRV (rMSSD 59 ± 29 ms vs. 84 ± 38 ms, P = 0.004) and increased leg vasoconstrictor responses to cold pressor test (Δ leg vascular conductance −45 ± 20% vs. −23 ± 26%; P = 0.023). In young men, an acute cortisol surge is accompanied by increases in HR and BP, as well as reductions in cardiovagal BRS and HRV, potentially providing a pro‐arrhythmic milieu that may precipitate ventricular arrhythmia or myocardial infarction and increase cardiovascular risk. - The Journal of Physiology, EarlyView.
    September 13, 2018   doi: 10.1113/JP276644   open full text
  • ‘Compliance’ to exercise: how much is really needed for a healthy heart (and mind)?
    Theodore M. DeConne, Joseph M. Stock, Kamila U. Migdal.
    The Journal of Physiology. September 13, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 13, 2018   doi: 10.1113/JP277013   open full text
  • Effect of coil orientation on motor‐evoked potentials in humans with tetraplegia.
    Hang Jin Jo, Vincenzo Di Lazzaro, Monica A. Perez.
    The Journal of Physiology. September 13, 2018
    --- - |2+ Key points Although corticospinal function changes following spinal cord injury (SCI), the extent to which we can activate the corticospinal tract after injury remains poorly understood. To address this question, we used transcranial magnetic stimulation over the hand representation of the primary motor cortex to elicit motor‐evoked potentials (MEPs) using posterior–anterior and anterior–posterior induced currents in the brain and compared them with responses evoked using lateral–medial currents in participants with and without cervical incomplete SCI during small levels of index finger abduction. We found prolonged MEP latencies in all coil orientations in SCI compared to control subjects. However, the latencies of MEPs elicited by posterior–anterior and anterior–posterior compared to lateral–medial stimulation were shorter in SCI compared to controls, particularly for MEPs elicited by anterior–posterior currents. Our findings demonstrate for the first time that corticospinal responses elicited by different directions of the induced current in the brain are differentially affected after SCI. Abstract The corticospinal tract undergoes reorganization following spinal cord injury (SCI). However, the extent to which we can activate corticospinal neurons using non‐invasive stimulation after injury remains poorly understood. To address this question, we used transcranial magnetic stimulation over the hand representation of the primary motor cortex to elicit motor‐evoked potentials (MEPs) using posterior–anterior (PA) and anterior–posterior (AP) induced currents in the brain and compared them with the responses evoked by direct activation of corticospinal axons using lateral–medial (LM) currents. Testing was completed during small levels of index finger abduction in humans with and without (controls) cervical incomplete SCI. We found prolonged MEP latencies in individuals with SCI in all coil orientations compared to controls. However, latencies of MEPs elicited by PA and AP stimulation relative to those elicited by LM stimulation were shorter in SCI compared to control subjects. Notably, the largest difference between SCI and control subjects was present in MEPs elicited by AP currents. Using a novel controllable pulse parameter transcranial magnetic stimulation, we also found that MEPs elicited by AP currents with 30 μs compared to 60 and 120 μs pulse width had increased latency in controls but not in SCI subjects. Our findings demonstrate that differences between corticospinal responses elicited by AP and PA induced currents were not preserved in humans with tetraplegia and suggest that neural structures activated by AP currents change largely after the injury. - The Journal of Physiology, EarlyView.
    September 13, 2018   doi: 10.1113/JP275798   open full text
  • GHSR constitutive activity impairs voltage‐gated calcium channel (CaV)‐dependent inhibitory neurotransmission in hippocampal neurons.
    Martínez Damonte Valentina, Rodríguez Silvia Susana, Raingo Jesica*.
    The Journal of Physiology. September 10, 2018
    --- - |2+ Key Points Presynaptic voltage‐gated calcium channels (CaV2) link action potentials arriving at the presynaptic terminal to neurotransmitter release. Hence, their regulation is essential to fine tuning brain circuitry. CaV2 are highly sensitive to G protein‐coupled receptor (GPCR) modulation. Our previous data indicated that ghrelin receptor GHSR constitutive activity impairs CaV2 by decreasing their surface density. We present compelling support for the impact of CaV2.2 inhibition by agonist‐independent GHSR activity exclusively on GABA release in hippocampal cultures. We found that this selectivity arises from a high reliance of GABA release on CaV2.2 rather than on CaV2.1. Our data provides new information on the effects of the ghrelin/GHSR system on synaptic transmission, suggesting a putative physiological role of the constitutive signaling of a GPCR that is expressed at high levels in brain areas with restricted access to its natural agonist. Abstract GHSR (growth hormone secretagogue receptor) displays high constitutive activity, independent from its endogenous ligand, ghrelin. Unlike ghrelin‐induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic voltage‐gated calcium channels (CaV2). Here we postulate that GHSR constitutive activity‐mediated modulation of CaV2 could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole‐cell patch‐clamp in hippocampal primary cultures from E16‐18 day old C57BL6 wild type and GHSR deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs CaV2.1 and CaV2.2 native calcium currents and that CaV2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher CaV2.2 over CaV2.1 contribution to GABA release (∼40% for CaV2.2 in wild type vs. ∼20% in wild type GHSR overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra‐granule cell inhibitory postsynaptic currents (IPSCs) size ∼ ‐67 pA in wild type vs. ∼ ‐100 pA in GHSR deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a CaV2‐dependent mechanism without affecting glutamatergic transmission. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 10, 2018   doi: 10.1113/JP276256   open full text
  • Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission.
    CA Cobine, M McKechnie, RJ Brookfield, KI Hannigan, KD Keef.
    The Journal of Physiology. September 10, 2018
    --- - |2+ Key points summary Inhibitory NMT was compared in the IAS and rectum of the Cynomolgus monkey; an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+/VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were similar. SK3 channel activation and inhibition caused greater/faster changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in the IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. Abstract Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey; an animal with high gene sequence identity to humans. Electrical field stimulation produced NOS‐dependent contractile inhibition in both muscles whereas P2Y1‐dependent purinergic NMT was restricted to rectum. An additional NOS‐independent, α‐Chymotrypsin‐sensitive component was identified in the IAS consistent with VIPergic NMT. Microelectrode recordings revealed slow NOS‐dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1‐dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+/SK3+ cells were closely aligned with nNOS+/VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2‐14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was 5 times faster in rectum than the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 10, 2018   doi: 10.1113/JP275437   open full text
  • To clot or not to clot? That is a free radical question.
    Daniel R. Crabtree, David Muggeridge, Stephen J. Leslie, Ian L. Megson, James N. Cobley.
    The Journal of Physiology. September 10, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 10, 2018   doi: 10.1113/JP276786   open full text
  • Nutritional programming by maternal obesity: insights into the development of non‐alcoholic fatty liver disease.
    Judy Ghalayini, Shin‐Hann Lee.
    The Journal of Physiology. September 10, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 10, 2018   doi: 10.1113/JP276965   open full text
  • Optimal dissection of a model circuit.
    Takeshi Sakaba.
    The Journal of Physiology. September 10, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 10, 2018   doi: 10.1113/JP276895   open full text
  • Chronic aerobic exercise reduces the aortic age of an elderly cohort.
    Mina Amin Iskandar, Tharmegan Tharmaratnam, Zunair Ahmad.
    The Journal of Physiology. September 10, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 10, 2018   doi: 10.1113/JP276961   open full text
  • Rac1 supports muscle glucose uptake independently of Akt.
    Daniel M. Marko, Hesham Shamshoum.
    The Journal of Physiology. September 09, 2018
    --- - - The Journal of Physiology, EarlyView.
    September 09, 2018   doi: 10.1113/JP276851   open full text
  • Sodium and potassium conductances in principal neurons of the mouse piriform cortex: A quantitative description.
    Kaori Ikeda, Norimitsu Suzuki, John M. Bekkers.
    The Journal of Physiology. September 08, 2018
    --- - |2+ Key points The primary olfactory (or piriform) cortex is a promising model system for understanding how the cerebral cortex processes sensory information, but study of the piriform cortex is hindered by a lack of detailed information about the intrinsic electrical properties of its component neurons. Here we quantify the properties of voltage‐dependent sodium currents and voltage‐ and calcium‐dependent potassium currents in two important classes of excitatory neurons in the main input layer of the piriform cortex. We identify several classes of these currents and show that their properties are similar to those found in better‐studied cortical regions. Our detailed quantitative descriptions of these currents will be valuable to computational neuroscientists wishing to build models that explain how the piriform cortex encodes odours. Abstract The primary olfactory cortex (or piriform cortex, PC) is an anatomically simple palaeocortex that is increasingly used as a model system for studying cortical sensory processing. However, little information is available on the intrinsic electrical conductances in neurons of the PC, hampering efforts to build realistic computational models of this cortex. Here we used nucleated macropatches and whole‐cell recordings to rigorously quantify the biophysical properties of voltage‐gated sodium (NaV), voltage‐gated potassium (KV) and calcium‐activated potassium (KCa) conductances in two major classes of glutamatergic neurons in layer 2 of the PC, semilunar (SL) cells and superficial pyramidal (SP) cells. We found that SL and SP cells both express a fast‐inactivating NaV current, two types of KV current (A‐type and delayed rectifier‐type) and three types of KCa current (fast‐, medium‐ and slow‐afterhyperpolarization currents). The kinetic and voltage dependent properties of the NaV and KV conductances were, with some exceptions, identical in SL and SP cells and similar to those found in neocortical pyramidal neurons. The KCa conductances were also similar across the different types of neurons. Our results are summarised in a series of empirical equations that should prove useful to computational neuroscientists seeking to model the PC. More broadly, our findings indicate that, at the level of single‐cell electrical properties, this palaeocortex is not so different from the neocortex, vindicating efforts to use the PC as a model of cortical sensory processing in general. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 08, 2018   doi: 10.1113/JP275824   open full text
  • The application of stable‐isotope tracers to study human musculoskeletal protein turnover: a tale of bag filling and bag enlargement.
    D. Joe Millward, Ken Smith.
    The Journal of Physiology. September 07, 2018
    --- - |2+ Abstract The nutritional regulation of protein and amino acid balance in human skeletal muscle carried out by the authors with Mike Rennie is reviewed in the context of a simple physiological model for the regulation of the maintenance and growth of skeletal muscle, the “Bag Theory”. Beginning in London in the late 1970s the work has involved the use of stable isotopes to probe muscle protein synthesis and breakdown with two basic experimental models, primed‐dose continuous tracer infusions combined with muscle biopsies and arterio‐venous (A‐V) studies across a limb, most often the leg, allowing both protein synthesis and breakdown as well as net balance to be measured. In this way, over a 30 year period, the way in which amino acids and insulin mediate the anabolic effect of a meal has been elaborated in great detail confirming the original concepts of bag filling within the muscle endomysial “bag”, which is limited by the “bag” size unless bag enlargement occurs requiring new collagen synthesis. Finally we briefly review some new developments involving 2H2O labelling of muscle proteins. - The Journal of Physiology, EarlyView.
    September 07, 2018   doi: 10.1113/JP275430   open full text
  • A systems perspective on placental amino acid transport.
    Jane K. Cleal, Emma M. Lofthouse, Bram G. Sengers, Rohan M. Lewis.
    The Journal of Physiology. September 07, 2018
    --- - |2+ Abstract Placental amino acid transfer is a complex process that is essential for fetal development. Impaired amino acid transfer causes fetal growth restriction, which may have lifelong health consequences. Transepithelial transfer of amino acids across the placental syncytiotrophoblast requires accumulative, exchange and facilitated transporters on the apical and basal membranes to work in concert. However, transporters alone do not determine amino acid transfer and factors that affect substrate availability, such as blood flow and metabolism, may also become rate‐limiting for transfer. In order to determine the rate‐limiting processes, it is necessary to take a systems approach which recognises the interdependence of these processes. New technologies have the potential to deliver targeted interventions to the placenta and help poorly growing fetuses. While many factors are necessary for amino acid transfer, novel therapies need to target the rate‐limiting factors if they are going to be effective. This review will outline the factors which determine amino acid transfer and describe how they become interdependent. It will also highlight the role of computational modelling as a tool to understand this process. - The Journal of Physiology, EarlyView.
    September 07, 2018   doi: 10.1113/JP274883   open full text
  • ERG3 potassium channel‐mediated suppression of neuronal intrinsic excitability and prevention of seizure generation in mice.
    Kuo Xiao, Zhiming Sun, Xueqin Jin, Weining Ma, Yan Song, Shirong Lai, Qian Chen, Minghua Fan, Jingliang Zhang, Weihua Yue, Zhuo Huang.
    The Journal of Physiology. September 07, 2018
    --- - |2+ Key points ERG3 channels have a high expression level in the central nervous system. Knockdown of ERG3 channels enhances neuronal intrinsic excitability (caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials) in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. The expression of ERG3 protein is reduced in human and mouse hippocampal epileptogenic foci. Knockdown of ERG3 channels in hippocampus enhanced seizure susceptibility, while mice treated with the ERG channel activator NS‐1643 were less prone to epileptogenesis. The results provide strong evidence that ERG3 channels have a crucial role in the regulation of neuronal intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells and are critically involved in the onset and development of epilepsy. Abstract The input–output relationship of neuronal networks depends heavily on the intrinsic properties of their neuronal elements. Profound changes in intrinsic properties have been observed in various physiological and pathological processes, such as learning, memory and epilepsy. However, the cellular and molecular mechanisms underlying acquired changes in intrinsic excitability are still not fully understood. Here, we demonstrate that ERG3 channels are critically involved in the regulation of intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Knock‐down of ERG3 channels significantly increases neuronal intrinsic excitability, which is mainly caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials. Interestingly, the expression level of ERG3 protein is significantly reduced in human and mouse brain tissues with temporal lobe epilepsy. Moreover, ERG3 channel knockdown in hippocampus significantly enhanced seizure susceptibility, while mice treated with the ERG channel activator NS‐1643 were less prone to epileptogenesis. Taken together, our results suggest ERG3 channels play an important role in determining the excitability of hippocampal neurons and dysregulation of these channels may be involved in the generation of epilepsy. ERG3 channels may thus be a novel therapeutic target for the prevention of epilepsy. - The Journal of Physiology, Volume 596, Issue 19, Page 4729-4752, 1 October 2018.
    September 07, 2018   doi: 10.1113/JP275970   open full text
  • A mother's gift: consequences of unhealthy diet for offspring metabolism.
    Elisa Karen Silva Ramos, Paola Visnardi Fassina, Michelle Andrade Lemos.
    The Journal of Physiology. September 07, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4575-4577, 1 October 2018.
    September 07, 2018   doi: 10.1113/JP276769   open full text
  • The importance of exercise intensity, volume and metabolic signalling events in the induction of mitochondrial biogenesis.
    Heather L. Petrick, Kaitlyn M. J. H. Dennis, Paula M. Miotto.
    The Journal of Physiology. September 07, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4571-4572, 1 October 2018.
    September 07, 2018   doi: 10.1113/JP276802   open full text
  • Oestrogen and a Goldilocks zone for post‐damage muscle inflammation and repair?
    Peter M. Tiidus.
    The Journal of Physiology. September 07, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4563-4564, 1 October 2018.
    September 07, 2018   doi: 10.1113/JP276870   open full text
  • Finding the metabolic stress ‘sweet spot’: implications for sprint interval training‐induced muscle remodelling.
    Lauren E. Skelly, Jenna B. Gillen.
    The Journal of Physiology. September 06, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4573-4574, 1 October 2018.
    September 06, 2018   doi: 10.1113/JP276912   open full text
  • Sex differences in diaphragmatic fatigue and the metaboreflex following inspiratory pressure‐threshold loading.
    Christina D. Bruce, Alexandra F. Yacyshyn, Luca Ruggiero.
    The Journal of Physiology. September 05, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4579-4580, 1 October 2018.
    September 05, 2018   doi: 10.1113/JP276978   open full text
  • The role of the C‐terminus of SUR in the differential regulation of β‐cell and cardiac KATP channels by MgADP and metabolism.
    Natascia Vedovato, Olof Rorsman, Konstantin Hennis, Frances Ashcroft, Peter Proks.
    The Journal of Physiology. September 04, 2018
    --- - |2+ Key points β‐cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration‐inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 then Kir6.2/SUR2A channels; an effect that declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg‐nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C‐terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg‐nucleotides, which is determined by the final 42 amino acids. Abstract ATP‐sensitive potassium (KATP) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to β‐cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. It is known this is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 μm MgADP immediately after patch excision (when the channel open probability (PO) is near maximal). The results were strikingly different: 100 μm MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and v.v.), and deleting this region abolished Mg‐nucleotide activation. This suggests the C‐terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, although, not sufficient for activation of the KATP channel in the intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of β‐cell and cardiac KATP channels. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 04, 2018   doi: 10.1113/JP276708   open full text
  • Neural memory of the genioglossus muscle during sleep is stage‐dependent in healthy subjects and obstructive sleep apnoea patients.
    Luigi Taranto‐Montemurro, Scott A. Sands, Kevin P. Grace, Ali Azarbarzin, Ludovico Messineo, Rebecca Salant, David P. White, D. Andrew Wellman.
    The Journal of Physiology. September 04, 2018
    --- - |2+ Key points In most patients with obstructive sleep apnoea (OSA), there is a spontaneous resolution of the breathing disorders during slow wave sleep (SWS) for yet unknown reasons related to non‐anatomical factors. Some recently identified forms of neural memory specific of upper airway muscles may play a role in this phenomenon. In the present study, we show for the first time that a form of memory of the genioglossus (tongue) muscle is greatly enhanced during SWS compared to non‐rapid eye movement stage 2 sleep. The present study represents a step forward in understanding the mechanisms responsible for the spontaneous development of stable breathing during SWS in OSA patients and may help the discovery of novel therapeutic strategies for this disease. Abstract Several studies have shown that obstructive sleep apnoea (OSA) improves during slow wave sleep (SWS) for reasons that remain unclear. Recent studies have identified forms of neural memory such as short‐term potentiation or after‐discharge that can occur in response to upper airway obstruction. Neural memory may play a role in the development of stable breathing during SWS by increasing upper airway muscles activity in this sleep stage. We hypothesize that the after‐discharge of the genioglossus muscle following upper airway obstruction is enhanced during SWS compared to non‐rapid eye movement stage 2 (N2). During sleep, we performed five‐breath drops in continuous positive airway pressure (CPAP‐drop) to simulate obstructive events and reflexively activate the genioglossus. Immediately afterwards, CPAP was returned to an optimal level. Once the post‐drop ventilation returned to eupnoea, the genioglossus after‐discharge was measured as the time it took for genioglossus activity to return to baseline levels. In total, 171 CPAP‐drops were analysed from a group of 16 healthy subjects and 19 OSA patients. A mixed‐model analysis showed that after‐discharge duration during SWS was 208% (95% confidence interval = 112% to 387%, P = 0.022) greater than during N2 after adjusting for covariates (ventilatory drive, CPAP levels). There was also a non‐significant trend for a –35% reduction in after‐discharge duration following an arousal vs. no‐arousal from sleep (95% confidence interval = –59.5% to 5%, P = 0.08). Genioglossus after‐discharge is two‐fold greater in SWS vs. N2, which could partly explain the breathing stabilization described in OSA patients during this sleep stage. - The Journal of Physiology, EarlyView.
    September 04, 2018   doi: 10.1113/JP276618   open full text
  • Deep continuous theta burst stimulation of the operculo‐insular cortex selectively affects Aδ‐fibre heat pain.
    Cédric Lenoir, Maxime Algoet, André Mouraux.
    The Journal of Physiology. September 04, 2018
    --- - |2+ Key points Deep continuous theta burst stimulation (cTBS) of the right operculo‐insular cortex delivered with a double cone coil selectively impairs the ability to perceive thermonociceptive input conveyed by Aδ‐fibre thermonociceptors without concomitantly affecting the ability to perceive innocuous warm, cold or vibrotactile sensations. Unlike deep cTBS, superficial cTBS of the right operculum delivered with a figure‐of‐eight coil does not affect the ability to perceive thermonociceptive input conveyed by Aδ‐fibre thermonociceptors. The effect of deep operculo‐insular cTBS on the perception of Aδ‐fibre input was present at both the contralateral and the ipsilateral hand. The magnitude of the increase in Aδ‐heat detection threshold induced by the deep cTBS was significantly correlated with the intensity of the cTBS pulses. Deep cTBS delivered over the operculo‐insular cortex is associated with a risk of transcranial magnetic stimulation‐induced seizure. Abstract Previous studies have suggested a pivotal role of the insular cortex in nociception and pain perception. Using a double‐cone coil designed for deep transcranial magnetic stimulation, our objective was to assess (1) whether continuous theta burst stimulation (cTBS) of the operculo‐insular cortex affects differentially the perception of different types of thermal and mechanical somatosensory inputs, (2) whether the induced after‐effects are lateralized relative to the stimulated hemisphere, and (3) whether the after‐effects are due to neuromodulation of the insula or neuromodulation of the more superficial opercular cortex. Seventeen participants took part in two experiments. In Experiment 1, thresholds and perceived intensity of Aδ‐ and C‐fibre heat pain elicited by laser stimulation, non‐painful cool sensations elicited by contact cold stimulation and mechanical vibrotactile sensations were assessed at the left hand before, immediately after and 20 min after deep cTBS delivered over the right operculo‐insular cortex. In Experiment 2, Aδ‐fibre heat pain and vibrotactile sensations elicited by stimulating the contralateral and ipsilateral hands were evaluated before and after deep cTBS or superficial cTBS delivered using a flat figure‐of‐eight coil. Only the threshold to detect Aδ‐fibre heat pain was significantly increased 20 min after deep cTBS. This effect was present at both hands. No effect was observed after superficial cTBS. Neuromodulation of the operculo‐insular cortex using deep cTBS induces a bilateral reduction of the ability to perceive Aδ‐fibre heat pain, without concomitantly affecting the ability to perceive innocuous warm, cold or vibrotactile sensations. - The Journal of Physiology, Volume 596, Issue 19, Page 4767-4787, 1 October 2018.
    September 04, 2018   doi: 10.1113/JP276359   open full text
  • In vivo evidence for reduced ion channel expression in motor axons of patients with amyotrophic lateral sclerosis.
    James Howells, José Manuel Matamala, Susanna B Park, Nidhi Garg, Steve Vucic, Hugh Bostock, David Burke, Matthew C Kiernan.
    The Journal of Physiology. September 03, 2018
    --- - |2+ Amyotrophic lateral sclerosis (ALS) is characterised by a progressive loss of motor units and reinnervation of denervated muscle fibres by surviving motor axons preserves muscle function until symptom onset, when some 60–80% of motor units have been lost. A disruption in protein homeostasis is thought to play a critical role in the pathogenesis of ALS. We have studied the changes in surviving motor neurons by comparing the nerve excitability properties of 31 single motor axons from patients with ALS with those from 21 single motor axons in control subjects. A mathematical model indicated that approximately 99% of the differences between the ALS and control units could be explained by a non‐selective reduction in the expression of all ion channels. These changes in ALS patients are best explained by a failure in the supply of ion channel and other membrane proteins from the diseased motor neuron. Abstract Amyotrophic lateral sclerosis (ALS) is characterised by a progressive loss of motor units and the reinnervation of denervated muscle fibres by surviving motor axons. This reinnervation preserves muscle function until symptom onset, when some 60–80% of motor units have been lost. We have studied the changes in surviving motor neurons by comparing the nerve excitability properties of 31 single motor axons from patients with ALS with those from 21 single motor axons in control subjects. ALS motor axons were classified as coming from moderately or severely affected muscles according to the compound muscle action potential amplitude of the parent muscle. Compared with control units, thresholds were increased, and there was reduced inward and outward rectification and greater superexcitability following a conditioning impulse. These abnormalities were greater in axons from severely affected muscles, and were correlated with loss of fine motor skills. A mathematical model indicated that 99.1% of the differences between the moderately affected ALS and control units could be explained by a reduction in the expression of all ion channels. For the severely affected units, modelling required, in addition, an increase in the current leak through and under the myelin sheath. This might be expected if the anchoring proteins responsible for the paranodal seal were reduced. We conclude that changes in axonal excitability identified in ALS patients are best explained by a failure in the supply of ion channel and other membrane proteins from the diseased motor neuron, a conclusion consistent with recent animal and in vitro human data. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.
    September 03, 2018   doi: 10.1113/JP276624   open full text
  • Evolving systems biology approaches to understanding non‐coding RNAs in pulmonary hypertension.
    Lloyd D. Harvey, Stephen Y. Chan.
    The Journal of Physiology. September 03, 2018
    --- - |2 Abstract Our appreciation of the roles of non‐coding RNAs, in particular microRNAs, in the manifestation of pulmonary hypertension (PH) has advanced considerably over the past decade. Comprised of small nucleotide sequences, microRNAs have demonstrated critical and broad regulatory roles in the pathogenesis of PH via the direct binding to messenger RNA transcripts for degradation or inhibition of translation, thereby exerting a profound influence on cellular activity. Yet, as inherently pleiotropic molecules, microRNAs have been difficult to study using traditional, reductionist approaches alone. With the advent of high‐throughput –omics technologies and more advanced computational modelling, the study of microRNAs and their multi‐faceted and complex functions in human disease serves as a fertile platform for the application of systems biology methodologies in combination with traditional experimental techniques. Here, we offer our viewpoint of past successes of systems biology in elucidating the otherwise hidden actions of microRNAs in PH, as well as areas for future development to integrate these strategies into the discovery of RNA pathobiology in this disease. We contend that such successful applications of systems biology in elucidating the functional architecture of microRNA regulation will further reveal the molecular mechanisms of disease, while simultaneously revealing potential diagnostic and therapeutic strategies in disease amelioration. - The Journal of Physiology, EarlyView.
    September 03, 2018   doi: 10.1113/JP275855   open full text
  • Ultrastructural basis of strong unitary inhibition in a binaural neuron.
    Enida Gjoni, Clémentine Aguet, Daniela A. Sahlender, Graham Knott, Ralf Schneggenburger.
    The Journal of Physiology. September 03, 2018
    --- - |2+ Key points Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1–11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. Abstract Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr2+ method, we find a constant quantal size but a variable quantal content between ‘weak’ and ‘strong’ unitary IPSCs. A 3‐D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7 μm), which contain several active zones (range 1–11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi‐active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons. - The Journal of Physiology, EarlyView.
    September 03, 2018   doi: 10.1113/JP276015   open full text
  • Fatigue‐related group III/IV muscle afferent feedback facilitates intracortical inhibition during locomotor exercise.
    Simranjit K. Sidhu, Joshua C. Weavil, Taylor S. Thurston, Dorothea Rosenberger, Jacob E. Jessop, Eivind Wang, Russell S. Richardson, Chris J. McNeil, Markus Amann.
    The Journal of Physiology. September 03, 2018
    --- - |2+ Key Points This study investigated the influence of group III/IV muscle afferents on corticospinal excitability during cycling exercise and focused on GABAB neuron‐mediated inhibition as a potential underlying mechanism. The study provides novel evidence to demonstrate that group III/IV muscle afferent feedback facilitates inhibitory intracortical neurons during whole body exercise. Firing of these interneurons probably contributes to the development of central fatigue during physical activity. Abstract We investigated the influence of group III/IV muscle afferents in determining corticospinal excitability during cycling exercise and focused on GABAB neuron‐mediated inhibition as a potential underlying mechanism. Both under control conditions (CTRL) and with lumbar intrathecal fentanyl (FENT) impairing feedback from group III/IV leg muscle afferents, subjects (n = 11) cycled at a comparable vastus‐lateralis EMG signal (∼0.26 mV) before (PRE; 100 W) and immediately after (POST; 90 ± 2 W) fatiguing constant‐load cycling exercise (80% Wpeak; 221 ± 10 W; ∼8 min). During, PRE and POST cycling, single and paired‐pulse (100 ms interstimulus interval) transcranial magnetic stimulations (TMS) were applied to elicit unconditioned and conditioned motor‐evoked potentials (MEPs), respectively. To distinguish between cortical and spinal contributions to the MEPs, cervicomedullary stimulations (CMS) were used to elicit unconditioned (CMS only) and conditioned (TMS+CMS, 100 ms interval) cervicomedullary motor‐evoked potentials (CMEPs). While unconditioned MEPs were unchanged from PRE to POST in CTRL, unconditioned CMEPs increased significantly, resulting in a decrease in unconditioned MEP/CMEP (P < 0.05). This paralleled a reduction in conditioned MEP (P < 0.05) and no change in conditioned CMEP. During FENT, unconditioned and conditioned MEPs and CMEPs were similar and comparable during PRE and POST (P > 0.2). These findings reveal that feedback from group III/IV muscle afferents innervating locomotor muscle decreases the excitability of the motor cortex during fatiguing cycling exercise. This impairment is, at least in part, determined by the facilitating effect of these sensory neurons on inhibitory GABAB intracortical interneurons. - The Journal of Physiology, Volume 596, Issue 19, Page 4789-4801, 1 October 2018.
    September 03, 2018   doi: 10.1113/JP276460   open full text
  • Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain.
    Shinichiro Kume, Takushi Shimomura, Michihiro Tateyama, Yoshihiro Kubo.
    The Journal of Physiology. September 03, 2018
    --- - |2+ Key points In the human ether‐a‐go‐go related gene (hERG) channel, both the ether‐a‐go‐go (EAG) domain in the N‐terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C‐terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C‐linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C‐linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage‐dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C‐terminal domains is a result of the lack of interaction between the EAG and CNBH domains. Abstract The human ether‐a‐go‐go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N‐terminal cytoplasmic ether‐a‐go‐go (EAG) domain and the C‐terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C‐linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter‐domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C‐linker domain, which is reported to be critical for the function of CN‐regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter‐domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage‐dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain. - The Journal of Physiology, Volume 596, Issue 19, Page 4629-4650, 1 October 2018.
    September 03, 2018   doi: 10.1113/JP276208   open full text
  • Phase waves and trigger waves: emergent properties of oscillating and excitable networks in the gut.
    Sean P. Parsons, Jan D. Huizinga.
    The Journal of Physiology. August 31, 2018
    --- - |2 Abstract The gut is enmeshed by a number of cellular networks, but there is only a limited understanding of how these networks generate the complex patterns of activity that drive gut contractile functions. Here we review two fundamental types of cell behaviour, excitable and oscillating, and the patterns that networks of such cells generate, trigger waves and phase waves, respectively. We use both the language of biophysics and the theory of nonlinear dynamics to define these behaviours and understand how they generate patterns. Based on this we look for evidence of trigger and phase waves in the gut, including some of our recent work on the small intestine. - The Journal of Physiology, EarlyView.
    August 31, 2018   doi: 10.1113/JP273425   open full text
  • Keto diets: good, bad or ugly?
    Mark Evans.
    The Journal of Physiology. August 31, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4561-4561, 1 October 2018.
    August 31, 2018   doi: 10.1113/JP276703   open full text
  • Blocking slow exocytosis with slow Ca2+ buffers slows recovery from depression.
    Skyler Jackman, Henrique Gersdorff.
    The Journal of Physiology. August 31, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4555-4557, 1 October 2018.
    August 31, 2018   doi: 10.1113/JP276673   open full text
  • Redox‐regulation of haemostasis in hypoxic exercising humans: a randomised double‐blind placebo‐controlled antioxidant study.
    Lewis Fall, Julien V. Brugniaux, Danielle Davis, Christopher J. Marley, Bruce Davies, Karl J. New, Jane McEneny, Ian S. Young, Damian M. Bailey.
    The Journal of Physiology. August 29, 2018
    --- - |2+ Key points In vitro evidence has identified that coagulation is activated by increased oxidative stress, though the link and underlying mechanism in humans have yet to be established. We conducted the first randomised controlled trial in healthy participants to examine if oral antioxidant prophylaxis alters the haemostatic responses to hypoxia and exercise given their synergistic capacity to promote free radical formation. Systemic free radical formation was shown to increase during hypoxia and was further compounded by exercise, responses that were attenuated by antioxidant prophylaxis. In contrast, antioxidant prophylaxis increased thrombin generation at rest in normoxia, and this was normalised only in the face of prevailing oxidation. Collectively, these findings suggest that human free radical formation is an adaptive phenomenon that serves to maintain vascular haemostasis. Abstract In vitro evidence suggests that blood coagulation is activated by increased oxidative stress although the link and underlying mechanism in humans have yet to be established. We conducted the first randomised controlled trial to examine if oral antioxidant prophylaxis alters the haemostatic responses to hypoxia and exercise. Healthy males were randomly assigned double‐blind to either an antioxidant (n = 20) or placebo group (n = 16). The antioxidant group ingested two capsules/day that each contained 500 mg of l‐ascorbic acid and 450 international units (IU) of dl‐α‐tocopherol acetate for 8 weeks. The placebo group ingested capsules of identical external appearance, taste and smell (cellulose). Both groups were subsequently exposed to acute hypoxia and maximal physical exercise with venous blood sampled pre‐supplementation (normoxia), post‐supplementation at rest (normoxia and hypoxia) and following maximal exercise (hypoxia). Systemic free radical formation (electron paramagnetic resonance spectroscopic detection of the ascorbate radical (A•−)) increased during hypoxia (15,152 ± 1193 AU vs. 14,076 ± 810 AU at rest, P < 0.05) and was further compounded by exercise (16,569 ± 1616 AU vs. rest, P < 0.05), responses that were attenuated by antioxidant prophylaxis. In contrast, antioxidant prophylaxis increased thrombin generation as measured by thrombin–antithrombin complex, at rest in normoxia (28.7 ± 6.4 vs. 4.3 ± 0.2 μg mL−1 pre‐intervention, P < 0.05) and was restored but only in the face of prevailing oxidation. Collectively, these findings are the first to suggest that human free radical formation likely reflects an adaptive response that serves to maintain vascular haemostasis. - The Journal of Physiology, EarlyView.
    August 29, 2018   doi: 10.1113/JP276414   open full text
  • Maternal obesity has sex‐dependent effects on insulin, glucose and lipid metabolism and the liver transcriptome in young adult rat offspring.
    Consuelo Lomas‐Soria, Luis A. Reyes‐Castro, Guadalupe L. Rodríguez‐González, Carlos A. Ibáñez, Claudia J. Bautista, Laura A. Cox, Peter W. Nathanielsz, Elena Zambrano.
    The Journal of Physiology. August 29, 2018
    --- - |2+ Key points Maternal high‐fat diet consumption predisposes to metabolic dysfunction in male and female offspring at young adulthood. Maternal obesity programs non‐alcoholic fatty liver disease (NAFLD) in a sex‐dependent manner. We demonstrate sex‐dependent liver transcriptome profiles in rat offspring of obese mothers. In this study, we focused on pathways related to insulin, glucose and lipid signalling. These results improve understanding of the mechanisms by which a maternal high‐fat diet affects the offspring. Abstract Maternal obesity (MO) predisposes offspring (F1) to obesity, insulin resistance (IR) and non‐alcoholic fatty liver disease (NAFLD). MO's effects on the F1 liver transcriptome are poorly understood. We used RNA‐seq to determine the liver transcriptome of male and female F1 of MO and control‐fed mothers. We hypothesized that MO‐F1 are predisposed to sex‐dependent adult liver dysfunction. Female Wistar rat mothers ate a control (C) or obesogenic (MO) diet from the time they were weaned through breeding at postnatal day (PND) 120, delivery and lactation. After weaning, all male and female F1 ate a control diet. At PND 110, F1 serum, liver and fat were collected to analyse metabolites, histology and liver differentially expressed genes. Male and female MO‐F1 showed increased adiposity index, triglycerides, insulin and homeostatic model assessment vs. C‐F1 with similar body weight and glucose serum concentrations. MO‐F1 males presented greater physiological and histological NAFLD characteristics than MO‐F1 females. RNA‐seq revealed 1365 genes significantly changed in male MO‐F1 liver and only 70 genes in female MO‐F1 compared with controls. GO and KEGG analysis identified differentially expressed genes related to metabolic processes. Male MO‐F1 liver showed the following altered pathways: insulin signalling (22 genes), phospholipase D signalling (14 genes), NAFLD (13 genes) and glycolysis/gluconeogenesis (7 genes). In contrast, few genes were altered in these pathways in MO‐F1 females. In summary, MO programs sex‐dependent F1 changes in insulin, glucose and lipid signalling pathways, leading to liver dysfunction and insulin resistance. - The Journal of Physiology, Volume 596, Issue 19, Page 4611-4628, 1 October 2018.
    August 29, 2018   doi: 10.1113/JP276372   open full text
  • The muscle anabolic effect of protein ingestion during a hyperinsulinaemic euglycaemic clamp in middle‐aged women is not caused by leucine alone.
    Stephan Vliet, Gordon I. Smith, Lane Porter, Raja Ramaswamy, Dominic N. Reeds, Adewole L. Okunade, Jun Yoshino, Samuel Klein, Bettina Mittendorfer.
    The Journal of Physiology. August 29, 2018
    --- - |2+ Key points It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic‐euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased muscle p‐eIF2αSer51 and increased muscle protein synthesis. Abstract It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR‐p70S6K signalling cascade. We tested this hypothesis by measuring muscle p‐mTORSer2448, p‐p70S6KThr389 and p‐eIF2αSer51, as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45 g protein kg−1 fat‐free mass (containing 0.0513 g leucine kg−1 fat‐free mass) or a control drink (n = 14) or 0.0513 g leucine kg−1 fat‐free mass or a control drink (n = 14) during a hyperinsulinaemic‐euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p‐mTORSer2448 and p‐p70S6KThr389 by >50% (all P < 0.05) but had no effect on p‐eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p‐mTORSer2448 and p‐p70S6KThr389, although only protein, and not leucine, ingestion decreased (by ∼35%) p‐eIF2αSer51 and increased (by ∼100%) MPS (all P < 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation. - The Journal of Physiology, Volume 596, Issue 19, Page 4681-4692, 1 October 2018.
    August 29, 2018   doi: 10.1113/JP276504   open full text
  • Legacy of excess: consequences of maternal obesity for the adult offspring.
    Alison J. Forhead.
    The Journal of Physiology. August 29, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4559-4560, 1 October 2018.
    August 29, 2018   doi: 10.1113/JP276762   open full text
  • Age‐dependent effects on sympathetic responsiveness in cardiac action potential conduction and calcium handling.
    Xavier Alexander Lee, Neal Ingraham Callaghan.
    The Journal of Physiology. August 29, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4569-4570, 1 October 2018.
    August 29, 2018   doi: 10.1113/JP276950   open full text
  • Spinal dorsal horn astrocytes release GABA in response to synaptic activation.
    Rasmus Kordt Christensen, Rodolfo Delgado‐Lezama, Raúl E. Russo, Barbara Lykke Lind, Emanuel Loeza Alcocer, Martin Fredensborg Rath, Gabriela Fabbiani, Nicole Schmitt, Martin Lauritzen, Anders Victor Petersen, Eva Meier Carlsen, Jean‐François Perrier.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Key points GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing. Abstract Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red‐eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABAA receptors. By positioning the sensor recorded in the whole‐cell patch‐clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca2+, we concluded that they originated from non‐neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca2+ increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca2+ in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs. - The Journal of Physiology, EarlyView.
    August 28, 2018   doi: 10.1113/JP276562   open full text
  • Hepatic mitochondrial adaptations to physical activity: impact of sexual dimorphism, PGC1α and BNIP3‐mediated mitophagy.
    Alex Schulze, Colin S. McCoin, Chiemela Onyekere, Julie Allen, Paige Geiger, Gerald W. Dorn, E. Matthew Morris, John P. Thyfault.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Key points Hepatic mitochondrial adaptations to physical activity may be regulated by mitochondrial biogenesis (PGC1α) and mitophagy (BNIP3). Additionally, these adaptations may be sex‐dependent. Chronic increase in physical activity lowers basal mitochondrial respiratory capacity in mice. Female mice have higher hepatic electron transport system protein content, elevated respiratory capacity, lowered mitophagic flux, and emit less mitochondrial H2O2 independent of physical activity. Males require chronic daily physical activity to attain a similar mitochondrial phenotype compared to females. In contrast, females have limited hepatic adaptations to chronic physical activity. Livers deficient in PGC1α and BNIP3 display similar mitochondrial adaptations to physical activity to those found in wild‐type mice. Abstract Hepatic mitochondrial adaptations to physical activity may be regulated by biogenesis‐ and mitophagy‐associated pathways in a sex‐dependent manner. Here, we tested if mice with targeted deficiencies in liver‐specific peroxisome proliferator‐activated receptor γ coactivator 1α (PGC1α; LPGC1α+/−) and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3)‐mediated mitophagy (BNIP3−/−) would have reduced physical activity‐induced adaptations in respiratory capacity, H2O2 emission and mitophagy compared to wild‐type (WT) controls and if these effects were impacted by sex. Male and female WT, LPGC1α+/− and BNIP3−/− C57BL6/J mice were divided into groups that remained sedentary or had access to daily physical activity via voluntary wheel running (VWR) (n = 6–10/group) for 4 weeks. Mice had ad libitum access to low‐fat diet and water. VWR reduced basal mitochondrial respiration, increased mitochondrial coupling and altered ubiquitin‐mediated mitophagy in a sex‐specific manner in WT mice. Female mice of all genotypes displayed higher electron transport system content, displayed increased ADP‐stimulated respiration, produced less mitochondrially derived reactive oxygen species, exhibited reduced mitophagic flux, and were less responsive to VWR compared to males. Males responded more robustly to VWR‐induced changes in hepatic mitochondrial function resulting in a match to adaptations found in females. Deficiencies in PGC1α and BNIP3 alone did not largely alter mitochondrial adaptations to VWR. However, VWR restored sex‐dependent abnormalities in mitophagic flux in LPGC1α+/−. Finally, BNIP3−/− mice had elevated mitochondrial content and increased mitochondrial respiration putatively through repressed mitophagic flux. In conclusion, hepatic mitochondrial adaptations to physical activity are more dependent on sex than PGC1α and BNIP3. - The Journal of Physiology, EarlyView.
    August 28, 2018   doi: 10.1113/JP276539   open full text
  • Short‐chain fatty acids: microbial metabolites that alleviate stress‐induced brain–gut axis alterations.
    Marcel de Wouw, Marcus Boehme, Joshua M. Lyte, Niamh Wiley, Conall Strain, Orla O'Sullivan, Gerard Clarke, Catherine Stanton, Timothy G. Dinan, John F. Cryan.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Key points Chronic (psychosocial) stress changes gut microbiota composition, as well as inducing behavioural and physiological deficits. The microbial metabolites short‐chain fatty acids (SCFAs) have been implicated in gastrointestinal functional, (neuro)immune regulation and host metabolism, but their role in stress‐induced behavioural and physiological alterations is poorly understood. Administration of SCFAs to mice undergoing psychosocial stress alleviates enduring alterations in anhedonia and heightened stress‐responsiveness, as well as stress‐induced increases in intestinal permeability. In contrast, chronic stress‐induced alterations in body weight gain, faecal SCFAs and the gene expression of the SCFA receptors FFAR2 and FFAR3 remained unaffected by SCFA supplementation. These results present novel insights into mechanisms underpinning the influence of the gut microbiota on brain homeostasis, behaviour and host metabolism, informing the development of microbiota‐targeted therapies for stress‐related disorders. Abstract There is a growing recognition of the involvement of the gastrointestinal microbiota in the regulation of physiology and behaviour. Microbiota‐derived metabolites play a central role in the communication between microbes and their host, with short‐chain fatty acids (SCFAs) being perhaps the most studied. SCFAs are primarily derived from fermentation of dietary fibres and play a pivotal role in host gut, metabolic and immune function. All these factors have previously been demonstrated to be adversely affected by stress. Therefore, we sought to assess whether SCFA supplementation could counteract the enduring effects of chronic psychosocial stress. C57BL/6J male mice received oral supplementation of a mixture of the three principle SCFAs (acetate, propionate and butyrate). One week later, mice underwent 3 weeks of repeated psychosocial stress, followed by a comprehensive behavioural analysis. Finally, plasma corticosterone, faecal SCFAs and caecal microbiota composition were assessed. SCFA treatment alleviated psychosocial stress‐induced alterations in reward‐seeking behaviour, and increased responsiveness to an acute stressor and in vivo intestinal permeability. In addition, SCFAs exhibited behavioural test‐specific antidepressant and anxiolytic effects, which were not present when mice had also undergone psychosocial stress. Stress‐induced increases in body weight gain, faecal SCFAs and the colonic gene expression of the SCFA receptors free fatty acid receptors 2 and 3 remained unaffected by SCFA supplementation. Moreover, there were no collateral effects on caecal microbiota composition. Taken together, these data show that SCFA supplementation alleviates selective and enduring alterations induced by repeated psychosocial stress and these data may inform future research into microbiota‐targeted therapies for stress‐related disorders. - The Journal of Physiology, EarlyView.
    August 28, 2018   doi: 10.1113/JP276431   open full text
  • Sensors and signals: the role of reactive oxygen species in hypoxic pulmonary vasoconstriction.
    Kimberly A. Smith, Paul T. Schumacker.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Abstract When lung cells experience hypoxia, the functional response, termed hypoxic pulmonary vasoconstriction, activates a multitude of pathways with the goal of optimizing gas exchange. While previously controversial, overwhelming evidence now suggests that increased reactive oxygen species – produced at complex III of the mitochondrial electron transport chain and released into the intermembrane space – is the cellular oxygen signal responsible for triggering hypoxic pulmonary vasoconstriction. The increased reactive oxygen species (ROS) activate many downstream targets that ultimately lead to increased intracellular ionized calcium concentration and contraction of pulmonary arterial smooth muscle cells. While the specific targets of ROS signals are not completely understood, it is clear that this signalling pathway is critical for development and for normal lung function in newborns and adults. - The Journal of Physiology, EarlyView.
    August 28, 2018   doi: 10.1113/JP275852   open full text
  • Specific synaptic input strengths determine the computational properties of excitation–inhibition integration in a sound localization circuit.
    Enida Gjoni, Friedemann Zenke, Brice Bouhours, Ralf Schneggenburger.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Key points During the computation of sound localization, neurons of the lateral superior olive (LSO) integrate synaptic excitation arising from the ipsilateral ear with inhibition from the contralateral ear. We characterized the functional connectivity of the inhibitory and excitatory inputs onto LSO neurons in terms of unitary synaptic strength and convergence. Unitary IPSCs can generate large conductances, although their strength varies over a 10‐fold range in a given recording. By contrast, excitatory inputs are relatively weak. The conductance associated with IPSPs needs to be at least 2‐fold stronger than the excitatory one to guarantee effective inhibition of action potential (AP) firing. Computational modelling showed that strong unitary inhibition ensures an appropriate slope and midpoint of the tuning curve of LSO neurons. Conversely, weak but numerous excitatory inputs filter out spontaneous AP firing from upstream auditory neurons. Abstract The lateral superior olive (LSO) is a binaural nucleus in the auditory brainstem in which excitation from the ipsilateral ear is integrated with inhibition from the contralateral ear. It is unknown whether the strength of the unitary inhibitory and excitatory inputs is adapted to allow for optimal tuning curves of LSO neuron action potential (AP) firing. Using electrical and optogenetic stimulation of afferent synapses, we found that the strength of unitary inhibitory inputs to a given LSO neuron can vary over a ∼10‐fold range, follows a roughly log‐normal distribution, and, on average, causes a large conductance (9 nS). Conversely, unitary excitatory inputs, stimulated optogenetically under the bushy‐cell specific promoter Math5, were numerous, and each caused a small conductance change (0.7 nS). Approximately five to seven bushy cell inputs had to be active simultaneously to bring an LSO neuron to fire. In double stimulation experiments, the effective inhibition window caused by IPSPs was short (1−3 ms) and its length depended on the inhibitory conductance; an ∼2‐fold stronger inhibition than excitation was needed to suppress AP firing. Computational modelling suggests that few, but strong, unitary IPSPs create a tuning curve of LSO neuron firing with an appropriate slope and midpoint. Furthermore, weak but numerous excitatory inputs reduce the spontaneous AP firing that LSO neurons would otherwise inherit from their upstream auditory neurons. Thus, the specific connectivity and strength of unitary excitatory and inhibitory inputs to LSO neurons is optimized for the computations performed by these binaural neurons. - The Journal of Physiology, EarlyView.
    August 28, 2018   doi: 10.1113/JP276012   open full text
  • Insulin transport across the blood–brain barrier can occur independently of the insulin receptor.
    Elizabeth M. Rhea, Christian Rask‐Madsen, William A. Banks.
    The Journal of Physiology. August 28, 2018
    --- - |2+ Key points Insulin enters the brain from the blood via a saturable transport system. It is unclear how insulin is transported across the blood–brain barrier (BBB). Using two models of the signalling‐related insulin receptor loss or inhibition, we show insulin transport can occur in vivo without the signalling‐related insulin receptor. Insulin in the brain has multiple roles including acting as a metabolic regulator and improving memory. Understanding how insulin is transported across the BBB will aid in developing therapeutics to further increase CNS concentrations. Abstract A saturable system transports insulin from blood across the blood–brain barrier (BBB) and into the central nervous system. Whether or not the classic or signalling‐related insulin receptor plays a role in mediating this transport in vivo is controversial. Here, we employed kinetics methods that distinguish between transport across the brain endothelial cell and reversible luminal surface receptor binding. Using a previously established line of mice with endothelial‐specific loss of the signalling‐related insulin receptor (EndoIRKO) or inhibiting the insulin receptor with the selective antagonist S961, we show insulin transport across the BBB is maintained. Rates of insulin transport were similar in all groups and transport was still saturable. Unlike transport, binding of insulin to the brain endothelial cell was decreased with the loss or inhibition of the signalling‐related insulin receptor. These findings demonstrate that the signalling‐related insulin receptor is not required for insulin transport across the BBB. - The Journal of Physiology, Volume 596, Issue 19, Page 4753-4765, 1 October 2018.
    August 28, 2018   doi: 10.1113/JP276149   open full text
  • Jack‐of‐many‐trades: discovering new roles for troponin C.
    Kerry S. McDonald.
    The Journal of Physiology. August 28, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4553-4554, 1 October 2018.
    August 28, 2018   doi: 10.1113/JP276790   open full text
  • Effects of living at moderate altitude on pulmonary vascular function and exercise capacity in mice with sickle cell anaemia.
    Scott K. Ferguson, Katherine Redinius, Ayla Yalamanoglu, Julie W. Harral, Jin Hyen Baek, David Pak, Zoe Loomis, Daniel Hassell, Paul Eigenberger, Eva Nozik‐Grayck, Rachelle Nuss, Kathryn Hassell, Kurt R. Stenmark, Paul W. Buehler, David C. Irwin.
    The Journal of Physiology. August 26, 2018
    --- - |2+ Key points Sickle cell disease (SCD) results in cardiopulmonary dysfunction, which may be exacerbated by prolonged exposure to environmental hypoxia. It is currently unknown whether exposure to mild and moderate altitude exacerbates SCD associated cardiopulmonary and systemic complications. Three months of exposure to mild (1609 m) and moderate (2438 m) altitude increased rates of haemolysis and right ventricular systolic pressures in mice with SCD compared to healthy wild‐type cohorts and SCD mice at sea level. The haemodynamic changes in SCD mice that had lived at mild and moderate altitude were accompanied by changes in the balance between pulmonary vascular endothelial nitric oxide synthase and endothelin receptor expression and impaired exercise tolerance. These data demonstrate that chronic altitude exposure exacerbates the complications associated with SCD and provides pertinent information for the clinical counselling of SCD patients. Abstract Exposure to high altitude worsens symptoms and crises in patients with sickle cell disease (SCD). However, it remains unclear whether prolonged exposure to low barometric pressures exacerbates SCD aetiologies or impairs quality of life. We tested the hypothesis that, relative to wild‐type (WT) mice, Berkley sickle cell mice (BERK‐SS) residing at sea level, mild (1609 m) and moderate (2438 m) altitude would have a higher rate of haemolysis, impaired cardiac function and reduced exercise tolerance, and that the level of altitude would worsen these decrements. Following 3 months of altitude exposure, right ventricular systolic pressure was measured (solid‐state transducer). In addition, the adaptive balance between pulmonary vascular endothelial nitric oxide synthase and endothelin was assessed in lung tissue to determine differences in pulmonary vascular adaptation and the speed/duration relationship (critical speed) was used to evaluate treadmill exercise tolerance. At all altitudes, BERK‐SS mice had a significantly lower percentage haemocrit and higher total bilirubin and free haemoglobin concentration (P < 0.05 for all). right ventricular systolic pressures in BERK‐SS were higher than WT at moderate altitude and also compared to BERK‐SS at sea level (P < 0.05, for both). Critical speed was significantly lower in BERK‐SS at mild and moderate altitude (P < 0.05). BERK‐SS demonstrated exacerbated SCD complications and reduced exercise capacity associated with an increase in altitude. These results suggest that exposure to mild and moderate altitude enhances the progression of SCD in BERK‐SS mice compared to healthy WT cohorts and BERK‐SS mice at sea level and provides crucial information for the clinical counselling of SCD patients. - The Journal of Physiology, EarlyView.
    August 26, 2018   doi: 10.1113/JP275810   open full text
  • Renewed excitement for paraventricular neurons and sympathetic nerve activity.
    Susan M. Barman.
    The Journal of Physiology. August 25, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4551-4552, 1 October 2018.
    August 25, 2018   doi: 10.1113/JP276813   open full text
  • Emerging views of how changes in blood pressure influence cerebral blood flow.
    Hannah G. Caldwell.
    The Journal of Physiology. August 25, 2018
    --- - - The Journal of Physiology, Volume 596, Issue 19, Page 4565-4567, 1 October 2018.
    August 25, 2018   doi: 10.1113/JP276800   open full text
  • Long‐term pulmonary vascular consequences of perinatal insults.
    Kara Goss.
    The Journal of Physiology. August 24, 2018
    --- - |2+ Abstract Development of the pulmonary circulation is a critical component of fetal lung development, and continues throughout infancy and childhood, marking an extended window of susceptibility to vascular maldevelopment and maladaptation. Perinatal vascular insults may result in abnormal vascular structure or function, including decreased angiogenic signaling and vascular endowment, impaired vasoreactivity through increased pulmonary artery endothelial dysfunction and remodeling, or enhanced genetic susceptibility to pulmonary vascular disease through epigenetic modifications or germline mutations. Although some infants develop early onset pulmonary hypertension, due to the unique adaptive capabilities of the immature host many do not have clinically evident early pulmonary vascular dysfunction. These individuals remain at increased risk for development of late‐onset pulmonary hypertension, and may be particularly susceptible to secondary insults. This review will address the role of perinatal vascular insults in the development of late pulmonary vascular dysfunction with an effort to highlight areas of critical research need. - The Journal of Physiology, EarlyView.
    August 24, 2018   doi: 10.1113/JP275859   open full text
  • A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated.
    Gengyun Le, Susan A. Novotny, Tara L. Mader, Sarah M. Greising, Sunny S. K. Chan, Michael Kyba, Dawn A. Lowe, Gordon L. Warren.
    The Journal of Physiology. August 24, 2018
    --- - |2+ Key points The female hormone oestrogen may protect muscle from injury by reducing inflammation but this is debatable. In this study, the inflammatory response of injured muscle from oestrogen‐replete mice was comprehensively compared to that from oestrogen‐deficient mice. We show that oestrogen markedly promotes movement of neutrophils, an inflammatory white blood cell type, into muscle over the first few days after injury but has only a minor effect on the movement of macrophages, another inflammatory cell type. Despite the enhancement of inflammation by oestrogen in injured muscle, we found strength in oestrogen‐replete mice to recover faster and to a greater extent than it does in oestrogen‐deficient mice. Our study and others indicate that lower doses of oestrogen, such as that used in our study, may affect muscle inflammation and injury differently from higher doses. Abstract Oestrogen has been shown to protect against skeletal muscle injury and a reduced inflammatory response has been suggested as a possible protective mechanism. There are, however, dissenting reports. Our objective was to conduct an unbiased, comprehensive study of the effect of oestradiol on the inflammatory response following muscle injury. Female C57BL6/J mice were ovariectomized and supplemented with and without oestradiol. Tibialis anterior muscles were freeze injured and studied primarily at 1–4 days post‐injury. Oestradiol supplementation increased injured muscle gene expression of neutrophil chemoattractants (Cxcl1 and Cxcl5) and to a lesser extent that of monocyte/macrophage chemoattractants (Ccl2 and Spp1). Oestradiol markedly increased gene expression of the neutrophil cell surface marker (Ly6g) but had less consistent effects on the monocyte/macrophage cell surface markers (Cd68, Cd163 and Cd206). These results were confirmed at the protein level by immunoblot with oestradiol increasing LY6G/C content and having no significant effect on CD163 content. These findings were confirmed with fluorescence‐activated cell sorting counts of neutrophils and macrophages in injured muscles; oestradiol increased the proportion of CD45+ cells that were neutrophils (LY6G+) but not the proportion that were macrophages (CD68+ or CD206+). Physiological impact of the oestradiol‐enhanced neutrophil response was assessed by strength measurements. There was no significant difference in strength between oestradiol‐supplemented and ‐unsupplemented mice until 2 weeks post‐injury; strength was 13–24% greater in supplemented mice at 2–6 weeks post‐injury. In conclusion, a moderate level of oestradiol supplementation enhances neutrophil infiltration in injured muscle and this is associated with a beneficial effect on strength recovery. - The Journal of Physiology, Volume 596, Issue 19, Page 4665-4680, 1 October 2018.
    August 24, 2018   doi: 10.1113/JP276432   open full text
  • Angiogenesis in the lung.
    Lindsey Eldridge, Elizabeth M. Wagner.
    The Journal of Physiology. August 18, 2018
    --- - |2+ Abstract Both systemic (tracheal and bronchial) and pulmonary circulations perfuse the lung. However, documentation of angiogenesis of either is complicated by the presence of the other. Well‐documented angiogenesis of the systemic circulations have been identified in asthma, cystic fibrosis, chronic thromboembolism and primary carcinomas. Angiogenesis of the vasa vasorum, which are branches of bronchial arteries, is seen in the walls of large pulmonary vessels after a period of chronic hypoxia. Documentation of increased pulmonary capillaries has been shown in models of chronic hypoxia, after pneumonectomy and in some carcinomas. Although endothelial cell proliferation may occur as part of the repair process in several pulmonary diseases, it is separate from the unique establishment of new functional perfusing networks defined as angiogenesis. Identification of the mechanisms driving the expansion of new vascular beds in the adult needs further investigation. Yet the growth factors and molecular mechanisms of lung angiogenesis remain difficult to separate from underlying disease sequelae. - The Journal of Physiology, EarlyView.
    August 18, 2018   doi: 10.1113/JP275860   open full text
  • The impact of immobilisation and inflammation on the regulation of muscle mass and insulin resistance: different routes to similar end‐points.
    Hannah Crossland, Sarah Skirrow, Zudin A. Puthucheary, Dumitru Constantin‐Teodosiu, Paul L. Greenhaff.
    The Journal of Physiology. August 18, 2018
    --- - |2 Abstract Loss of muscle mass and insulin sensitivity are common phenotypic traits of immobilisation and increased inflammatory burden. The suppression of muscle protein synthesis is the primary driver of muscle mass loss in human immobilisation, and includes blunting of post‐prandial increases in muscle protein synthesis. However, the mechanistic drivers of this suppression are unresolved. Immobilisation also induces limb insulin resistance in humans, which appears to be attributable to the reduction in muscle contraction per se. Again mechanistic insight is missing such that we do not know how muscle senses its “inactivity status” or whether the proposed drivers of muscle insulin resistance are simply arising as a consequence of immobilisation. A heightened inflammatory state is associated with major and rapid changes in muscle protein turnover and mass, and dampened insulin‐stimulated glucose disposal and oxidation in both rodents and humans. A limited amount of research has attempted to elucidate molecular regulators of muscle mass loss and insulin resistance during increased inflammatory burden, but rarely concurrently. Nevertheless, there is evidence that Akt (protein kinase B) signalling and FOXO transcription factors form part of a common signalling pathway in this scenario, such that molecular cross‐talk between atrophy and insulin signalling during heightened inflammation is believed to be possible. To conclude, whilst muscle mass loss and insulin resistance are common end‐points of immobilisation and increased inflammatory burden, a lack of understanding of the mechanisms responsible for these traits exists such that a substantial gap in understanding of the pathophysiology in humans endures. - The Journal of Physiology, EarlyView.
    August 18, 2018   doi: 10.1113/JP275444   open full text
  • Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.
    Satoshi Koba, Eri Hanai, Nao Kumada, Naoya Kataoka, Kazuhiro Nakamura, Tatsuo Watanabe.
    The Journal of Physiology. August 18, 2018
    --- - |2+ Key points Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN‐RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN‐RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. Abstract The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN‐RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN‐RVLM neurons. Male rats received microinjections into the PVN with an adeno‐associated virus (AAV) vector that encoded a hybrid of channelrhodopsin‐2/1 with the reporter tdTomato (ChIEF‐tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2‐GFPretro). Under anaesthesia with urethane and α‐chloralose, photostimulation (473 nm wavelength) of PVN‐RVLM neurons, achieved by laser illumination of either RVLM of ChIEF‐tdTomato rats (n = 8) or PVN of ChR2‐GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF‐tdTomato rats were closely associated with tdTomato‐labelled, PVN‐derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF‐tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN‐RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons. - The Journal of Physiology, Volume 596, Issue 19, Page 4581-4595, 1 October 2018.
    August 18, 2018   doi: 10.1113/JP276223   open full text
  • Novel mechanisms regulating endothelial barrier function in the pulmonary microcirculation.
    Szandor Simmons, Lasti Erfinanda, Christoph Bartz, Wolfgang M. Kuebler.
    The Journal of Physiology. August 14, 2018
    --- - |2 Abstract The pulmonary epithelial and vascular endothelial cell layers provide two sequential physical and immunological barriers that together form a semi‐permeable interface and prevent alveolar and interstitial oedema formation. In this review, we focus specifically on the continuous endothelium of the pulmonary microvascular bed that warrants strict control of the exchange of gases, fluid, solutes and circulating cells between the plasma and the interstitial space. The present review provides an overview of emerging molecular mechanisms that permit constant transcellular exchange between the vascular and interstitial compartment, and cause, prevent or reverse lung endothelial barrier failure under experimental conditions, yet with a clinical perspective. Based on recent findings and at times seemingly conflicting results we discuss emerging paradigms of permeability regulation by altered ion transport as well as shifts in the homeostasis of sphingolipids, angiopoietins and prostaglandins. - The Journal of Physiology, EarlyView.
    August 14, 2018   doi: 10.1113/JP276245   open full text
  • The choroid plexus sodium‐bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.
    Henriette L. Christensen, Dagne Barbuskaite, Aleksandra Rojek, Hans Malte, Inga B. Christensen, Annette C. Füchtbauer, Ernst‐Martin Füchtbauer, Tobias Wang, Jeppe Praetorius, Helle H. Damkier.
    The Journal of Physiology. August 12, 2018
    --- - |2+ Key points Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2. The low pH, however, is harmful and bases such as HCO3− are imported across the brain barriers in order to normalize brain pH. We show that the HCO3− transporter NBCe2 in the choroid plexus of the blood‐cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2. This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid‐base disturbances, such as chronic lung disease. Abstract The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE‐mediated CSF pH regulation by extrusion of acid‐base equivalents was tested by determining the contribution of the electrogenic Na+‐HCO3− cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE‐targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia‐induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild‐type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating‐induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia‐induced acidosis. - The Journal of Physiology, Volume 596, Issue 19, Page 4709-4728, 1 October 2018.
    August 12, 2018   doi: 10.1113/JP275489   open full text
  • Short‐term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high‐fat diet.
    Gerald Grandl, Leon Straub, Carla Rudigier, Myrtha Arnold, Stephan Wueest, Daniel Konrad, Christian Wolfrum.
    The Journal of Physiology. August 08, 2018
    --- - |2+ Key points A ketogenic diet is known to lead to weight loss and is considered metabolically healthy; however there are conflicting reports on its effect on hepatic insulin sensitivity. KD fed animals appear metabolically healthy in the fasted state after 3 days of dietary challenge, whereas obesogenic high‐fat diet (HFD) fed animals show elevated insulin levels. A glucose challenge reveals that both KD and HFD fed animals are glucose intolerant. Glucose intolerance correlates with increased lipid oxidation and lower respiratory exchange ratio (RER); however, all animals respond to glucose injection with an increase in RER. Hyperinsulinaemic–euglycaemic clamps with double tracer show that the effect of KD is a result of hepatic insulin resistance and increased glucose output but not impaired glucose clearance or tissue glucose uptake in other tissues. Abstract Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high‐fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high‐fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short‐term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP‐derived glucose. - The Journal of Physiology, Volume 596, Issue 19, Page 4597-4609, 1 October 2018.
    August 08, 2018   doi: 10.1113/JP275173   open full text
  • Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension.
    Cheng‐Jun Hu, Hui Zhang, Aya Laux, Soni S. Pullamsetti, Kurt R. Stenmark.
    The Journal of Physiology. August 07, 2018
    --- - |2+ Abstract Chronic pulmonary hypertension (PH) is characterized by the accumulation of persistently activated cell types in the pulmonary vessel exhibiting aberrant expression of genes involved in apoptosis resistance, proliferation, inflammation and extracellular matrix (ECM) remodelling. Current therapies for PH, focusing on vasodilatation, do not normalize these activated phenotypes. Furthermore, current approaches to define additional therapeutic targets have focused on determining the initiating signals and their downstream effectors that are important in PH onset and development. Although these approaches have produced a large number of compelling PH treatment targets, many promising human drugs have failed in PH clinical trials. Herein, we propose that one contributing factor to these failures is that processes important in PH development may not be good treatment targets in the established phase of chronic PH. We hypothesize that this is due to alterations of chromatin structure in PH cells, resulting in functional differences between the same factor or pathway in normal or early PH cells versus cells in chronic PH. We propose that the high expression of genes involved in the persistently activated phenotype of PH vascular cells is perpetuated by an open chromatin structure and multiple transcription factors (TFs) via the recruitment of high levels of epigenetic regulators including the histone acetylases P300/CBP, histone acetylation readers including BRDs, the Mediator complex and the positive transcription elongation factor (Abstract figure). Thus, determining how gene expression is controlled by examining chromatin structure, TFs and epigenetic regulators associated with aberrantly expressed genes in pulmonary vascular cells in chronic PH, may uncover new PH therapeutic targets. - The Journal of Physiology, EarlyView.
    August 07, 2018   doi: 10.1113/JP275857   open full text
  • Apparent calcium dependence of vesicle recruitment.
    Andreas Ritzau‐Jost, Lukasz Jablonski, Julio Viotti, Noa Lipstein, Jens Eilers, Stefan Hallermann.
    The Journal of Physiology. August 07, 2018
    --- - |2+ Key points Synaptic transmission relies on the recruitment of neurotransmitter‐filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium‐dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium‐dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium‐independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast‐recruited vesicles but does not change the vesicle recruitment rates themselves. Abstract During sustained synaptic transmission, recruitment of new transmitter‐filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity‐dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin–Munc13 pathway, which has been proposed to mediate Ca2+‐dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+‐independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin–Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+‐dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well‐established two‐pool models predict an apparent Ca2+‐dependence of vesicle recruitment. Thus, previous conclusions of Ca2+‐dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution. - The Journal of Physiology, Volume 596, Issue 19, Page 4693-4707, 1 October 2018.
    August 07, 2018   doi: 10.1113/JP275911   open full text
  • Sickle cell vasculopathy: vascular phenotype on fire!
    Gregory J. Kato.
    The Journal of Physiology. August 03, 2018
    --- - - The Journal of Physiology, EarlyView.
    August 03, 2018   doi: 10.1113/JP276705   open full text
  • The impact of exercise and nutrition on the regulation of skeletal muscle mass.
    Chris McGlory, Stephan Vliet, Tanner Stokes, Bettina Mittendorfer, Stuart M. Phillips.
    The Journal of Physiology. August 02, 2018
    --- - |2 Abstract The maintenance of skeletal muscle mass and strength throughout life is a key determinant of human health and well‐being. There is a gradual loss of both skeletal muscle mass and strength with ageing (a process termed sarcopenia) that increases the risk of functional dependence, morbidity and mortality. Understanding the factors that regulate the size of human muscle mass, particularly during the later years of life, has therefore become an area of intense scientific inquiry. The amount of muscle mass is determined by coordinated changes in muscle protein synthesis (MPS) and muscle protein breakdown (MPB). In this review, we assess both classical and contemporary work that has examined how resistance exercise and nutrition impact on MPS and MPB. Special consideration is given to the role of different sources of dietary protein (food vs. supplements) and non‐protein nutrients such as omega‐3 fatty acids in regulating MPS. We also critically evaluate recent studies that have employed novel ‘omic’ technologies such as dynamic protein profiling to probe for changes in rates of MPS and MPB at the individual protein level following exercise. Finally, we provide suggestions for future research that we hope will yield important information for the development of exercise and nutritional strategies to counteract muscle loss in a variety of clinical settings. - The Journal of Physiology, EarlyView.
    August 02, 2018   doi: 10.1113/JP275443   open full text
  • Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.
    Svetlana Tikunova, Natalya Belevych, Kelly Doan, Peter J. Reiser.
    The Journal of Physiology. August 02, 2018
    --- - |2+ Key points The Ca2+‐desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (‐log [Ca]) relationship in slow‐twitch mouse fibres. The D73N mutation led to a rightward shift in the force–stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross‐sectional area of slow‐twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. Abstract The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow‐twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force–stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross‐sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation. - The Journal of Physiology, Volume 596, Issue 19, Page 4651-4663, 1 October 2018.
    August 02, 2018   doi: 10.1113/JP276296   open full text
  • Mechanoadaptation: articular cartilage through thick and thin.
    Tonia L. Vincent, Angus K. T. Wann.
    The Journal of Physiology. July 29, 2018
    --- - |2 Abstract The articular cartilage is exquisitely sensitive to mechanical load. Its structure is largely defined by the mechanical environment and destruction in osteoarthritis is the pathophysiological consequence of abnormal mechanics. It is often overlooked that disuse of joints causes profound loss of volume in the articular cartilage, a clinical observation first described in polio patients and stroke victims. Through the 1980s, the results of studies exploiting experimental joint immobilisation supported this. Importantly, this substantial body of work was also the first to describe metabolic changes that resulted in decreased synthesis of matrix molecules, especially sulfated proteoglycans. The molecular mechanisms that underlie disuse atrophy are poorly understood despite the identification of multiple mechanosensing mechanisms in cartilage. Moreover, there has been a tendency to equate cartilage loss with osteoarthritic degeneration. Here, we review the historic literature and clarify the structural, metabolic and clinical features that clearly distinguish cartilage loss due to disuse atrophy and those due to osteoarthritis. We speculate on the molecular sensing pathways in cartilage that may be responsible for cartilage mechanoadaptation. - The Journal of Physiology, EarlyView.
    July 29, 2018   doi: 10.1113/JP275451   open full text
  • Inspiratory pre‐motor potentials during quiet breathing in ageing and chronic obstructive pulmonary disease.
    David A. T. Nguyen, Claire L. Boswell‐Ruys, Rachel A. McBain, Danny J. Eckert, Simon C. Gandevia, Jane E. Butler, Anna L. Hudson.
    The Journal of Physiology. July 29, 2018
    --- - |2+ Key points A cortical contribution to breathing, as indicated by a Bereitschaftspotential (BP) in averaged electroencephalographic signals, occurs in healthy individuals when external inspiratory loads are applied. Chronic obstructive pulmonary disease (COPD) is a condition where changes in the lung, chest wall and respiratory muscles produce an internal inspiratory load. These changes also occur in normal ageing, although to a lesser extent. In the present study, we determined whether BPs are present during quiet breathing and breathing with an external inspiratory load in COPD compared to age‐matched and young healthy controls. We demonstrated that increased age, rather than COPD, is associated with a cortical contribution to quiet breathing. A cortical contribution to inspiratory loading is associated with more severe dyspnoea (i.e. the sensation of breathlessness). We propose that cortical mechanisms may be engaged to defend ventilation in ageing with dyspnoea as a consequence. Abstract A cortical contribution to breathing is determined by the presence of a Bereitschaftspotential, a low amplitude negativity in the averaged electroencephalographic (EEG) signal, which begins ∼1 s before inspiration. It occurs in healthy individuals when external inspiratory loads to breathing are applied. In chronic obstructive pulmonary disease (COPD), changes in the lung, chest wall and respiratory muscles produce an internal inspiratory load. We hypothesized that there would be a cortical contribution to quiet breathing in COPD and that a cortical contribution to breathing with an inspiratory load would be linked to dyspnoea, a major symptom of COPD. EEG activity was analysed in 14 participants with COPD (aged 57–84 years), 16 healthy age‐matched (57–87 years) and 15 young (18–26 years) controls during quiet breathing and inspiratory loading. The presence of Bereitschaftspotentials, from ensemble averages of EEG epochs at Cz and FCz, were assessed by blinded assessors. Dyspnoea was rated using the Borg scale. The incidence of a cortical contribution to quiet breathing was significantly greater in participants with COPD (6/14) compared to the young (0/15) (P = 0.004) but not the age‐matched controls (6/16) (P = 0.765). A cortical contribution to inspiratory loading was associated with higher Borg ratings (P = 0.007), with no effect of group (P = 0.242). The data show that increased age, rather than COPD, is associated with a cortical contribution to quiet breathing. A cortical contribution to inspiratory loading is associated with more severe dyspnoea. We propose that cortical mechanisms may be engaged to defend ventilation with dyspnoea as a consequence. - The Journal of Physiology, EarlyView.
    July 29, 2018   doi: 10.1113/JP275764   open full text
  • Maternal exercise in rats upregulates the placental insulin‐like growth factor system with diet‐ and sex‐specific responses: minimal effects in mothers born growth restricted.
    Yeukai T. M. Mangwiro, James S. M. Cuffe, Jessica F. Briffa, Dayana Mahizir, Kristina Anevska, Andrew J. Jefferies, Sogand Hosseini, Tania Romano, Karen M. Moritz, Mary E. Wlodek.
    The Journal of Physiology. July 26, 2018
    --- - |2+ Key points The placental insulin‐like growth factor (IGF) system is critical for normal fetoplacental growth, which is dysregulated following several pregnancy perturbations including uteroplacental insufficiency and maternal obesity. We report that the IGF system was altered in placentae of mothers born growth restricted compared to normal birth weight mothers, with maternal diet‐ and fetal sex‐specific responses. Additionally, we report increased body weight and plasma IGF1 concentrations in fetuses from chow‐fed normal birth weight mothers that exercised prior to and continued during pregnancy compared to sedentary mothers. Exercise initiated during pregnancy, on the other hand, resulted in placental morphological alterations and increased IGF1 and IGF1R protein expression, which may in part be modulated by reduced Let 7f‐1 miRNA abundance. Growth restriction of mothers before birth and exercise differentially regulate the placental IGF system with diet‐ and sex‐specific responses, probably as a means to improve fetoplacental growth and development, and hence neonatal survival. This increased neonatal survival may prevent adult disease onset. Abstract The insulin‐like growth factor (IGF) system regulates fetoplacental growth and plays a role in disease programming. Dysregulation of the IGF system is implicated in several pregnancy perturbations associated with altered fetal growth, including intrauterine growth restriction and maternal obesity. Limited human studies have demonstrated that maternal exercise enhances fetoplacental growth and decreases cord IGF ligands, which may restore the placental IGF system in complicated pregnancies. This study investigated the impact maternal exercise has on the placental IGF system in placentae from mothers born growth restricted and if these outcomes are dependent on maternal diet or fetal sex. Uteroplacental insufficiency (Restricted) or sham (Control) surgery was induced on embryonic day (E) 18 in Wistar–Kyoto rats. F1 offspring were fed a chow or high‐fat diet from weaning, and at 16 weeks were randomly allocated an exercise protocol: Sedentary, Exercised prior to and during pregnancy (Exercise), or Exercised during pregnancy only (PregEx). Females were mated (20 weeks) with placentae associated with F2 fetuses collected at E20. The placental IGF system mRNA abundance and placental morphology was altered in mothers born growth restricted. Exercise increased fetal weight and Control plasma IGF1 concentrations, and decreased female placental weight. PregEx did not influence fetoplacental growth but increased placental IGF1 and IGF1R (potentially modulated by reduced Let 7f‐1 miRNA) and decreased placental IGF2 protein. Importantly, these placental IGF system changes occurred with sex‐specific responses. These data highlight that exercise differently influences fetoplacental growth and the placental IGF system depending on maternal exercise initiation, which may prevent the transgenerational transmission of deficits and dysfunction. - The Journal of Physiology, EarlyView.
    July 26, 2018   doi: 10.1113/JP275758   open full text
  • Antenatal prevention of cerebral palsy and childhood disability: is the impossible possible?
    Stacey J. Ellery, Meredith Kelleher, Peta Grigsby, Irina Burd, Jan B. Derks, Jon Hirst, Suzanne L. Miller, Larry S. Sherman, Mary Tolcos, David W. Walker.
    The Journal of Physiology. July 22, 2018
    --- - |2+ Abstract This review covers our current knowledge of the causes of perinatal brain injury leading to cerebral palsy‐like outcomes, and argues that much of this brain damage is preventable. We review the experimental evidence that there are treatments that can be safely administered to women in late pregnancy that decrease the likelihood and extent of perinatal brain damage that occurs because of acute and severe hypoxia that arises during some births, and the additional impact of chronic fetal hypoxia, infection, inflammation, growth restriction and preterm birth. We discuss the types of interventions required to ameliorate or even prevent apoptotic and necrotic cell death, and the vulnerability of all the major cell types in the brain (neurons, astrocytes, oligodendrocytes, microglia, cerebral vasculature) to hypoxia/ischaemia, and whether a pan‐protective treatment given to the mother before birth is a realistic prospect. - The Journal of Physiology, EarlyView.
    July 22, 2018   doi: 10.1113/JP275595   open full text
  • The impact of loading, unloading, ageing and injury on the human tendon.
    S. Peter Magnusson, Michael Kjaer.
    The Journal of Physiology. July 20, 2018
    --- - |2+ Abstract A tendon transfers force from the contracting muscle to the skeletal system to produce movement and is therefore a crucial component of the entire muscle‐tendon complex and its function. However, tendon research has for some time focused on mechanical properties without any major appreciation of potential cellular and molecular changes. At the same time, methodological developments have permitted determination of the mechanical properties of human tendons in vivo, which was previously not possible. Here we review the current understanding of how tendons respond to loading, unloading, ageing and injury from cellular, molecular and mechanical points of view. A mechanistic understanding of tendon tissue adaptation will be vital for development of adequate guidelines in physical training and rehabilitation, as well as for optimal injury treatment. - The Journal of Physiology, EarlyView.
    July 20, 2018   doi: 10.1113/JP275450   open full text
  • Omecamtiv Mercabil and Blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.
    Thomas Kampourakis, Xuemeng Zhang, Yin‐Biao Sun, Malcolm Irving.
    The Journal of Physiology. October 20, 2017
    Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules‐ Omecamtiv Mecarbil (OM) and Blebbistatin (BS) ‐ that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. This article is protected by copyright. All rights reserved
    October 20, 2017   doi: 10.1113/JP275050   open full text
  • Heteromeric α/β glycine receptors regulate excitability in parvalbumin‐expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.
    M. A. Gradwell, K. A. Boyle, R. J. Callister, D. I. Hughes, B. A. Graham.
    The Journal of Physiology. October 19, 2017
    Key points Spinal parvalbumin‐expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn. This study assessed the inhibitory regulation of the parvalbumin‐expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability. Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin‐expressing interneuron excitability. Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/β subunit‐containing receptors underlie both synaptic and tonic glycinergic currents. Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin‐expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. Abstract The dorsal horn (DH) of the spinal cord is an important site for modality‐specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin‐expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch‐clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine‐mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/β glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.
    October 19, 2017   doi: 10.1113/JP274926   open full text
  • Molecular mechanism for muscarinic M1 receptor‐mediated endocytosis of TWIK‐related acid‐sensitive K+ 1 channels in rat adrenal medullary cells.
    Hidetada Matsuoka, Masumi Inoue.
    The Journal of Physiology. October 19, 2017
    Key points The muscarinic acetylcholine receptor (mAChR)‐mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)‐related acid‐sensitive K+ (TASK)1 channels. In this study we focused on the molecular mechanism of mAChR‐mediated inhibition of TASK1 channels. Exposure to muscarine resulted in a clathrin‐dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R). This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non‐receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1. The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin‐dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. Abstract Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK‐related acid‐sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR‐mediated inhibition. TASK1‐like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine‐induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose–response curves for muscarinic agonist‐induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine‐induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1‐like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1R stimulation results in internalization of TASK1 channels through the PLC–PKC–Src pathway with the consequent phosphorylation of tyrosine and that this M1R‐mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.
    October 19, 2017   doi: 10.1113/JP275039   open full text
  • Endomorphins potentiate ASIC currents and enhance the lactic acid‐mediated increase in arterial blood pressure—effects amplified in hindlimb ischemia.
    Mohamed Farrag, Julie K. Drobish, Henry L. Puhl, Joyce S. Kim, Paul B. Herold, Marc P. Kaufman, Victor Ruiz‐Velasco.
    The Journal of Physiology. October 16, 2017
    Chronic muscle ischemia leads to accumulation of lactic acid and other inflammatory mediators with a subsequent drop in interstitial pH. Acid‐sensing ion channels (ASICs), expressed in thin muscle afferents, sense the decrease in pH and evoke a pressor reflex known to increase mean arterial pressure. The naturally occurring endomorphins are also released by primary afferents under ischemic conditions. We examined whether high affinity mu opioid receptor (MOR) agonists, endomorphin‐1 (E‐1) and ‐2 (E‐2), modulate ASIC currents and the lactic acid‐mediated pressor reflex. In rat dorsal root ganglion (DRG) neurons, exposure to E‐2 in acidic solutions significantly potentiated ASIC currents when compared to acidic solutions alone. The potentiation was significantly greater in DRG neurons isolated from rats whose femoral arteries were ligated for 72 hr. Sustained ASIC current potentiation was also observed in neurons pretreated with pertussis toxin, an uncoupler of G proteins and MOR. The endomorphin‐mediated potentiation was a result of a leftward shift of the activation curve to more basic pH values and a slight shift of the inactivation curve to more acidic pH values. Intra‐arterial co‐administration of lactic acid and E‐2 led to a significantly greater pressor reflex than lactic acid alone in the presence of naloxone. Finally, E‐2 effects were inhibited by pretreatment with the ASIC3 blocker (APETx2) and enhanced by pretreatment with the ASIC1a blocker psalmotoxin‐1. These findings have uncovered a novel role of endomorphins by which the opioids can enhance the lactic acid‐mediated reflex increase in arterial pressure that is MOR stimulation‐independent and APETx2‐sensitive. This article is protected by copyright. All rights reserved
    October 16, 2017   doi: 10.1113/JP275058   open full text
  • Altered NMDA receptor‐evoked intracellular Ca2+ dynamics in magnocellular neurosecretory neurons of hypertensive rats.
    Meng Zhang, Javier E. Stern.
    The Journal of Physiology. October 15, 2017
    A growing body of evidence supports an elevated NMDA receptor‐mediated glutamate excitatory function in the SON and PVN of hypertensive rats that contributes to neurohumoral activation in this disease. Still, the precise mechanisms underlying altered NMDAR signalling in hypertension remains to be elucidated. In this study, we performed simultaneous electrophysiology and fast confocal Ca2+ imaging to determine whether an altered NMDAR‐mediated changes in intracellular Ca2+ levels (NMDAR‐ΔCa2+) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in renovascular hypertensive (RVH) rats. We found that despite evoking a similar excitatory inward current, activation of NMDARs resulted in a larger and prolonged ΔCa2+ in MNCs from RVH rats. Changes in NMDAR‐ΔCa2+ dynamics were observed both in somatic and dendritic compartments. Inhibition of the ER SERCA pump activity with thapsigargin prolonged NMDAR‐ΔCa2+ responses in MNCs of sham rats, but this effect was occluded in RVH rats, thus equalizing the magnitude and time course of the NMDA‐ΔCa2 responses between the two experimental groups. Taken together, our results support (1) an exacerbated NMDAR‐ΔCa2+ response in somatodendritic compartments of MNCs of RVH rats, and (2) that a blunted ER Ca2+ buffering capacity contributes to the altered NMDAR‐ΔCa2+ dynamics in this condition. Thus, an altered spatiotemporal dynamics of NMDAR‐ΔCa2+ response stands as an underlying mechanisms contributing to neurohumoral activation in neurogenic hypertension. This article is protected by copyright. All rights reserved
    October 15, 2017   doi: 10.1113/JP275169   open full text
  • Asymmetry between ON and OFF α ganglion cells of mouse retina: integration of signal and noise from synaptic inputs.
    Michael A. Freed.
    The Journal of Physiology. October 15, 2017
    Key points Bipolar and amacrine cells presynaptic to the ON sustained α cell of mouse retina provide currents with a higher signal‐to‐noise power ratio (SNR) than those presynaptic to the OFF sustained α cell. Yet the ON cell loses proportionately more SNR from synaptic inputs to spike output than the OFF cell does. The higher SNR of ON bipolar cells at the beginning of the ON pathway compensates for losses incurred by the ON ganglion cell, and improves the processing of positive contrasts. Abstract ON and OFF pathways in the retina include functional pairs of neurons that, at first glance, appear to have symmetrically similar responses to brightening and darkening, respectively. Upon careful examination, however, functional pairs exhibit asymmetries in receptive field size and response kinetics. Until now, descriptions of how light‐adapted retinal circuitry maintains a preponderance of signal over the noise have not distinguished between ON and OFF pathways. Here I present evidence of marked asymmetries between members of a functional pair of sustained α ganglion cells in the mouse retina. The ON cell exhibited a proportionately greater loss of signal‐to‐noise power ratio (SNR) from its presynaptic arrays to its postsynaptic currents. Thus the ON cell combines signal and noise from its presynaptic arrays of bipolar and amacrine cells less efficiently than the OFF cell does. Yet the inefficiency of the ON cell is compensated by its presynaptic arrays providing a higher SNR than the arrays presynaptic to the OFF cell, apparently to improve visual processing of positive contrasts. Dynamic clamp experiments were performed that introduced synaptic conductances into ON and OFF cells. When the amacrine‐modulated conductance was removed, the ON cell's spike train exhibited an increase in SNR. The OFF cell, however, showed the opposite effect of removing amacrine input, which was a decrease in SNR. Thus ON and OFF cells have different modes of synaptic integration with direct effects on the SNR of the spike output.
    October 15, 2017   doi: 10.1113/JP274736   open full text
  • Laminar‐specific encoding of texture elements in rat barrel cortex.
    Benjamin J. Allitt, Dasuni S. Alwis, Ramesh Rajan.
    The Journal of Physiology. October 15, 2017
    Key points For rats texture discrimination is signalled by the large face whiskers by stick‐slip events. Neural encoding of repetitive stick‐slip events will be influenced by intrinsic properties of adaptation. We show that texture coding in the barrel cortex is laminar specific and follows a power function. Our results also show layer 2 codes for novel feature elements via robust firing rates and temporal fidelity. We conclude that texture coding relies on a subtle neural ensemble to provide important object information. Abstract Texture discrimination by rats is exquisitely guided by fine‐grain mechanical stick‐slip motions of the face whiskers as they encounter, stick to and slip past successive texture‐defining surface features such as bumps and grooves. Neural encoding of successive stick‐slip texture events will be shaped by adaptation, common to all sensory systems, whereby receptor and neural responses to a stimulus are affected by responses to preceding stimuli, allowing resetting to signal novel information. Additionally, when a whisker is actively moved to contact and brush over surfaces, that motion itself generates neural responses that could cause adaptation of responses to subsequent stick‐slip events. Nothing is known about encoding in the rat whisker system of stick‐slip events defining textures of different grain or the influence of adaptation from whisker protraction or successive texture‐defining stick‐slip events. Here we recorded responses from halothane‐anaesthetized rats in response to texture‐defining stimuli applied to passive whiskers. We demonstrate that: across the columnar network of the whisker‐recipient barrel cortex, adaptation in response to repetitive stick‐slip events is strongest in uppermost layers and equally lower thereafter; neither whisker protraction speed nor stick‐slip frequency impede encoding of stick‐slip events at rates up to 34.08 Hz; and layer 2 normalizes responses to whisker protraction to resist effects on texture signalling. Thus, within laminar‐specific response patterns, barrel cortex reliably encodes texture‐defining elements even to high frequencies.
    October 15, 2017   doi: 10.1113/JP274865   open full text
  • N1366S mutation of human skeletal muscle sodium channel causes paramyotonia congenita.
    Qing Ke, Jia Ye, Siyang Tang, Jin Wang, Benyan Luo, Fang Ji, Xu Zhang, Ye Yu, Xiaoyang Cheng, Yuezhou Li.
    The Journal of Physiology. October 15, 2017
    Key points Paramyotonia congenita is a hereditary channelopathy caused by missense mutations in the SCN4A gene, which encodes the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. Affected individuals suffered from myotonia and paralysis of muscles, which were aggravated by exposure to cold. We report a three‐generation Chinese family with patients presenting paramyotonia congenita and identify a novel N1366S mutation of NaV1.4. Whole‐cell electrophysiological recordings of the N1366S channel reveal a gain‐of‐function change of gating in response to cold. Modelling and molecular dynamic simulation data suggest that an arginine‐to‐serine substitution at position 1366 increases the distance from N1366 to R1454 and disrupts the hydrogen bond formed between them at low temperature. We demonstrate that N1366S is a disease‐causing mutation and that the temperature‐sensitive alteration of N1366S channel activity may be responsible for the pronounced paramyotonia congenita symptoms of these patients. Abstract Paramyotonia congenita is an autosomal dominant skeletal muscle channelopathy caused by missense mutations in SCN4A, the gene encoding the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. We report a three‐generation family in which six members present clinical symptoms of paramyotonia congenita characterized by a marked worsening of myotonia by cold and by the presence of clear episodes of paralysis. We identified a novel mutation in SCN4A (Asn1366Ser, N1366S) in all patients in the family but not in healthy relatives or in 500 normal control subjects. Functional analysis of the channel protein expressed in HEK293 cells by whole‐cell patch clamp recording revealed that the N1366S mutation led to significant alterations in the gating process of the NaV1.4 channel. The N1366S mutant displayed a cold‐induced hyperpolarizing shift in the voltage dependence of activation and a depolarizing shift in fast inactivation, as well as a reduced rate of fast inactivation and accelerated recovery from fast inactivation. In addition, homology modelling and molecular dynamic simulation of N1366S and wild‐type NaV1.4 channels indicated that the arginine‐to‐serine substitution disrupted the hydrogen bond formed between N1366 and R1454. Together, our results suggest that N1366S is a gain‐of‐function mutation of NaV1.4 at low temperature and the mutation may be responsible for the clinical symptoms of paramyotonia congenita in the affected family and constitute a basis for studies into its pathogenesis.
    October 15, 2017   doi: 10.1113/JP274877   open full text
  • Dietary sodium induces a redistribution of the tubular metabolic workload.
    Khalil Udwan, Ahmed Abed, Isabelle Roth, Eva Dizin, Marc Maillard, Carla Bettoni, Johannes Loffing, Carsten A. Wagner, Aurélie Edwards, Eric Feraille.
    The Journal of Physiology. October 15, 2017
    Key points Body Na+ content is tightly controlled by regulated urinary Na+ excretion. The intrarenal mechanisms mediating adaptation to variations in dietary Na+ intake are incompletely characterized. We confirmed and expanded observations in mice that variations in dietary Na+ intake do not alter the glomerular filtration rate but alter the total and cell‐surface expression of major Na+ transporters all along the kidney tubule. Low dietary Na+ intake increased Na+ reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments. High dietary Na+ intake decreased Na+ reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency. The abundance of apical transporters and Na+ delivery are the main determinants of Na+ reabsorption along the kidney tubule. Tubular O2 consumption and the efficiency of sodium reabsorption are dependent on sodium diet. Abstract Na+ excretion by the kidney varies according to dietary Na+ intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na+ reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na+, a normal sodium diet (NSD) containing 0.18% Na+ and a moderately high sodium diet (HSD) containing 1.25% Na+. As expected, LSD did not alter measured GFR and increased the abundance of total and cell‐surface NHE3, NKCC2, NCC, α‐ENaC and cleaved γ‐ENaC compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na+ delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O2 consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) was related to the changes in tubular Na+ reabsorption. Our data show that fractional Na+ reabsorption is distributed differently according to dietary Na+ intake and induces changes in tubular O2 consumption and sodium transport efficiency.
    October 15, 2017   doi: 10.1113/JP274927   open full text
  • Task‐dependent output of human parasternal intercostal motor units across spinal levels.
    Anna L. Hudson, Simon C. Gandevia, Jane E. Butler.
    The Journal of Physiology. October 13, 2017
    Key points During breathing, there is differential activity in the human parasternal intercostal muscles and the activity is tightly coupled to the known mechanical advantages for inspiration of the same regions of muscles. It is not known whether differential activity is preserved for the non‐respiratory task of ipsilateral trunk rotation. In the present study, we compared single motor units during resting breathing and axial rotation of the trunk during apnoea. We not only confirmed non‐uniform recruitment of motor units across parasternal intercostal muscles in breathing, but also demonstrated that the same motor units show an altered pattern of recruitment in the non‐respiratory task of trunk rotation. The output of parasternal intercostal motoneurones is modulated differently across spinal levels depending on the task and these results help us understand the mechanisms that may govern task‐dependent differences in motoneurone output. Abstract During inspiration, there is differential activity in the human parasternal intercostal muscles across interspaces. We investigated whether the earlier recruitment of motor units in the rostral interspaces compared to more caudal spaces during inspiration is preserved for the non‐respiratory task of ipsilateral trunk rotation. Single motor unit activity (SMU) was recorded from the first, second and fourth parasternal interspaces on the right side in five participants in two tasks: resting breathing and ‘isometric’ axial rotation of the trunk during apnoea. Recruitment of the same SMUs was compared between tasks (n = 123). During resting breathing, differential activity was indicated by earlier recruitment of SMUs in the first and second interspaces compared to the fourth space in inspiration (P < 0.01). By contrast, during trunk rotation, the same motor units showed an altered pattern of recruitment because SMUs in the first interspace were recruited later and at a higher rotation torque than those in the second and fourth interspaces (P < 0.05). Tested for a subset of SMUs, the reliability of the breathing and rotation tasks, as well as the SMU recruitment measures, was good–excellent [intraclass correlation (2,1): 0.69–0.91]. Thus, the output of parasternal intercostal motoneurones is modulated differently across spinal levels depending on the task. Given that the differential inspiratory output of parasternal intercostal muscles is linked to their relative mechanical effectiveness for inspiration and also that this output is altered in trunk rotation, we speculate that a mechanism matching neural drive to muscle mechanics underlies the task‐dependent differences in output of axial motoneurone pools.
    October 13, 2017   doi: 10.1113/JP274866   open full text
  • Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport, and osmotic enhancement of delivery.
    Michelle E. Pizzo, Daniel J. Wolak, Niyanta N. Kumar, Eric Brunette, Christina L. Brunnquell, Melanie‐Jane Hannocks, N. Joan Abbott, M. Elizabeth Meyerand, Lydia Sorokin, Danica B. Stanimirovic, Robert G. Thorne.
    The Journal of Physiology. October 12, 2017
    The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance as well as several ongoing intrathecal clinical trials. Here, we clarify how immunoglobulin G (IgG) and smaller single‐domain antibodies (sdAb) distribute throughout the whole brain in a size‐dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo 3D magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along perivascular spaces of all vessel types, with sdAb accessing 4–7 times greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size‐dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points for substances in the CSF to enter the perivascular space. Osmolyte co‐infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by approximately 50%. Our results reveal potential distribution mechanisms for endogenous IgG, one of the most abundant proteins in the CSF, as well as provide new insights needed to understand and improve drug delivery of macromolecules to the central nervous system via the intrathecal route. This article is protected by copyright. All rights reserved
    October 12, 2017   doi: 10.1113/JP275105   open full text
  • An investigation of fetal behavioural states during maternal sleep in healthy late gestation pregnancy: an observational study.
    Peter R. Stone, Wendy Burgess, Jordan McIntyre, Alistair J. Gunn, Christopher A. Lear, Laura Bennet, Edwin A Mitchell, John M. D. Thompson,.
    The Journal of Physiology. October 11, 2017
    Background Fetal behavioural states (FBS) are measures of fetal wellbeing. Maternal position affects FBS with supine being associated with an increased likelihood of fetal quiescence consistent with the human fetus adapting to a lower oxygen consuming state. A number of studies now confirm the association of sleep position with risk of late intrauterine death. We designed this study to observe the effects of maternal sleep positions overnight in healthy late gestation pregnancy. Method Twenty nine healthy women had continuous fetal ECG recordings overnight. Two blinded observers, assigned fetal states in 5 minute blocks. Measures of fetal heart rate variability (FHRV) were calculated from ECG beat to beat data. Maternal position was determined from infrared video recording. Results Compared to state 2F (active sleep), 4F (active awake‐high activity) occurred almost exclusively when the mother was in a left or right lateral position. State 1F (quiet sleep) was more common when mother was supine (OR 1.30, 95%CI, 1.11‐1.52) and less common on maternal right side with the left being referent position (OR 0.81, 95%CI, 0.70‐0.93). State 4F was more common between 2100 and 0100 than between 0100 and 0700 (OR 2 2.83, 95%CI, 2.32‐3.47). In each fetal state, maternal position had significant effects on fetal heart rate (FHR) and measures of FHRV. Conclusion In healthy late gestation pregnancy, maternal sleep position affects FBS and heart rate variability. These effects are likely fetal adaptations to positions which may produce a mild hypoxic stress. This article is protected by copyright. All rights reserved
    October 11, 2017   doi: 10.1113/JP275084   open full text
  • Physiological tremor increases when skeletal muscle is shortened: implications for fusimotor control.
    Kian Jalaleddini, Akira Nagamori, Christopher M. Laine, Mahsa A. Golkar, Robert E. Kearney, Francisco J. Valero‐Cuevas.
    The Journal of Physiology. October 11, 2017
    The involuntary force fluctuations associated with physiological (as distinct from pathological) tremor are an unavoidable component of human motor control. While the origins of physiological tremor are known to depend on muscle afferentation, it is possible that the mechanical properties of muscle‐tendon systems also affect its generation, amplification and maintenance. In this paper, we investigated the dependence of physiological tremor on muscle length in healthy individuals. We measured physiological tremor during tonic, isometric plantarflexion torque at 30% of maximum at three ankle angles. The amplitude of physiological tremor increased as calf muscles shortened in contrast to the stretch reflex whose amplitude decreases as muscle shortens. We used a published closed‐loop simulation model of afferented muscle to explore the mechanisms responsible for this behaviour. We demonstrate that changing muscle lengths does not suffice to explain our experimental findings. Rather, the model consistently required the modulation of γ‐static fusimotor drive to produce increases in physiological tremor with muscle shortening—while successfully replicating the concomitant reduction in stretch reflex amplitude. This need to control γ‐static fusimotor drive explicitly as a function of muscle length has important implications. First, it permits the amplitudes of physiological tremor and stretch reflex to be decoupled. Second, it postulates neuromechanical interactions that require length‐dependent γ drive modulation to be independent from α drive to the parent muscle. Lastly, it suggests that physiological tremor can be used as a simple, non‐invasive measure of the afferent mechanisms underlying healthy motor function, and their disruption in neurological conditions. This article is protected by copyright. All rights reserved
    October 11, 2017   doi: 10.1113/JP274899   open full text
  • KLF2 mediates enhanced chemoreflex sensitivity, disordered breathing and autonomic dysregulation in heart failure.
    Noah J. Marcus, Rodrigo Del Rio, Yanfeng Ding, Harold D. Schultz.
    The Journal of Physiology. October 11, 2017
    Key points Enhanced carotid body chemoreflex activity contributes to development of disordered breathing patterns, autonomic dysregulation and increases in incidence of arrhythmia in animal models of reduced ejection fraction heart failure. Chronic reductions in carotid artery blood flow are associated with increased carotid body chemoreceptor activity. Krüppel‐like Factor 2 (KLF2) is a shear stress‐sensitive transcription factor that regulates the expression of enzymes which have previously been shown to play a role in increased chemoreflex sensitivity. We investigated the impact of restoring carotid body KLF2 expression on chemoreflex control of ventilation, sympathetic nerve activity, cardiac sympatho‐vagal balance and arrhythmia incidence in an animal model of heart failure. The results indicate that restoring carotid body KLF2 in chronic heart failure reduces sympathetic nerve activity and arrhythmia incidence, and improves cardiac sympatho‐vagal balance and breathing stability. Therapeutic approaches that increase KLF2 in the carotid bodies may be efficacious in the treatment of respiratory and autonomic dysfunction in heart failure. Abstract Oscillatory breathing and increased sympathetic nerve activity (SNA) are associated with increased arrhythmia incidence and contribute to mortality in chronic heart failure (CHF). Increased carotid body chemoreflex (CBC) sensitivity plays a role in this process and can be precipitated by chronic blood flow reduction. We hypothesized that downregulation of a shear stress‐sensitive transcription factor, Krüppel‐like Factor 2 (KLF2), mediates increased CBC sensitivity in CHF and contributes to associated autonomic, respiratory and cardiac sequelae. Ventilation (Ve), renal SNA (RSNA) and ECG were measured at rest and during CBC activation in sham and CHF rabbits. Oscillatory breathing was quantified as the apnoea–hypopnoea index (AHI) and respiratory rate variability index (RRVI). AHI (control 6 ± 1/h, CHF 25 ± 1/h), RRVI (control 9 ± 3/h, CHF 29 ± 3/h), RSNA (control 22 ± 2% max, CHF 43 ± 5% max) and arrhythmia incidence (control 50 ± 10/h, CHF 300 ± 100/h) were increased in CHF at rest (FIO2 21%), as were CBC responses (Ve, RSNA) to 10% FIO2 (all P < 0.05 vs. control). In vivo adenoviral transfection of KLF2 to the carotid bodies in CHF rabbits restored KLF2 expression, and reduced AHI (7 ± 2/h), RSNA (18 ± 2% max) and arrhythmia incidence (46 ± 13/h) as well as CBC responses to hypoxia (all P < 0.05 vs. CHF empty virus). Conversely, lentiviral KLF2 siRNA in the carotid body decreased KLF2 expression, increased chemoreflex sensitivity, and increased AHI (6 ± 2/h vs. 14 ± 3/h), RRVI (5 ± 3/h vs. 20 ± 3/h) and RSNA (24 ± 4% max vs. 34 ± 5% max) relative to scrambled‐siRNA rabbits. In conclusion, down‐regulation of KLF2 in the carotid body increases CBC sensitivity, oscillatory breathing, RSNA and arrhythmia incidence during CHF.
    October 11, 2017   doi: 10.1113/JP273805   open full text
  • Plasma membrane Ca2+ ATPase 1 is required for maintaining atrial Ca2+ homeostasis and electrophysiological stability in the mouse.
    Yanwen Wang, Claire Wilson, Elizabeth J. Cartwright, Ming Lei.
    The Journal of Physiology. October 10, 2017
    To determine the role of PMCA1 in maintaining Ca2+ homeostasis and electrical stability in the atrium under physiological and stress conditions, mice with a cardiomyocyte‐specific deletion of PMCA1 (PMCA1cko) and their control littermates (PMCA1loxP/loxP) were studied at the organ and cellular levels.   At the organ level, the PMCA1cko hearts became more susceptible to atrial arrhythmias under rapid programmed electrical stimulation (PES) compared with the PMCA1loxP/loxP hearts, and such arrhythmic events became more severe under Ca2+ overload conditions. At the cellular level, the occurrence of irregular‐type APs of PMCA1cko atrial myocytes increased significantly under Ca2+ overload conditions and/or at higher frequency of stimulation. The decay of Na+‐Ca2+ exchanger (NCX) current that followed a stimulation protocol was significantly prolonged in PMCA1cko atrial myocytes under basal conditions, with Ca2+ overload leading to even greater prolongation. In conclusion, PMCA1 is required for maintaining Ca2+ homeostasis and electrical stability in the atrium. This is particularly critical during fast removal of Ca2+ from the cytosol which is required under stress conditions. This article is protected by copyright. All rights reserved
    October 10, 2017   doi: 10.1113/JP274110   open full text
  • Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus.
    Ellen Paula Santos da Conceição, Christopher J. Madden, Shaun F. Morrison.
    The Journal of Physiology. October 10, 2017
    We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling brown adipose tissue (BAT) thermogenesis. Nanoinjections of the muscarinic acetylcholine receptor (mAChR) receptor agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold‐evoked BAT sympathetic nerve activity (SNA, nadirs: −72%, and −95%), BAT temperature (TBAT, −0.5°C and −0.6°C), expired CO2 (Exp. CO2, −0.3% and −0.5%), and heart rate (HR, −22 bpm and −41 bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), TBAT (+1.8°C), Exp. CO2 (+0.7%), core temperature (TCORE, +0.5°C), and HR (+54 bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically‐active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa. This article is protected by copyright. All rights reserved
    October 10, 2017   doi: 10.1113/JP275299   open full text
  • Calcium and electrical dynamics in lymphatic endothelium.
    Erik J. Behringer, Joshua P. Scallan, Mohammad Jafarnejad, Jorge A. Castorena‐Gonzalez, Scott D. Zawieja, James E. Moore, Michael J. Davis, Steven S. Segal.
    The Journal of Physiology. October 09, 2017
    Subsequent to a rise in intracellular Ca2+ ([Ca2+]i), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼−70 mV. Acetylcholine (ACh) increased [Ca2+]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained “plateau”). In striking contrast to the endothelium‐derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+‐activated K+ channels (KCa) as confirmed with PCR, persisted in the absence of extracellular Ca2+, was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4−/− mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion. This article is protected by copyright. All rights reserved
    October 09, 2017   doi: 10.1113/JP274842   open full text
  • Leukoencephalopathy‐causing CLCN2 mutations are associated with impaired Cl− channel function and trafficking.
    Héctor Gaitán‐Peñas, Pirjo M Apaja, Tanit Arnedo, Aida Castellanos, Xabier Elorza‐Vidal, David Soto, Xavier Gasull, Gergely L Lukacs, Raúl Estévez.
    The Journal of Physiology. October 09, 2017
    Key points Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC‐2. GlialCAM, a regulatory subunit of ClC‐2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC‐2 mutant by affecting ClC‐2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC‐2 at the plasma membrane by GlialCAM depends on its localisation at cell–cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC‐2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC‐2 form a protein complex in glial cells. Abstract Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC‐2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V‐ClC‐2 functional expression. Co‐expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC‐2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC‐2 localisation to cell–cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC‐2 at the PM. Wild‐type, but not mutant, ClC‐2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy‐causing CLCN2 mutations reduce the functional expression of ClC‐2, which is partly counteracted by GlialCAM/MLC1‐mediated increase in the gating and stability of the channel.
    October 09, 2017   doi: 10.1113/JP275087   open full text
  • Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.
    David P. Burns, Arijit Roy, Eric F. Lucking, Fiona B. McDonald, Sam Gray, Richard J. Wilson, Deirdre Edge, Ken D. O'Halloran.
    The Journal of Physiology. October 09, 2017
    Key points Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO2 production is equivalent in wild‐type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild‐type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. Abstract Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8‐week‐old male wild‐type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force‐generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO2 production was not different between wild‐type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild‐type and mdx mice. Diaphragm force was severely depressed in mdx mice, with evidence of fibre remodelling and damage. Diaphragm EMG responses to chemoactivation were enhanced in mdx mice. We conclude that there is evidence of chronic hypoventilation in young mdx mice. Diaphragm dysfunction confers mechanical deficiency in mdx resulting in impaired capacity to generate normal tidal volume at rest and decreased absolute ventilation during chemoactivation. Enhanced mdx diaphragm EMG responsiveness suggests compensatory neuroplasticity facilitating respiratory motor output, which may extend to accessory muscles of breathing. Our results may have relevance to emerging treatments for human DMD aiming to preserve ventilatory capacity.
    October 09, 2017   doi: 10.1113/JP274792   open full text
  • Empowering human cardiac progenitor cells by P2Y14 nucleotide receptor overexpression.
    Farid G. Khalafalla, Waqas Kayani, Arwa Kassab, Kelli Ilves, Megan M. Monsanto, Roberto Alvarez, Monica Chavarria, Benjamin Norman, Walter P. Dembitsky, Mark A. Sussman.
    The Journal of Physiology. October 05, 2017
    Autologous cardiac progenitor cell (hCPC) therapy is a promising alternative approach to current inefficient therapies for heart failure (HF). However, ex vivo expansion and pharmacological/genetic modification of hCPCs are necessary interventions to rejuvenate aged/diseased cells and improve their regenerative capacities. This study was designed to assess the potential of improving hCPC functional capacity by targeting P2Y14 purinergic receptor (P2Y14R), which has been previously reported to induce regenerative and anti‐senescence responses in a variety of experimental models. c‐Kit+ hCPCs were isolated from cardiac biopsies of multiple HF patients undergoing left ventricular assist device (LVAD) implantation surgery. Significant correlations existed between expression of P2Y14R in hCPCs and clinical parameters of HF patients. P2Y14R was downregulated in hCPCs derived from patients with relatively lower ejection fraction and patients diagnosed with diabetes. hCPC lines with lower P2Y14R expression did not respond to P2Y14R agonist UDP‐glucose (UDP‐Glu) while hCPCs with higher P2Y14R expression showed enhanced proliferation in response to UDP‐Glu stimulation. Mechanistically, UDP‐Glu stimulation enhanced activation of canonical growth signalling pathways ERK1/2 and AKT. Restoring P2Y14R expression levels in functionally compromised hCPCs via lentiviral‐mediated overexpression improved proliferation, migration and survival under stress stimuli. Additionally, P2Y14R overexpression reversed senescence‐associated morphology and reduced levels of molecular markers of senescence p16INK4a, p53, p21 and mitochondrial reactive oxygen species (ROS). Findings from this study unveil novel biological roles of the UDP‐sugar receptor P2Y14 in hCPCs and suggest purinergic signalling modulation as a promising strategy to improve phenotypic properties of functionally impaired hCPCs. This article is protected by copyright. All rights reserved
    October 05, 2017   doi: 10.1113/JP274980   open full text
  • Rapid versus slow ascending vasodilatation: intercellular conduction versus flow‐mediated signalling with tetanic versus rhythmic muscle contractions.
    Shenghua Y. Sinkler, Steven S. Segal.
    The Journal of Physiology. October 05, 2017
    In response to exercise, vasodilatation initiated within the microcirculation of skeletal muscle ascends the resistance network into upstream feed arteries (FAs) located external to the tissue. Ascending vasodilatation (AVD) is essential to reducing FA resistance that otherwise restricts blood flow into the microcirculation. We tested the hypothesis that signalling events underlying AVD vary with the intensity and duration of muscle contraction. In the gluteus maximus muscle of anaesthetized male C57BL/6 mice (age, 3‐4 months), brief tetanic contraction (100 Hz, 500 ms) evoked rapid onset vasodilatation (ROV) in FAs that peaked within 4 s. In contrast, during rhythmic twitch contractions (4 Hz), slow onset vasodilatation (SOV) of FAs began after ∼10 s and plateaued within 30 s. Selectively damaging the endothelium with light‐dye treatment midway between a FA and its primary arteriole eliminated ROV in the FA along with conducted vasodilatation of the FA initiated on the arteriole using ACh microiontophoresis. Superfusion of SKCa and IKCa channel blockers UCL 1684 + TRAM 34 attenuated ROV, implicating endothelial hyperpolarization as the underlying signal. Nevertheless, SOV of FAs during rhythmic contractions persisted until superfusion of NO synthase with L‐NAME. Thus, ROV of FAs reflects hyperpolarization of downstream arterioles that conducts along the endothelium into proximal FAs. In contrast, SOV of FAs reflects local production of NO by the endothelium in response to luminal shear stress, which increases secondary to arteriolar dilatation downstream. Thus, AVD ensures increased oxygen delivery to active muscle fibres by reducing upstream resistance via complementary signalling pathways that reflect the intensity and duration of muscle contraction. This article is protected by copyright. All rights reserved
    October 05, 2017   doi: 10.1113/JP275186   open full text
  • Chronic beta2‐adrenoceptor agonist treatment alters muscle proteome and functional adaptations induced by high intensity training in young men.
    Morten Hostrup, Johan Onslev, Glenn Jacobson, Richard Wilson, Jens Bangsbo.
    The Journal of Physiology. October 05, 2017
    Although the effects of training have been studied for decades, data on muscle proteome signature remodelling induced by high intensity training in relation to functional changes in humans remains incomplete. Likewise, β2‐agonists are frequently used to counteract exercise‐induced bronchoconstriction, but the effects β2‐agonist treatment on muscle remodelling and adaptations to training are unknown. In a placebo‐controlled parallel study, we randomized 21 trained men to four weeks of high intensity training with (HIT + β2A) or without (HIT) daily inhalation of β2‐agonist (terbutaline, 4 mg d−1). Of 486 proteins identified by mass‐spectrometry proteomics of muscle biopsies sampled before and after the intervention, 32 and 85 were changing (FDR ≤ 5%) with the intervention in HIT and HIT + β2A. Proteome signature changes were different in HIT and HIT + β2A (P = 0.005), wherein β2‐agonist caused a repression of 25 proteins in HIT + β2A compared to HIT, and an upregulation of 7 proteins compared to HIT. β2‐agonist repressed or even downregulated training‐induced enrichment of pathways related to oxidative phosphorylation and glycogen metabolism, but upregulated pathways related to histone trimethylation and the nucleosome. Muscle contractile phenotype changed differently in HIT and HIT + β2A (P ≤ 0.001), with a fast‐to‐slow twitch transition in HIT and a slow‐to‐fast twitch transition in HIT + β2A. β2‐agonist attenuated training‐induced enhancements in maximal oxygen consumption (P ≤ 0.01) and exercise performance (11.6 vs. 6.1%, P ≤ 0.05) in HIT + β2A compared to HIT. These findings indicate that daily β2‐agonist treatment attenuates the beneficial effects of high intensity training on exercise performance and oxidative capacity, and causes remodelling of muscle proteome signature towards a fast‐twitch phenotype. This article is protected by copyright. All rights reserved
    October 05, 2017   doi: 10.1113/JP274970   open full text
  • Post‐exercise recovery of contractile function and endurance in humans and mice is accelerated by heating and slowed by cooling skeletal muscle.
    Arthur J. Cheng, Sarah J. Willis, Christoph Zinner, Thomas Chaillou, Niklas Ivarsson, Niels Ørtenblad, Johanna T. Lanner, Hans‐Christer Holmberg, Håkan Westerblad.
    The Journal of Physiology. October 04, 2017
    Manipulation of muscle temperature is believed to improve post‐exercise recovery, with cooling being especially popular among athletes. However, it is unclear whether such temperature manipulations actually have positive effects. Accordingly, we studied the effect of muscle temperature on the acute recovery of force and fatigue resistance after endurance exercise. One hour of moderate‐intensity arm cycling exercise in humans was followed by two hours recovery in which the upper arms were either heated to 38°C, not treated (33°C), or cooled to ∼15°C. Fatigue resistance after the recovery period was assessed by performing 3 × 5 min sessions of all‐out arm cycling at physiological temperature for all conditions (i.e. not heated or cooled). Power output during the all‐out exercise was better maintained when muscles were heated during recovery, whereas cooling had the opposite effect. Mechanisms underlying the temperature‐dependent effect on recovery were tested in mouse intact single muscle fibres, which were exposed to ∼12 min of glycogen‐depleting fatiguing stimulation (350 ms tetani given at 10 s interval until force decreased to 30% of the starting force). Fibres were subsequently exposed to the same fatiguing stimulation protocol after 1–2 h of recovery at 16–36°C. Recovery of submaximal force (30 Hz), the tetanic myoplasmic free [Ca2+] (measured with the fluorescent indicator indo‐1), and fatigue resistance were all impaired by cooling (16‐26°C) and improved by heating (36°C). In addition, glycogen resynthesis was faster at 36°C than 26°C in whole FDB muscles. We conclude that recovery from exhaustive endurance exercise is accelerated by raising and slowed by lowering muscle temperature. This article is protected by copyright. All rights reserved
    October 04, 2017   doi: 10.1113/JP274870   open full text
  • Rapid decline in MyHC I(β) mRNA expression in rat soleus during hindlimb unloading is associated with the AMPK dephosphorylation.
    Natalia A. Vilchinskaya, Ekaterina P. Mochalova, Tatiana L. Nemirovskaya, Timur M. Mirzoev, Olga V. Turtikova, Boris S. Shenkman.
    The Journal of Physiology. October 03, 2017
    One of the key events that occurs during skeletal muscle inactivation is a change in myosin phenotype, i.e. increased expression of fast isoforms and decreased expression of slow isoform of myosin heavy chain (MyHC). It is known that calcineurin/NFAT and AMP‐activated protein kinase (AMPK) can regulate the expression of genes encoding MyHC slow isoform. Earlier, we found a significant decrease in phosphorylated AMPK in rat soleus after 24 h of hindlimb unloading (HU). We hypothesized that a decrease in AMPK phosphorylation and subsequent histone deacetylase (HDAC) nuclear translocation can be one of the triggering events leading to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with AMPK activator (AICAR) for 6 d before HU as well as during 24‐h HU. We discovered that AICAR treatment prevented a decrease in pre‐mRNA and mRNA expression of MyHC I as well as MyHC IIa mRNA expression. 24‐h HS resulted in HDAC4 accumulation in the nuclei of rat soleus but AICAR pretreatment prevented such an accumulation. The results of the study indicate that AMPK dephosphorylation after 24‐h HU had a significant impact on the MyHC I and MyHC IIa mRNA expression in rat soleus. AMPK dephosphorylation also contributed to the HDAC4 translocation to the nuclei of soleus muscle fibres, suggesting an important role of HDAC4 as an epigenetic regulator in the process of myosin phenotype transformation. This article is protected by copyright. All rights reserved
    October 03, 2017   doi: 10.1113/JP275184   open full text
  • δ‐ and β‐cells are electrically coupled and regulate α‐cell activity via somatostatin.
    L. J. B. Briant, T. M. Reinbothe, I. Spiliotis, C. Miranda, B. Rodriguez, P. Rorsman.
    The Journal of Physiology. October 03, 2017
    Glucagon, the body's principal hyperglycaemic hormone, is released from the α‐cells of the pancreatic islet. The secretion of this hormone is dysregulated in type 2 diabetes mellitus but the mechanisms controlling secretion are not well understood. Regulation of glucagon secretion by factors secreted by neighbouring β‐ and δ‐cells (paracrine regulation) have been proposed to be important. In this study, we explored the importance of paracrine regulation by using an optogenetic strategy. Specific light‐induced activation of β‐cells in mouse islets expressing the light‐gated channelrhodopsin‐2 resulted in stimulation of electrical activity in δ‐cells but suppression of α‐cell activity. The activation of the δ‐cells was rapid and sensitive to the gap junction inhibitor carbenoxolone, whereas the effect on electrical activity in α‐cells was blocked by CYN 154806, an antagonist of the somatostatin‐2 receptor. These observations indicate that optogenetic activation of the β‐cells propagates to the δ‐cells via gap junctions, and the consequential stimulation of somatostatin secretion inhibits α‐cell electrical activity by a paracrine mechanism. To explore whether this pathway is important for regulating α‐cell activity and glucagon secretion in human islets, we constructed computational models of human islets. These models had detailed architectures based on human islets and consisted of a collection of >500 α‐, β‐ and δ‐cells. Simulations of these models revealed that this gap junctional/paracrine mechanism accounts for up to 23% of the suppression of glucagon secretion by high glucose. This article is protected by copyright. All rights reserved
    October 03, 2017   doi: 10.1113/JP274581   open full text
  • Maternal exercise modifies body composition and energy substrates handling in high‐fat/high‐sucrose diet fed male offspring.
    Charline Quiclet, Hervé Dubouchaud, Phanélie Berthon, Hervé Sanchez, Guillaume Vial, Farida Siti, Eric Fontaine, Cécile Batandier, Karine Couturier.
    The Journal of Physiology. October 03, 2017
    Maternal exercise during gestation has been reported to modify offspring metabolism and health. Whether these effects are exacerbated when offspring is under high‐fat diet remains rather unclear. Our purpose was to evaluate the effect of maternal exercise before and during gestation on the offspring fed a high‐fat/high‐sucrose diet (HF), by assessing its body composition, pancreatic function and the energy substrates handling by two major glucose‐utilizing tissues: liver and muscle. Fifteen week‐old nulliparous female Wistar rats exercised 4 weeks before as well as during gestation at a constant submaximal intensity (TR) or remained sedentary (CT). At weaning, pups from each group were fed either a standard diet (TRCD or CTCD) or a high‐fat/high‐sucrose diet (TRHF or CTHF) for 10 weeks. Offspring from TR dams gained less weight compared to those from CT dams. Selected fat depots were larger with HF diet compared to CD but significantly smaller in TRHF compared to CTHF. Surprisingly, insulin secretion index was higher in islets from HF offspring compared to CD. TR offspring showed a higher muscle insulin sensitivity estimated by the pPKB/PKB ratio compared with CT offspring (+48%, P < 0.05). With CD, permeabilized isolated muscle fibres from TR rats displayed a lower apparent affinity constant (Km) for pyruvate and palmitoyl Co‐A as substrates compared to the CT group (−46% and −58% respectively, P < 0.05). These results suggest that maternal exercise has positive effects on young adult offspring body composition and on muscle carbohydrate and lipid metabolism depending on the nutritional status. This article is protected by copyright. All rights reserved
    October 03, 2017   doi: 10.1113/JP274739   open full text
  • Visuo‐manual tracking: does intermittent control with aperiodic sampling explain linear power and non‐linear remnant without sensorimotor noise?
    Henrik Gollee, Peter J. Gawthrop, Martin Lakie, Ian D. Loram.
    The Journal of Physiology. October 01, 2017
    Key points A human controlling an external system is described most easily and conventionally as linearly and continuously translating sensory input to motor output, with the inevitable output remnant, non‐linearly related to the input, attributed to sensorimotor noise. Recent experiments show sustained manual tracking involves repeated refractoriness (insensitivity to sensory information for a certain duration), with the temporary 200–500 ms periods of irresponsiveness to sensory input making the control process intrinsically non‐linear. This evidence calls for re‐examination of the extent to which random sensorimotor noise is required to explain the non‐linear remnant. This investigation of manual tracking shows how the full motor output (linear component and remnant) can be explained mechanistically by aperiodic sampling triggered by prediction error thresholds. Whereas broadband physiological noise is general to all processes, aperiodic sampling is associated with sensorimotor decision making within specific frontal, striatal and parietal networks; we conclude that manual tracking utilises such slow serial decision making pathways up to several times per second. Abstract The human operator is described adequately by linear translation of sensory input to motor output. Motor output also always includes a non‐linear remnant resulting from random sensorimotor noise from multiple sources, and non‐linear input transformations, for example thresholds or refractory periods. Recent evidence showed that manual tracking incurs substantial, serial, refractoriness (insensitivity to sensory information of 350 and 550 ms for 1st and 2nd order systems respectively). Our two questions are: (i) What are the comparative merits of explaining the non‐linear remnant using noise or non‐linear transformations? (ii) Can non‐linear transformations represent serial motor decision making within the sensorimotor feedback loop intrinsic to tracking? Twelve participants (instructed to act in three prescribed ways) manually controlled two systems (1st and 2nd order) subject to a periodic multi‐sine disturbance. Joystick power was analysed using three models, continuous‐linear‐control (CC), continuous‐linear‐control with calculated noise spectrum (CCN), and intermittent control with aperiodic sampling triggered by prediction error thresholds (IC). Unlike the linear mechanism, the intermittent control mechanism explained the majority of total power (linear and remnant) (77–87% vs. 8–48%, IC vs. CC). Between conditions, IC used thresholds and distributions of open loop intervals consistent with, respectively, instructions and previous measured, model independent values; whereas CCN required changes in noise spectrum deviating from broadband, signal dependent noise. We conclude that manual tracking uses open loop predictive control with aperiodic sampling. Because aperiodic sampling is inherent to serial decision making within previously identified, specific frontal, striatal and parietal networks we suggest that these structures are intimately involved in visuo‐manual tracking.
    October 01, 2017   doi: 10.1113/JP274288   open full text
  • Inducible satellite cell depletion attenuates skeletal muscle regrowth following a scald‐burn injury.
    Celeste C. Finnerty, Colleen F. McKenna, Lauren A. Cambias, Camille R. Brightwell, Anesh Prasai, Ye Wang, Amina El Ayadi, David N. Herndon, Oscar E. Suman, Christopher S. Fry.
    The Journal of Physiology. October 01, 2017
    Key points Severe burns result in significant skeletal muscle cachexia that impedes recovery. Activity of satellite cells, skeletal muscle stem cells, is altered following a burn injury and likely hinders regrowth of muscle. Severe burn injury induces satellite cell proliferation and fusion into myofibres with greater activity in muscles proximal to the injury site. Conditional depletion of satellite cells attenuates recovery of myofibre area and volume following a scald burn injury in mice. Skeletal muscle regrowth following a burn injury requires satellite cell activity, underscoring the therapeutic potential of satellite cells in the prevention of prolonged frailty in burn survivors. Abstract Severe burns result in profound skeletal muscle atrophy; persistent muscle atrophy and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma, and we have previously shown concurrent activation and apoptosis of muscle satellite cells following a burn injury in paediatric patients. To determine the necessity of satellite cells during muscle recovery following a burn injury, we utilized a genetically modified mouse model (Pax7CreER‐DTA) that allows for the conditional depletion of satellite cells in skeletal muscle. Additionally, mice were provided 5‐ethynyl‐2′‐deoxyuridine to determine satellite cell proliferation, activation and fusion. Juvenile satellite cell‐wild‐type (SC‐WT) and satellite cell‐depleted (SC‐Dep) mice (8 weeks of age) were randomized to sham or burn injury consisting of a dorsal scald burn injury covering 30% of total body surface area. Both hindlimb and dorsal muscles were studied at 7, 14 and 21 days post‐burn. SC‐Dep mice had >93% depletion of satellite cells compared to SC‐WT (P < 0.05). Burn injury induced robust atrophy in muscles located both proximal and distal to the injury site (∼30% decrease in fibre cross‐sectional area, P < 0.05). Additionally, burn injury induced skeletal muscle regeneration, satellite cell proliferation and fusion. Depletion of satellite cells impaired post‐burn recovery of both muscle fibre cross‐sectional area and volume (P < 0.05). These findings support an integral role for satellite cells in the aetiology of lean tissue recovery following a severe burn injury.
    October 01, 2017   doi: 10.1113/JP274841   open full text
  • Defining the neural fulcrum for chronic vagus nerve stimulation: implications for integrated cardiac control.
    Jeffrey L. Ardell, Heath Nier, Matthew Hammer, E. Marie Southerland, Christopher L. Ardell, Eric Beaumont, Bruce H. KenKnight, J. Andrew Armour.
    The Journal of Physiology. September 30, 2017
    Key points The evoked cardiac response to bipolar cervical vagus nerve stimulation (VNS) reflects a dynamic interaction between afferent mediated decreases in central parasympathetic drive and suppressive effects evoked by direct stimulation of parasympathetic efferent axons to the heart. The neural fulcrum is defined as the operating point, based on frequency–amplitude–pulse width, where a null heart rate response is reproducibly evoked during the on‐phase of VNS. Cardiac control, based on the principal of the neural fulcrum, can be elicited from either vagus. Beta‐receptor blockade does not alter the tachycardia phase to low intensity VNS, but can increase the bradycardia to higher intensity VNS. While muscarinic cholinergic blockade prevented the VNS‐induced bradycardia, clinically relevant doses of ACE inhibitors, beta‐blockade and the funny channel blocker ivabradine did not alter the VNS chronotropic response. While there are qualitative differences in VNS heart control between awake and anaesthetized states, the physiological expression of the neural fulcrum is maintained. Abstract Vagus nerve stimulation (VNS) is an emerging therapy for treatment of chronic heart failure and remains a standard of therapy in patients with treatment‐resistant epilepsy. The objective of this work was to characterize heart rate (HR) responses (HRRs) during the active phase of chronic VNS over a wide range of stimulation parameters in order to define optimal protocols for bidirectional bioelectronic control of the heart. In normal canines, bipolar electrodes were chronically implanted on the cervical vagosympathetic trunk bilaterally with anode cephalad to cathode (n = 8, ‘cardiac’ configuration) or with electrode positions reversed (n = 8, ‘epilepsy’ configuration). In awake state, HRRs were determined for each combination of pulse frequency (2–20 Hz), intensity (0–3.5 mA) and pulse widths (130–750 μs) over 14 months. At low intensities and higher frequency VNS, HR increased during the VNS active phase owing to afferent modulation of parasympathetic central drive. When functional effects of afferent and efferent fibre activation were balanced, a null HRR was evoked (defined as ‘neural fulcrum’) during which HRR ≈ 0. As intensity increased further, HR was reduced during the active phase of VNS. While qualitatively similar, VNS delivered in the epilepsy configuration resulted in more pronounced HR acceleration and reduced HR deceleration during VNS. At termination, under anaesthesia, transection of the vagi rostral to the stimulation site eliminated the augmenting response to VNS and enhanced the parasympathetic efferent‐mediated suppressing effect on electrical and mechanical function of the heart. In conclusion, VNS activates central then peripheral aspects of the cardiac nervous system. VNS control over cardiac function is maintained during chronic therapy.
    September 30, 2017   doi: 10.1113/JP274678   open full text
  • Pre‐clinical evaluation of N‐acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy.
    Gavin J. Pinniger, Jessica R. Terrill, Evanna B. Assan, Miranda D. Grounds, Peter G. Arthur.
    The Journal of Physiology. September 30, 2017
    Key points Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N‐acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. Abstract Duchenne muscular dystrophy (DMD) is a fatal X‐linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N‐acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non‐dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC‐treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw)−1; P < 0.001] and C57 mice [3.90 (0.32) vs 5.32 (0.60) g bw−1; P < 0.001]. Maximum specific force was significantly greater in NAC‐treated mdx muscles [9.80 (2.27) vs 13.07 (3.37) N cm−2; P = 0.038]. Increased force in mdx mice was associated with reduced thiol oxidation and inflammation in fast muscles, and increased citrate synthase activity in slow muscle. Importantly, NAC significantly impaired body weight gain in both strains of young growing mice, and reduced liver weight in C57 mice and muscle weight in mdx mice. These potentially adverse effects of NAC emphasise the need for caution when interpreting improvements in muscle function based on normalised force measures, and that careful consideration be given to these effects when proposing NAC as a potential treatment for young DMD boys.
    September 30, 2017   doi: 10.1113/JP274229   open full text
  • Interstitial IgG antibody pharmacokinetics assessed by combined in vivo‐ and physiologically‐based pharmacokinetic modelling approaches.
    Miro J. Eigenmann, Tine V. Karlsen, Ben‐Fillippo Krippendorff, Olav Tenstad, Ludivine Fronton, Michael B. Otteneder, Helge Wiig.
    The Journal of Physiology. September 27, 2017
    For most therapeutic antibodies, the interstitium is the target space. Although experimental methods for measuring antibody pharmacokinetics (PK) in this space are not well established, making quantitative assessment difficult, the interstitial antibody concentration is assumed to be low. Here, we combined direct quantification of antibodies in the interstitial fluid with a physiologically‐based PK (PBPK) modelling approach with the goal of better describing the PK of monoclonal antibodies in the interstitial space of different tissues. We isolated interstitial fluid by tissue centrifugation, and conducted an antibody biodistribution study in mice, measuring total tissue‐ and interstitial concentrations in selected tissues. Residual plasma, interstitial volumes and lymph flows, which are important PBPK model parameters, were assessed in vivo. We could thereby refine PBPK modelling of monoclonal antibodies, better interpret antibody biodistribution data and more accurately predict their PK in the different tissue spaces. Our results indicate that in tissues with discontinuous capillaries (liver and spleen), interstitial concentrations are reflected by plasma concentration. In tissues with continuous capillaries (e.g. skin and muscle), ∼50‐60% of plasma concentration is found in the interstitial space. In brain and kidney, on the other hand, antibodies are restricted to the vascular space. Our data may significantly impact the interpretation of biodistribution data of monoclonal antibodies and might be important when relating measured concentrations to a therapeutic effect. Opposing the view that antibodies distribution to the interstitial space is limited, we show by direct measurements and model‐based data interpretation that high antibody interstitial concentrations are reached in most tissues. This article is protected by copyright. All rights reserved
    September 27, 2017   doi: 10.1113/JP274819   open full text
  • The α2A adrenoceptor suppresses excitatory synaptic transmission to both excitatory and inhibitory neurons in layer 4 barrel cortex.
    Minoru Ohshima, Chiaki Itami, Fumitaka Kimura.
    The Journal of Physiology. September 26, 2017
    The mammalian neocortex is widely innervated by noradrenergic (NA) fibres from the locus coeruleus. To determine the effects of NA on vertical synaptic inputs to layer 4 (L4) cells from the ventrobasal (VB) thalamus and layer 2/3 (L2/3), thalamocortical slices were prepared and whole‐cell recordings were made from L4 cells. Excitatory synaptic responses were evoked by electrical stimulation of the thalamus or L2/3 immediately above. Recorded cells were identified as regular spiking (RS), regular spiking non‐pyramidal (RSNP) or fast spiking (FS) cells through their firing patterns in response to current injections. NA suppressed (∼50% of control) excitatory vertical inputs to all cell types in a dose‐dependent manner. The presynaptic site of action of NA was suggested by three independent studies. First, responses caused by iontophoretically applied glutamate were not suppressed by NA. Second, paired pulse ratio was increased during NA suppression. Finally, a CV−2 (CV: coefficient of variation) analysis was performed. The resultant diagonal alignment of the ratio of CV−2 plotted against the ratio of the amplitude of postsynaptic responses suggests a presynaptic mechanism for the suppression. Experiments with phenylephrine (α1 agonist), prazosin (α1 antagonist), yohimbine (α2 antagonist) and propranolol (β antagonist) indicated that suppression was mediated by α2 adrenoceptor. To determine whether the α2A adrenoceptor subtype was involved, α2A adrenoceptor knockout mice were used. NA failed to suppress EPSCs in all cell types, suggesting an involvement of α2A adrenoceptor. Altogether, we concluded that NA suppresses vertical excitatory synaptic connections in L4 excitatory and inhibitory cells through presynaptic α2A adrenoceptor. This article is protected by copyright. All rights reserved
    September 26, 2017   doi: 10.1113/JP275142   open full text
  • Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells.
    Tam T. T. Phuong, Sarah N. Redmon, Oleg Yarishkin, Jacob M. Winter, Dean Y. Li, David Križaj.
    The Journal of Physiology. September 26, 2017
    The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Taking advantage of immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca2+/cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVECs (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca2+]i and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F‐actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE‐cadherin) and β−catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type, but not in TRPV4 knockout mice. Agonist‐evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells (CECs) showed lower TRPV4 mRNA/protein levels and less pronounced agonist‐evoked calcium signals compared to MVECs. These findings demonstrate a major role for TRPV4 in Ca2+ homeostasis and barrier function in the human retinal microvascular endothelial barrier and suggest TRPV4 may differentially contribute to the inner vis à vis outer blood‐retinal barrier function. This article is protected by copyright. All rights reserved
    September 26, 2017   doi: 10.1113/JP275052   open full text
  • Cortical thickness is associated with altered autonomic function in cognitively impaired and non‐impaired older adults.
    Feng Lin, Ping Ren, Xixi Wang, Mia Anthony, Duje Tadin, Kathi L. Heffner.
    The Journal of Physiology. September 26, 2017
    Background Parasympathetic nervous system (PNS) is critical for adaptation to environment demands. PNS can reflect an individual's regulatory capacity of frontal brain regions and has been linked to cognitive capacity. Yet, the relationship of PNS function with cognitive decline and abnormal frontal function that characterize preclinical progression toward Alzheimer's disease (AD) is unclear. Here, we aimed to elucidate the relationship between PNS function and AD‐associated neurodegeneration by testing two competing hypotheses involving frontal regions’ activity (neurodegeneration vs. compensation). Methods In 38 older human adults with amnestic mild cognitive impairment (aMCI) or normative cognition, we measured AD‐associated neurodegeneration (AD signature cortical thickness; ADSCT), resting‐state fMRI of frontal regions’ spontaneous activation, and an electrocardiography measure of PNS (high frequency heart rate variability; HF‐HRV). HF‐HRV was assessed at rest and during a cognitive task protocol designed to capture HF‐HRV reactivity. Results Higher HF‐HRV at rest was significantly related to both more severe AD‐associated neurodegeneration (lower ADSCT scores) and worse cognitive ability. Cognitive impairments were also related to greater suppression of HF‐HRV reactivity. High activities of the anterior cingulate cortex significantly mediated relationships between ADSCT and both HF‐HRV at rest and HF‐HRV reactivity. Notably, these relationships were not affected by the clinical phenotype. Conclusions We show that AD‐associated neurodegeneration is associated with altered PNS regulation and that compensatory processes linked to frontal overactivation might be responsible for those alterations. This finding provides the first line of evidence in a new framework for understanding how early‐stage AD‐associated neurodegeneration affects autonomic regulation. This article is protected by copyright. All rights reserved
    September 26, 2017   doi: 10.1113/JP274714   open full text
  • An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular type I hair cells.
    Paolo Spaiardi, Elisa Tavazzani, Marco Manca, Veronica Milesi, Giancarlo Russo, Ivo Prigioni, Walter Marcotti, Jacopo Magistretti, Sergio Masetto.
    The Journal of Physiology. September 24, 2017
    Key points Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low‐voltage‐activated outward rectifying K+ current, IK,L, whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L. We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L. Abstract Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low‐voltage‐activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch‐clamp whole‐cell technique, we examined the voltage‐ and time‐dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half‐activation at –79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The ‘early’ open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the ‘native’ biophysical properties of IK,L in adult mouse vestibular type I hair cells.
    September 24, 2017   doi: 10.1113/JP274202   open full text
  • Sex differences in the role of phospholipase A2‐dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.
    Abdulla A. Ahmad, Michael D. Randall, Richard E. Roberts.
    The Journal of Physiology. September 24, 2017
    Key points The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT‐induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT‐induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. Abstract Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase‐derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re‐added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re‐addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT‐induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT‐induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT‐induced vasoconstriction in male and female PCAs, respectively.
    September 24, 2017   doi: 10.1113/JP274831   open full text
  • Do right‐ventricular trabeculae gain energetic advantage from having a greater velocity of shortening?
    Toan Pham, June‐Chiew Han, Andrew Taberner, Denis Loiselle.
    The Journal of Physiology. September 24, 2017
    Key points We designed a study to test whether velocity of shortening in right‐ventricular tissue preparations is greater than that of the left side under conditions mimicking those encountered by the heart in vivo. Our experiments allowed us to explore whether greater velocity of shortening results in any energetic advantage. We found that velocity of shortening was higher in the rat right‐ventricular trabeculae. These results at the tissue level seem paradoxical to the velocity of ventricular ejection at the organ level, and are not always in accord with shortening of unloaded cells. Despite greater velocity of shortening in right‐ventricular trabeculae, they neither gained nor lost advantage with respect to both mechanical efficiency and the heat generated during shortening. Abstract Our study aimed to ascertain whether the interventricular difference of shortening velocity, reported for isolated cardiac tissues in vitro, affects interventricular mechano‐energetic performance when tested under physiological conditions using a shortening protocol designed to mimic those in vivo. We isolated trabeculae from both ventricles of the rat, mounted them in a calorimeter, and performed experiments at 37°C and 5 Hz stimulus frequency to emulate conditions of the rat heart in vivo. Each trabecula was subjected to two experimental protocols: (i) isotonic work‐loop contractions at a variety of afterloads, and (ii) isometric contractions at a variety of preloads. Velocity of shortening was calculated from the former protocol during the isotonic shortening phase of the contraction. Simultaneous measurements of force–length work and heat output allowed calculation of mechanical efficiency. The shortening‐dependent thermal component was quantified from the difference in heat output between the two protocols. Our results show that both extent of shortening and velocity of shortening were higher in trabeculae from the right ventricle. Despite these differences, trabeculae from both ventricles developed the same stress, performed the same work, liberated the same amount of heat, and hence operated at the same mechanical efficiency. Shortening heat was also ventricle independent. The interventricular differences in velocity of shortening and extent of shortening of isolated trabeculae were not manifested in any index of energetics. These collective results underscore the absence of any mechano‐energetic advantage or disadvantage conferred on right‐ventricular trabeculae arising from their superior velocity of shortening.
    September 24, 2017   doi: 10.1113/JP274837   open full text
  • Elevated resting H+ current in the R1239H type 1 hypokalaemic periodic paralysis mutated Ca2+ channel.
    Clarisse Fuster, Jimmy Perrot, Christine Berthier, Vincent Jacquemond, Bruno Allard.
    The Journal of Physiology. September 24, 2017
    Key points Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage‐gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. Acute expression of human wild‐type and R1239H HypoPP1 mutant α1 subunits in mature mouse muscles showed that R1239H fibres displayed Ca2+ currents of reduced amplitude and larger resting leak inward current increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data suggest that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore and could explain why paralytic attacks preferentially occur during the recovery period following muscle exercise. Abstract Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage‐gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+. The present study aimed at identifying the changes in muscle fibre electrical properties induced by acute expression of the R1239H hypokalaemic periodic paralysis human mutant α1 subunit of Ca2+ channels in a mature muscle environment to better understand the pathophysiological mechanisms involved in this disorder. We transferred genes encoding wild‐type and R1239H mutant human Ca2+ channels into hindlimb mouse muscle by electroporation and combined voltage‐clamp and intracellular pH measurements on enzymatically dissociated single muscle fibres. As compared to fibres expressing wild‐type α1 subunits, R1239H mutant‐expressing fibres displayed Ca2+ currents of reduced amplitude and a higher resting leak inward current that was increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents. These data indicate that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore created by the mutation and that external acidification favours onset of muscle paralysis by potentiating H+ depolarizing currents and inhibiting resting inward rectifier K+ currents. Our results could thus explain why paralytic attacks preferentially occur during the recovery period following intense muscle exercise.
    September 24, 2017   doi: 10.1113/JP274638   open full text
  • Feto‐ and utero‐placental vascular adaptations to chronic maternal hypoxia in the mouse.
    Lindsay S. Cahill, Monique Y. Rennie, Johnathan Hoggarth, Lisa X. Yu, Anum Rahman, John C. Kingdom, Mike Seed, Christopher K. Macgowan, John G. Sled.
    The Journal of Physiology. September 24, 2017
    Key points Chronic fetal hypoxia is one of the most common complications of pregnancy and is known to cause fetal growth restriction. The structural adaptations of the placental vasculature responsible for growth restriction with chronic hypoxia are not well elucidated. Using a mouse model of chronic maternal hypoxia in combination with micro‐computed tomography and scanning electron microscopy, we found several placental adaptations that were beneficial to fetal growth including capillary expansion, thinning of the interhaemal membrane and increased radial artery diameters, resulting in a large drop in total utero‐placental vascular resistance. One of the mechanisms used to achieve the rapid increase in capillaries was intussusceptive angiogenesis, a strategy used in human placental development to form terminal gas‐exchanging villi. These results contribute to our understanding of the structural mechanisms of the placental vasculature responsible for fetal growth restriction and provide a baseline for understanding adaptive physiological responses of the placenta to chronic hypoxia. Abstract The fetus and the placenta in eutherian mammals have a unique set of compensatory mechanisms to respond to several pregnancy complications including chronic maternal hypoxia. This study examined the structural adaptations of the feto‐ and utero‐placental vasculature in an experimental mouse model of chronic maternal hypoxia (11% O2 from embryonic day (E) 14.5–E17.5). While placental weights were unaffected by exposure to chronic hypoxia, using micro‐computed tomography, we found a 44% decrease in the absolute feto‐placental arterial vascular volume and a 30% decrease in total vessel segments in the chronic hypoxia group compared to control group. Scanning electron microscopy imaging showed significant expansion of the capillary network; consequently, the interhaemal membrane was 11% thinner to facilitate maternal–fetal exchange in the chronic hypoxia placentas. One of the mechanisms for the rapid capillary expansion was intussusceptive angiogenesis. Analysis of the utero‐placental arterial tree showed significant increases (24%) in the diameter of the radial arteries, resulting in a decrease in the total utero‐placental resistance by 2.6‐fold in the mice exposed to chronic maternal hypoxia. Together these adaptations acted to preserve placental weight whereas fetal weight was decreased.
    September 24, 2017   doi: 10.1113/JP274845   open full text
  • Volume loading augments cutaneous vasodilatation and cardiac output of heat stressed older adults.
    Daniel Gagnon, Steven A. Romero, Hai Ngo, Satyam Sarma, William K. Cornwell, Paula Y. S. Poh, Douglas Stoller, Benjamin D. Levine, Craig G. Crandall.
    The Journal of Physiology. September 22, 2017
    Key points Age‐related changes in cutaneous microvascular and cardiac functions limit the extent of cutaneous vasodilatation and the increase in cardiac output that healthy older adults can achieve during passive heat stress. However, it is unclear if these age‐related changes in microvascular and cardiac functions maximally restrain the levels of cutaneous vasodilatation and cardiac output that healthy older adults can achieve during heat stress. We observed that rapid volume loading, performed during passive heat stress, augments both cutaneous vasodilatation and cardiac output in healthy older humans. These findings demonstrate that the microcirculation of healthy aged skin can further dilate during passive heat exposure, despite peripheral limitations to vasodilatation. Furthermore, healthy older humans can augment cardiac output when cardiac pre‐load is increased during heat stress. Abstract Primary ageing markedly attenuates cutaneous vasodilatation and the increase in cardiac output during passive heating. However, it remains unclear if these responses are maximally restrained by age‐related changes in cutaneous microvascular and cardiac functions. We hypothesized that rapid volume loading performed during heat stress would increase cardiac output in older adults without parallel increases in cutaneous vasodilatation. Twelve young (Y: 26 ± 5 years) and ten older (O: 69 ± 3 years) healthy adults were passively heated until core temperature increased by 1.5°C. Cardiac output (thermodilution), forearm vascular conductance (FVC, venous occlusion plethysmography) and cutaneous vascular conductance (CVC, laser‐Doppler) were measured before and after rapid infusion of warmed saline (15 mL kg−1, ∼7 min). While heat stressed, but prior to saline infusion, cardiac output (O: 6.8 ± 0.4 vs. Y: 9.4 ± 0.6 L min−1), FVC (O: 0.08 ± 0.01 vs. Y: 0.17 ± 0.02 mL (100 mL min−1 mmHg−1)−1), and CVC (O: 1.29 ± 0.34 vs. Y: 1.93 ± 0.30 units mmHg−1) were lower in older adults (all P < 0.01). Rapid saline infusion increased cardiac output (O: +1.9 ± 0.3, Y: +1.8 ± 0.7 L min−1), FVC (O: +0.015 ± 0.007, Y: +0.048 ± 0.013 mL (100 mL min−1 mmHg−1)−1), and CVC (O: +0.28 ± 0.10, Y: +0.29 ± 0.16 units mmHg−1) in both groups (all P < 0.01). The absolute increase in cardiac output and CVC were similar between groups, whereas FVC increased to a greater extent in young adults (P < 0.01). These results demonstrate that healthy older adults can achieve greater levels of cutaneous vasodilatation and cardiac output during passive heating.
    September 22, 2017   doi: 10.1113/JP274742   open full text
  • Subunit‐dependent oxidative stress sensitivity of LRRC8 volume‐regulated anion channels.
    Antonella Gradogna, Paola Gavazzo, Anna Boccaccio, Michael Pusch.
    The Journal of Physiology. September 22, 2017
    Key points Swelling‐activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel‐forming proteins or on regulatory factors. We found that LRRC8A–LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A–LRRC8C and LRRC8A–LRRC8D heteromers are inhibited by oxidation. Volume‐regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells. Our results show that LRRC8 channel proteins are directly modulated by oxidation in a subunit‐specific manner. Abstract The volume‐regulated anion channel (VRAC) is formed by heteromers of LRRC8 proteins containing the essential LRRC8A subunit and at least one among the LRRC8B–E subunits. Reactive oxygen species (ROS) play physiological and pathophysiological roles and VRAC channels are highly ROS sensitive. However, it is unclear if ROS act directly on the channels or on molecules involved in the activation pathway. We used fluorescently tagged LRRC8 proteins that yield large constitutive currents to test direct effects of oxidation. We found that 8A/8E heteromers are dramatically potentiated (more than 10‐fold) by oxidation of intracellular cysteine residues by chloramine‐T or tert‐butyl hydroperoxide. Oxidation was, however, not necessary for hypotonicity‐induced activation. In contrast, 8A/8C and 8A/8D heteromers were strongly inhibited by oxidation. Endogenous VRAC currents in Jurkat T lymphocytes were similarly inhibited by oxidation, in agreement with the finding that LRRC8C and LRRC8D subunits were more abundantly expressed than LRRC8E in Jurkat cells. Our results show that LRRC8 channels are directly modulated by oxidation in a subunit‐dependent manner.
    September 22, 2017   doi: 10.1113/JP274795   open full text
  • Co‐ordination of the upper and lower limbs for vestibular control of balance.
    Craig P. Smith, Jonathan E. Allsop, Michael Mistry, Raymond F. Reynolds.
    The Journal of Physiology. September 22, 2017
    Key points When standing and holding an earth‐fixed object, galvanic vestibular stimulation (GVS) can evoke upper limb responses to maintain balance. In the present study, we determined how these responses are affected by grip context (no contact, light grip and firm grip), as well as how they are co‐ordinated with the lower limbs to maintain balance. When GVS was applied during firm grip, hand and ground reaction forces were generated. The directions of these force vectors were co‐ordinated such that the overall body sway response was always aligned with the inter‐aural axis (i.e. craniocentric). When GVS was applied during light grip (< 1 N), hand forces were secondary to body movement, suggesting that the arm performed a mostly passive role. These results demonstrate that a minimum level of grip is required before the upper limb becomes active in balance control and also that the upper and lower limbs co‐ordinate for an appropriate whole‐body sway response. Abstract Vestibular stimulation can evoke responses in the arm when it is used for balance. In the present study, we determined how these responses are affected by grip context, as well as how they are co‐ordinated with the rest of the body. Galvanic vestibular stimulation (GVS) was used to evoke balance responses under three conditions of manual contact with an earth‐fixed object: no contact, light grip (< 1 N) (LG) and firm grip (FG). As grip progressed along this continuum, we observed an increase in GVS‐evoked hand force, with a simultaneous reduction in ground reaction force (GRF) through the feet. During LG, hand force was secondary to the GVS‐evoked body sway response, indicating that the arm performed a mostly passive role. By contrast, during FG, the arm became actively involved in driving body sway, as revealed by an early force impulse in the opposite direction to that seen in LG. We then examined how the direction of this active hand vector was co‐ordinated with the lower limbs. Consistent with previous findings on sway anisotropy, FG skewed the direction of the GVS‐evoked GRF vector towards the axis of baseline postural instability. However, this was effectively cancelled by the hand force vector, such that the whole‐body sway response remained aligned with the inter‐aural axis, maintaining the craniocentric principle. These results show that a minimum level of grip is necessary before the upper limb plays an active role in vestibular‐evoked balance responses. Furthermore, they demonstrate that upper and lower‐limb forces are co‐ordinated to produce an appropriate whole‐body sway response.
    September 22, 2017   doi: 10.1113/JP274272   open full text
  • Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth restricted fetal sheep.
    Paul J. Rozance, Laura Zastoupil, Stephanie R. Wesolowski, David A. Goldstrohm, Brittany Strahan, Melanie Cree‐Green, Melinda Sheffield‐Moore, Giacomo Meschia, William W. Hay, Randall B. Wilkening, Laura D. Brown.
    The Journal of Physiology. September 22, 2017
    Reduced skeletal muscle mass in the IUGR fetus persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake, and protein accretion rates in skeletal muscle, late gestation CON (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate, and fractional synthetic rate were lower in IUGR compared to CON (P < 0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9 ± 5.6, CON: 60.9 ± 6.5 ml·min−1; P < 0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4 ± 1.4, CON: 14.7 ± 1.3 μmol·min−1·100 g−1; P < 0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3 ± 0.5, CON: 2.9 ± 0.2 μmol·min−1·100 g−1; P < 0.05). Blood O2 saturation (R2 = 0.80, P < 0.0001) and plasma glucose (R2 = 0.68, P < 0.0001), insulin (R2 = 0.40, P < 0.005), and IGF‐1 (R2 = 0.80, P < 0.0001) were positively associated and norepinephrine (R2 = 0.59, P < 0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are likely hormonally mediated by mechanisms that include increased fetal norepinephrine and reduced IGF‐1 and insulin. This article is protected by copyright. All rights reserved
    September 22, 2017   doi: 10.1113/JP275230   open full text
  • Kindlin‐2 interacts with endothelial adherens junctions to support vascular barrier integrity.
    Elzbieta Pluskota, Kamila M. Bledzka, Katarzyna Bialkowska, Dorota Szpak, Dmitry A. Soloviev, Sidney V. Jones, Dmitriy Verbovetskiy, Edward F. Plow.
    The Journal of Physiology. September 21, 2017
    Key points A reduction in Kindlin‐2 levels in endothelial cells compromises vascular barrier function. Kindlin‐2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with β‐ or γ‐catenin and cortical actin filaments, Kindlin‐2 stabilizes adherens junctions. The Kindlin‐2 binding sites for β‐ and γ‐catenin reside within its F1 and F3 subdomains. Although Kindlin‐2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin‐2+/− mice. Abstract Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin‐2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin‐2 in regulating vascular integrity using several different approaches to decrease Kindlin‐2 levels in EC. Reduced levels of Kindlin‐2 in Kindlin‐2+/– mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin‐2 siRNA showed enhanced basal and platelet‐activating factor (PAF) or lipopolysaccharide‐stimulated vascular leakage compared to wild‐type (WT) counterparts. PAF preferentially disrupted the Kindlin‐2+/− MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin‐2 co‐localized and co‐immunoprecipitated with vascular endothelial cadherin‐based complexes, including β‐ and γ‐catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin‐2 with β‐ and γ‐catenin and actin was demonstrated in co‐immunoprecipitation and surface plasmon resonance experiments. In thrombin‐stimulated HUVECs, Kindlin‐2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The β‐ and γ‐catenin binding site resides within the F1 and F3 subdomains of Kindlin‐2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin‐2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin‐based complexes to cortical actin and thereby stabilizing AJ.
    September 21, 2017   doi: 10.1113/JP274380   open full text
  • Adenosine and dopamine oppositely modulate a hyperpolarization‐activated current Ih in chemosensory neurons of the rat carotid body in co‐culture.
    Min Zhang, Cathy Vollmer, Colin A. Nurse.
    The Journal of Physiology. September 21, 2017
    Key points Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co‐cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization‐activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih. Abstract Adenosine and dopamine (DA) act as neurotransmitters or neuromodulators at the carotid body (CB) chemosensory synapse, but their mechanisms of action are not fully understood. Using a functional co‐culture model of rat CB chemoreceptor (type I) cell clusters and juxtaposed afferent petrosal neurons (PNs), we tested the hypothesis that adenosine and DA act postsynaptically to modulate a hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) cation current (Ih). In whole‐cell recordings from hypoxia‐responsive PNs, cAMP mimetics enhanced Ih whereas the HCN blocker ZD7288 (2 μm) reversibly inhibited Ih. Adenosine caused a potentiation of Ih (EC50 ∼ 35 nm) that was sensitive to the A2a blocker SCH58261 (5 nm), and an ∼16 mV depolarizing shift in V½ for voltage dependence of Ih activation. By contrast, DA (10 μm) caused an inhibition of Ih that was sensitive to the D2 blocker sulpiride (1–10 μm), and an ∼11 mV hyperpolarizing shift in V½. Sulpiride potentiated Ih in neurons adjacent to, but not distant from, type I cell clusters. DA also decreased PN action potential frequency whereas adenosine had the opposite effect. During simultaneous paired recordings, SCH58261 inhibited both the presynaptic hypoxia‐induced receptor potential in type I cells and the postsynaptic PN response. By contrast, SCH58261 inhibited only the postsynaptic PN response induced by isohydric hypercapnia. Confocal immunofluorescence confirmed the localization of HCN4 subunits in tyrosine hydroxylase‐positive chemoafferent neurons in tissue sections of rat petrosal ganglia. These data suggest that adenosine and DA, acting through A2a and D2 receptors respectively, regulate PN excitability via their opposing actions on Ih.
    September 21, 2017   doi: 10.1113/JP274743   open full text
  • Synaptic excitation by climbing fibre collaterals in the cerebellar nuclei of juvenile and adult mice.
    Marion Najac, Indira M. Raman.
    The Journal of Physiology. September 20, 2017
    Key points The inferior olive sends instructive motor signals to the cerebellum via the climbing fibre projection, which sends collaterals directly to large premotor neurons of the mouse cerebellar nuclei (CbN cells). Optogenetic activation of inferior olivary axons in vitro evokes EPSCs in CbN cells of several hundred pA to more than 1 nA. The inputs are three‐fold larger at younger ages, 12 to 14 days old, than at 2 months old, suggesting a strong functional role for this pathway earlier in development. The EPSCs are multipeaked, owing to burst firing in several olivary afferents that fire asynchronously. The convergence of climbing fibre collaterals onto CbN cells decreases from ∼40 to ∼8, which is consistent with the formation of closed‐loop circuits in which each CbN neuron receives input from 4–7 collaterals from inferior olivary neurons as well as from all 30–50 Purkinje cells that are innervated by those olivary neurons. Abstract The inferior olive conveys instructive signals to the cerebellum that drive sensorimotor learning. Inferior olivary neurons transmit their signals via climbing fibres, which powerfully excite Purkinje cells, evoking complex spikes and depressing parallel fibre synapses. Additionally, however, these climbing fibres send collaterals to the cerebellar nuclei (CbN). In vivo and in vitro data suggest that climbing fibre collateral excitation is weak in adult mice, raising the question of whether the primary role of this pathway may be developmental. We therefore examined climbing fibre collateral input to large premotor CbN cells over development by virally expressing channelrhodopsin in the inferior olive. In acute cerebellar slices from postnatal day (P)12–14 mice, light‐evoked EPSCs were large (> 1 nA at −70 mV). The amplitude of these EPSCs decreased over development, reaching a plateau of ∼350 pA at P20–60. Trains of EPSCs (5 Hz) depressed strongly throughout development, whereas convergence estimates indicated that the total number of functional afferents decreased with age. EPSC waveforms consisted of multiple peaks, probably resulting from action potential bursts in single collaterals and variable times to spike threshold in converging afferents. Activating climbing fibre collaterals evoked well‐timed increases in firing probability in CbN neurons, especially in younger mice. The initially strong input, followed by the decrement in synaptic strength coinciding with the pruning of climbing fibres in the cerebellar cortex, implicates the climbing fibre collateral pathway in early postnatal development. Additionally, the persistence of substantial synaptic input at least to P60 suggests that this pathway may function in cerebellar processing into adulthood.
    September 20, 2017   doi: 10.1113/JP274598   open full text
  • Persistent aberrant cortical phase–amplitude coupling following seizure treatment in absence epilepsy models.
    Atul Maheshwari, Abraham Akbar, Mai Wang, Rachel L. Marks, Katherine Yu, Suhyeorn Park, Brett L. Foster, Jeffrey L. Noebels.
    The Journal of Physiology. September 19, 2017
    Key points In two monogenic models of absence epilepsy, interictal beta/gamma power is augmented in homozygous stargazer (stg/stg) but not homozygous tottering (tg/tg) mice. There are distinct gene‐linked patterns of aberrant phase–amplitude coupling in the interictal EEG of both stg/stg and tg/tg mice, compared to +/+ and stg/+ mice. Treatment with ethosuximide significantly blocks seizures in both genotypes, but the abnormal phase–amplitude coupling remains. Seizure‐free stg/+ mice have normal power and phase–amplitude coupling, but beta/gamma power is significantly reduced with NMDA receptor blockade, revealing a latent cortical network phenotype that is separable from, and therefore not a result of, seizures. Altogether, these findings reveal gene‐linked quantitative electrographic biomarkers free from epileptiform activity, and provide a potential network correlate for persistent cognitive deficits in absence epilepsy despite effective treatment. Abstract In childhood absence epilepsy, cortical seizures are brief and intermittent; however there are extended periods without behavioural or electrographic ictal events. This genetic disorder is associated with variable degrees of cognitive dysfunction, but no consistent functional biomarkers that might provide insight into interictal cortical function have been described. Previous work in monogenic mouse models of absence epilepsy have shown that the interictal EEG displays augmented beta/gamma power in homozygous stargazer (stg/stg) mice bearing a presynaptic AMPA receptor defect, but not homozygous tottering (tg/tg) mice with a P/Q type calcium channel mutation. To further evaluate the interictal EEG, we quantified phase–amplitude coupling (PAC) in stg/stg, stg/+, tg/tg and wild‐type (+/+) mice. We found distinct gene‐linked patterns of aberrant PAC in stg/stg and tg/tg mice compared to +/+ and stg/+ mice. Treatment with ethosuximide significantly blocks seizures in both stg/stg and tg/tg, but the abnormal PAC remains. Stg/+ mice are seizure free with normal baseline beta/gamma power and normal theta‐gamma PAC, but like stg/stg mice, beta/gamma power is significantly reduced by NMDA receptor blockade, a treatment that paradoxically enhances seizures in stg/stg mice. Stg/+ mice, therefore, have a latent cortical network phenotype that is veiled by NMDA‐mediated neurotransmission. Altogether, these findings reveal gene‐linked quantitative electrographic biomarkers in the absence of epileptiform activity and provide a potential network correlate for persistent cognitive deficits in absence epilepsy despite effective treatment.
    September 19, 2017   doi: 10.1113/JP274696   open full text
  • Median preoptic glutamatergic neurons promote thermoregulatory heat loss and water consumption in mice.
    Stephen B. G. Abbott, Clifford B. Saper.
    The Journal of Physiology. September 13, 2017
    Key points Glutamatergic neurons in the median preoptic area were stimulated using genetically targeted Channelrhodopsin 2 in transgenic mice. Stimulation of glutamatergic median preoptic area neurons produced a profound hypothermia due to cutaneous vasodilatation. Stimulation also produced drinking behaviour that was inhibited as water was ingested, suggesting pre‐systemic feedback gating of drinking. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap. Abstract The median preoptic nucleus (MnPO) serves an important role in the integration of water/electrolyte homeostasis and thermoregulation, but we have a limited understanding these functions at a cellular level. Using Cre–Lox genetic targeting of Channelrhodospin 2 in VGluT2 transgenic mice, we examined the effect of glutamatergic MnPO neuron stimulation in freely behaving mice while monitoring drinking behaviour and core temperature. Stimulation produced a strong hypothermic response in 62% (13/21) of mice (core temperature: −4.6 ± 0.5°C, P = 0.001 vs. controls) caused by cutaneous vasodilatation. Stimulating glutamatergic MnPO neurons also produced robust drinking behaviour in 82% (18/22) of mice. Mice that drank during stimulation consumed 912 ± 163 μl (n = 8) during a 20 min trial in the dark phase, but markedly less during the light phase (421 ± 83 μl, P = 0.0025). Also, drinking during stimulation was inhibited as water was ingested, suggesting pre‐systemic feedback gating of drinking. Both hypothermia and drinking during stimulation occurred in 50% of mice tested. Anatomical mapping of the stimulation sites showed that sites associated with hypothermia were more anteroventral than those associated with drinking, although there was substantial overlap. Thus, activation of separate but overlapping populations of glutamatergic MnPO neurons produces effects on drinking and autonomic thermoregulatory mechanisms, providing a structural basis for their frequently being coordinated (e.g. during hyperthermia).
    September 13, 2017   doi: 10.1113/JP274667   open full text
  • Molecular composition and heterogeneity of the LRRC8‐containing swelling‐activated osmolyte channels in primary rat astrocytes.
    Alexandra L. Schober, Corinne S. Wilson, Alexander A. Mongin.
    The Journal of Physiology. September 12, 2017
    Key points The volume‐regulated anion channel (VRAC) is a swelling‐activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners. We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling‐activated release of diverse organic osmolytes in rat astrocytes. Efflux of uncharged osmolytes (myo‐inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E. Conversely, release of charged osmolytes (d‐aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D. Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D‐containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules. Abstract The volume‐regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl−/anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d‐[14C]aspartate and [3H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [3H]taurine and myo‐[3H]inositol, without major impact on the simultaneously measured efflux of the charged d‐[14C]aspartate. In contrast, the release of d‐[14C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D‐containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules.
    September 12, 2017   doi: 10.1113/JP275053   open full text
  • Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.
    Natalie M. Hawken, Elena I. Zaika, Terunaga Nakagawa.
    The Journal of Physiology. September 12, 2017
    Key points The AMPA‐type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid‐accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex. Abstract During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid‐exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR–CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg‐ and CNIH3‐bound AMPARs were compared. Specifically, in detergent the GluA2‐A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2‐A793F–CNIH3 complex expressed a gain of function. In contrast, the GluA2‐A793F–Stg complex was stable, but had diminished gating modulation. GluA2‐C528L destabilized the AMPAR–CNIH3 complex but stabilized the AMPAR–Stg complex, with overall loss of function in gating modulation. Furthermore, loss‐of‐function mutations in this TMD region cancelled the effects of a gain‐of‐function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit‐specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant‐phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.
    September 12, 2017   doi: 10.1113/JP274897   open full text
  • Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.
    Jessica D. Clarke, Jessica L. Caldwell, Charles M. Pearman, David A. Eisner, Andrew W. Trafford, Katharine M. Dibb.
    The Journal of Physiology. September 11, 2017
    Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. Abstract Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via ICa‐L but decreased peak ICa‐L. Decreased peak ICa‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak ICa‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.
    September 11, 2017   doi: 10.1113/JP274053   open full text
  • Pulmonary artery wave propagation and reservoir function in conscious man: impact of pulmonary vascular disease, respiration and dynamic stress tests.
    Junjing Su, Charlotte Manisty, Ulf Simonsen, Luke S. Howard, Kim H. Parker, Alun D. Hughes.
    The Journal of Physiology. September 11, 2017
    Key points Wave travel plays an important role in cardiovascular physiology. However, many aspects of pulmonary arterial wave behaviour remain unclear. Wave intensity and reservoir‐excess pressure analyses were applied in the pulmonary artery in subjects with and without pulmonary hypertension during spontaneous respiration and dynamic stress tests. Arterial wave energy decreased during expiration and Valsalva manoeuvre due to decreased ventricular preload. Wave energy also decreased during handgrip exercise due to increased heart rate. In pulmonary hypertension patients, the asymptotic pressure at which the microvascular flow ceases, the reservoir pressure related to arterial compliance and the excess pressure caused by waves increased. The reservoir and excess pressures decreased during Valsalva manoeuvre but remained unchanged during handgrip exercise. This study provides insights into the influence of pulmonary vascular disease, spontaneous respiration and dynamic stress tests on pulmonary artery wave propagation and reservoir function. Abstract Detailed haemodynamic analysis may provide novel insights into the pulmonary circulation. Therefore, wave intensity and reservoir‐excess pressure analyses were applied in the pulmonary artery to characterize changes in wave propagation and reservoir function during spontaneous respiration and dynamic stress tests. Right heart catheterization was performed using a pressure and Doppler flow sensor tipped guidewire to obtain simultaneous pressure and flow velocity measurements in the pulmonary artery in control subjects and patients with pulmonary arterial hypertension (PAH) at rest. In controls, recordings were also obtained during Valsalva manoeuvre and handgrip exercise. The asymptotic pressure at which the flow through the microcirculation ceases, the reservoir pressure related to arterial compliance and the excess pressure caused by arterial waves increased in PAH patients compared to controls. The systolic and diastolic rate constants also increased, while the diastolic time constant decreased. The forward compression wave energy decreased by ∼8% in controls and ∼6% in PAH patients during expiration compared to inspiration, while the wave speed remained unchanged throughout the respiratory cycle. Wave energy decreased during Valsalva manoeuvre (by ∼45%) and handgrip exercise (by ∼27%) with unaffected wave speed. Moreover, the reservoir and excess pressures decreased during Valsalva manoeuvre but remained unaltered during handgrip exercise. In conclusion, reservoir‐excess pressure analysis applied to the pulmonary artery revealed distinctive differences between controls and PAH patients. Variations in the ventricular preload and afterload influence pulmonary arterial wave propagation as demonstrated by changes in wave energy during spontaneous respiration and dynamic stress tests.
    September 11, 2017   doi: 10.1113/JP274385   open full text
  • Mouse retinal ganglion cell signalling is dynamically modulated through parallel anterograde activation of cannabinoid and vanilloid pathways.
    Andrew O. Jo, Jennifer M. Noel, Monika Lakk, Oleg Yarishkin, Daniel A. Ryskamp, Koji Shibasaki, Maureen A. McCall, David Križaj.
    The Journal of Physiology. September 07, 2017
    Key points Retinal cells use vanilloid transient receptor potential (TRP) channels to integrate light‐evoked signals with ambient mechanical, chemical and temperature information. Localization and function of the polymodal non‐selective cation channel TRPV1 (transient receptor potential vanilloid isoform 1) remains elusive. TRPV1 is expressed in a subset of mouse retinal ganglion cells (RGCs) with peak expression in the mid‐peripheral retina. Endocannabinoids directly activate TRPV1 and inhibit it through cannabinoid type 1 receptors (CB1Rs) and cAMP pathways. Activity‐dependent endocannabinoid release may modulate signal gain in RGCs through simultaneous manipulation of calcium and cAMP signals mediated by TRPV1 and CB1R. Abstract How retinal ganglion cells (RGCs) process and integrate synaptic, mechanical, swelling stimuli with light inputs is an area of intense debate. The nociceptive cation channel TRPV1 (transient receptor potential vanilloid type 1) modulates RGC Ca2+ signals and excitability yet the proportion of RGCs that express it remains unclear. Furthermore, TRPV1's response to endocannabinoids (eCBs), the putative endogenous retinal activators, is unknown, as is the potential modulation by cannabinoid receptors (CBRs). The density of TRPV1‐expressing RGCs in the Ai9:Trpv1 reporter mouse peaked in the mid‐peripheral retina. TRPV1 agonists including capsaicin (CAP) and the eCBs anandamide and N‐arachidonoyl‐dopamine elevated [Ca2+]i in 30–40% of wild‐type RGCs, with effects suppressed by TRPV1 antagonists capsazepine (CPZ) and BCTC ((4‐(3‐chloro‐2‐pyridinyl)‐N‐[4‐(1,1‐dimethylethyl)phenyl]‐1‐piperazinecarboxamide), and lacking in Trpv1−/− cells. The cannabinoid receptor type 1 (CB1R) colocalized with TRPV1:tdTomato expression. Its agonists 2‐arachidonoylglycerol (2‐AG) and WIN55,122 inhibited CAP‐induced [Ca2+]i signals in adult, but not early postnatal, RGCs. The suppressive effect of 2‐AG on TRPV1 activation was emulated by positive modulators of the protein kinase A (PKA) pathway, inhibited by the CB1R antagonist rimonabant and Gi uncoupler pertussis toxin, and absent in Cnr1−/− RGCs. We conclude that TRPV1 is a modulator of Ca2+ homeostasis in a subset of RGCs that show non‐uniform distribution across the mouse retina. Non‐retrograde eCB‐mediated modulation of RGC signalling involves a dynamic push–pull between direct TRPV1 activation and PKA‐dependent regulation of channel inactivation, with potential functions in setting the bandwidth of postsynaptic responses, sensitivity to mechanical/excitotoxic stress and neuroprotection.
    September 07, 2017   doi: 10.1113/JP274562   open full text
  • Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.
    Adrianne R. Wilson‐Poe, Hyo‐Jin Jeong, Christopher W. Vaughan.
    The Journal of Physiology. September 07, 2017
    Key points Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. Abstract The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ‐opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO‐induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ‐opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.
    September 07, 2017   doi: 10.1113/JP274157   open full text
  • Hypercapnia‐induced active expiration increases in sleep and enhances ventilation in unanaesthetized rats.
    Isabela P. Leirão, Carlos A. Silva, Luciane H. Gargaglioni, Glauber S. F. da Silva.
    The Journal of Physiology. September 02, 2017
    Key points Expiratory muscles (abdominal and thoracic) can be recruited when respiratory drive increases under conditions of increased respiratory demand such as hypercapnia. Studying hypercapnia‐induced active expiration in unanaesthetized rats importantly contributes to the understanding of how the control system is integrated in vivo in freely moving animals. In unanaesthetized rats, hypercapnia‐induced active expiration was not always recruited either in wakefulness or in sleep, suggesting that additional factors influence the recruitment of active expiration. The pattern of abdominal muscle recruitment varied in a state‐dependent manner with active expiration being more predominant in the sleep state than in quiet wakefulness. Pulmonary ventilation was enhanced in periods with active expiration compared to periods without it. Abstract Expiration is passive at rest but becomes active through recruitment of abdominal muscles under increased respiratory drive. Hypercapnia‐induced active expiration has not been well explored in unanaesthetized rats. We hypothesized that (i) CO2‐evoked active expiration is recruited in a state‐dependent manner, i.e. differently in sleep or wakefulness, and (ii) recruitment of active expiration enhances ventilation, hence having an important functional role in meeting metabolic demand. To test these hypotheses, Wistar rats (280–330 g) were implanted with electrodes for EEG and electromyography EMG of the neck, diaphragm (DIA) and abdominal (ABD) muscles. Active expiratory events were considered as rhythmic ABDEMG activity interposed to DIAEMG. Animals were exposed to room air followed by hypercapnia (7% CO2) with EEG, EMG and ventilation (V̇E) recorded throughout the experimental protocol. No active expiration was observed during room air exposure. During hypercapnia, CO2‐evoked active expiration was predominantly recruited during non‐rapid eye movement sleep. Its increased occurrence during sleep was evidenced by the decreased DIA‐to‐ADB ratio (1:1 ratio means that each DIA event is followed by an ABD event, indicating a high occurrence of ABD activity). Moreover, V̇E was also enhanced (P < 0.05) in periods with active expiration. V̇E had a positive correlation (P < 0.05) with the peak amplitude of ABDEMG activity. The data demonstrate strongly that hypercapnia‐induced active expiration increases during sleep and provides an important functional role to support V̇E in conditions of increased respiratory demand.
    September 02, 2017   doi: 10.1113/JP274726   open full text
  • Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons.
    Cedric Yapo, Anu G. Nair, Lorna Clement, Liliana R. Castro, Jeanette Hellgren Kotaleski, Pierre Vincent.
    The Journal of Physiology. September 02, 2017
    Key points Brief dopamine events are critical actors of reward‐mediated learning in the striatum; the intracellular cAMP–protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub‐micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA‐dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA‐dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase‐1 (PP1) inhibition by DARPP‐32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. Abstract The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA‐dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium‐sized spiny neurons (MSNs), which express either D1 or D2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub‐micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D2 than on D1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA‐dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP‐32. Simulations further suggested that D2 MSNs can also operate in a ‘tone‐sensing’ mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought.
    September 02, 2017   doi: 10.1113/JP274475   open full text
  • Dissociating external power from intramuscular exercise intensity during intermittent bilateral knee‐extension in humans.
    Matthew J. Davies, Alan P. Benson, Daniel T. Cannon, Simon Marwood, Graham J. Kemp, Harry B. Rossiter, Carrie Ferguson.
    The Journal of Physiology. September 02, 2017
    Key points Continuous high‐intensity constant‐power exercise is unsustainable, with maximal oxygen uptake (V̇O2 max ) and the limit of tolerance attained after only a few minutes. Performing the same power intermittently reduces the O2 cost of exercise and increases tolerance. The extent to which this dissociation is reflected in the intramuscular bioenergetics is unknown. We used pulmonary gas exchange and 31P magnetic resonance spectroscopy to measure whole‐body V̇O2, quadriceps phosphate metabolism and pH during continuous and intermittent exercise of different work:recovery durations. Shortening the work:recovery durations (16:32 s vs. 32:64 s vs. 64:128 s vs. continuous) at a work rate estimated to require 110% peak aerobic power reduced V̇O2, muscle phosphocreatine breakdown and muscle acidification, eliminated the glycolytic‐associated contribution to ATP synthesis, and increased exercise tolerance. Exercise intensity (i.e. magnitude of intramuscular metabolic perturbations) can be dissociated from the external power using intermittent exercise with short work:recovery durations. Abstract Compared with work‐matched high‐intensity continuous exercise, intermittent exercise dissociates pulmonary oxygen uptake (V̇O2) from the accumulated work. The extent to which this reflects differences in O2 storage fluctuations and/or contributions from oxidative and substrate‐level bioenergetics is unknown. Using pulmonary gas‐exchange and intramuscular 31P magnetic resonance spectroscopy, we tested the hypotheses that, at the same power: ATP synthesis rates are similar, whereas peak V̇O2 amplitude is lower in intermittent vs. continuous exercise. Thus, we expected that: intermittent exercise relies less upon anaerobic glycolysis for ATP provision than continuous exercise; shorter intervals would require relatively greater fluctuations in intramuscular bioenergetics than in V̇O2 compared to longer intervals. Six men performed bilateral knee‐extensor exercise (estimated to require 110% peak aerobic power) continuously and with three different intermittent work:recovery durations (16:32, 32:64 and 64:128 s). Target work duration (576 s) was achieved in all intermittent protocols; greater than continuous (252 ± 174 s; P < 0.05). Mean ATP turnover rate was not different between protocols (∼43 mm min−1 on average). However, the intramuscular phosphocreatine (PCr) component of ATP generation was greatest (∼30 mm min−1), and oxidative (∼10 mm min−1) and anaerobic glycolytic (∼1 mm min−1) components were lowest for 16:32 and 32:64 s intermittent protocols, compared to 64:128 s (18 ± 6, 21 ± 10 and 10 ± 4 mm min−1, respectively) and continuous protocols (8 ± 6, 20 ± 9 and 16 ± 14 mm min−1, respectively). As intermittent work duration increased towards continuous exercise, ATP production relied proportionally more upon anaerobic glycolysis and oxidative phosphorylation, and less upon PCr breakdown. However, performing the same high‐intensity power intermittently vs. continuously reduced the amplitude of fluctuations in V̇O2 and intramuscular metabolism, dissociating exercise intensity from the power output and work done.
    September 02, 2017   doi: 10.1113/JP274589   open full text
  • Cortical control of object‐specific grasp relies on adjustments of both activity and effective connectivity: a common marmoset study.
    Banty Tia, Mitsuaki Takemi, Akito Kosugi, Elisa Castagnola, Alberto Ansaldo, Takafumi Nakamura, Davide Ricci, Junichi Ushiba, Luciano Fadiga, Atsushi Iriki.
    The Journal of Physiology. September 02, 2017
    Key points The cortical mechanisms of grasping have been extensively studied in macaques and humans; here, we investigated whether common marmosets could rely on similar mechanisms despite strong differences in hand morphology and grip diversity. We recorded electrocorticographic activity over the sensorimotor cortex of two common marmosets during the execution of different grip types, which allowed us to study cortical activity (power spectrum) and physiologically inferred connectivity (phase‐slope index). Analyses were performed in beta (16–35 Hz) and gamma (75–100 Hz) frequency bands and our results showed that beta power varied depending on grip type, whereas gamma power displayed clear epoch‐related modulation. Strength and direction of inter‐area connectivity varied depending on grip type and epoch. These findings suggest that fundamental control mechanisms are conserved across primates and, in future research, marmosets could represent an adequate model to investigate primate brain mechanisms. Abstract The cortical mechanisms of grasping have been extensively studied in macaques and humans. Here, we investigated whether common marmosets could rely on similar mechanisms despite striking differences in manual dexterity. Two common marmosets were trained to grasp‐and‐pull three objects eliciting different hand configurations: whole‐hand, finger and scissor grips. The animals were then chronically implanted with 64‐channel electrocorticogram arrays positioned over the left premotor, primary motor and somatosensory cortex. Power spectra, reflecting predominantly cortical activity, and phase‐slope index, reflecting the direction of information flux, were studied in beta (16–35 Hz) and gamma (75–100 Hz) bands. Differences related to grip type, epoch (reach, grasp) and cortical area were statistically assessed. Results showed that whole‐hand and scissor grips triggered stronger beta desynchronization than finger grip. Task epochs clearly modulated gamma power, especially for finger and scissor grips. Considering effective connectivity, finger and scissor grips evoked stronger outflow from primary motor to premotor cortex, whereas whole‐hand grip displayed the opposite pattern. These findings suggest that fundamental control mechanisms, relying on adjustments of cortical activity and connectivity, are conserved across primates. Consistently, marmosets could represent a good model to investigate primate brain mechanisms.
    September 02, 2017   doi: 10.1113/JP274629   open full text
  • Rearing‐environment‐dependent hippocampal local field potential differences in wild‐type and inositol trisphosphate receptor type 2 knockout mice.
    Mika Tanaka, Xiaowen Wang, Katsuhiko Mikoshiba, Hajime Hirase, Yoshiaki Shinohara.
    The Journal of Physiology. August 27, 2017
    Key points Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca2+ surges are involved in this experience‐dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP3R2)‐knockout (KO) mice, in which IP3/Ca2+ signalling in astrocytes is largely diminished. We found that this experience‐dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP3R2 deficiency. Abstract Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression‐like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane‐anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non‐theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP3R2)‐knockout (KO) mice, in which astrocytic Ca2+ elevations are largely diminished. We found that the gamma power was also higher in IP3R2‐KO‐ENR mice compared to IP3R2‐KO‐ISO mice, suggesting that the rearing‐environment‐dependent gamma power alteration does not necessarily require the astrocytic IP3/Ca2+ pathway. By contrast, ripple events showed genotype‐dependent changes, as well as rearing condition‐dependent changes: ISO housing and IP3R2 deficiency both lead to longer inter‐ripple intervals. Moreover, we found that ripple magnitude in the right CA1 tended to be smaller in IP3R2‐KO. Because IP3R2‐KO mice have been reported to have depression phenotypes, our results suggest that ripple events and the mood of animals may be broadly correlated.
    August 27, 2017   doi: 10.1113/JP274573   open full text
  • Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.
    Ning Gao, Ming‐Ho Tsai, Audrey N. Chang, Weiqi He, Cai‐Ping Chen, Minsheng Zhu, Kristine E. Kamm, James T. Stull.
    The Journal of Physiology. August 24, 2017
    Key points Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+/calmodulin‐dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve‐mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock‐in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Abstract Ca2+/calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation‐dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI‐17 (PKC‐potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI‐17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal‐force responses were similar in trachea from MYPT1SM+/+, MYPT1SM‐/− and the knock‐in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.
    August 24, 2017   doi: 10.1113/JP274715   open full text
  • Differential effects of late gestation maternal overnutrition on the regulation of surfactant maturation in fetal and postnatal life.
    Mitchell C. Lock, Erin V. McGillick, Sandra Orgeig, I. Caroline McMillen, Beverly S. Mühlhäusler, Song Zhang, Janna L. Morrison.
    The Journal of Physiology. August 24, 2017
    Key points Offspring of overweight and obese women are at greater risk for respiratory complications at birth. We determined the effect of late gestation maternal overnutrition (LGON) in sheep on surfactant maturation, glucose transport and fatty acid metabolism in the lung in fetal and postnatal life. There were significant decreases in surfactant components and numerical density of surfactant producing cells in the alveolar epithelium due to LGON in the fetal lung. However, there were no differences in the levels of these surfactant components between control and LGON lambs at 30 days of age. The reduced capacity for surfactant production in fetuses as a result of LGON may affect the transition to air breathing at birth. There was altered glucose transport and fatty acid metabolism in the lung as a result of LGON in postnatal life. However, there is a normalisation of surfactant components that suggests accelerated maturation in the lungs after birth. Abstract With the increasing incidence of obesity worldwide, the proportion of women entering pregnancy overweight or obese has increased dramatically. The fetus of an overnourished mother experiences numerous metabolic changes that may modulate lung development and hence successful transition to air breathing at birth. We used a sheep model of maternal late gestation overnutrition (LGON; from 115 days’ gestation, term 147 ± 3 days) to determine the effect of exposure to an increased plane of nutrition in late gestation on lung development in the fetus (at 141 days’ gestation) and the lamb (30 days after birth). We found a decrease in the numerical density of surfactant protein positive cells, as well as a reduction in mRNA expression of surfactant proteins (SFTP‐A, ‐B and ‐C), a rate limiting enzyme in surfactant phospholipid synthesis (phosphate cytidylyltransferase 1, choline, α; PCYT1A), and glucose transporters (SLC2A1 and SLC2A4) in the fetal lung. In lambs at 30 days after birth, there were no differences between Control and LGON groups in the surfactant components that were downregulated in the LGON fetuses. However, mRNA expression of SFTP‐A, PCYT1A, peroxisome proliferator activated receptor‐γ, fatty acid synthase and fatty acid transport protein were increased in LGON lambs compared to controls. These results indicate a reduced capacity for surfactant production in late gestation. While these deficits are normalised by 30 days after birth, the lungs of LGON lambs exhibited altered glucose transport and fatty acid metabolism, which is consistent with an enhanced capacity for surfactant synthesis and restoration of surfactant maturity in these animals.
    August 24, 2017   doi: 10.1113/JP274528   open full text
  • Experimental and modelling evidence of shortening heat in cardiac muscle.
    Kenneth Tran, June‐Chiew Han, Edmund John Crampin, Andrew James Taberner, Denis Scott Loiselle.
    The Journal of Physiology. August 22, 2017
    Key points Heat associated with muscle shortening has been repeatedly demonstrated in skeletal muscle, but its existence in cardiac muscle remains contentious after five decades of study. By iterating between experiments and computational modelling, we show compelling evidence for the existence of shortening heat in cardiac muscle and reveal, mechanistically, the source of this excess heat. Our results clarify a long‐standing uncertainty in the field of cardiac muscle energetics. We provide a revised partitioning of cardiac muscle energy expenditure to include this newly revealed thermal component. Abstract When a muscle shortens against an afterload, the heat that it liberates is greater than that produced by the same muscle contracting isometrically at the same level of force. This excess heat is defined as ‘shortening heat’, and has been repeatedly demonstrated in skeletal muscle but not in cardiac muscle. Given the micro‐structural similarities between these two muscle types, and since we imagine that shortening heat is the thermal accompaniment of cross‐bridge cycling, we have re‐examined this issue. Using our flow‐through microcalorimeter, we measured force and heat generated by isolated rat trabeculae undergoing isometric contractions at different muscle lengths and work‐loop (shortening) contractions at different afterloads. We simulated these experimental protocols using a thermodynamically constrained model of cross‐bridge cycling and probed the mechanisms underpinning shortening heat. Predictions generated by the model were subsequently validated by a further set of experiments. Both our experimental and modelling results show convincing evidence for the existence of shortening heat in cardiac muscle. Its magnitude is inversely related to the afterload or, equivalently, directly related to the extent of shortening. Computational simulations reveal that the heat of shortening arises from the cycling of cross‐bridges, and that the rate of ATP hydrolysis is more sensitive to change of muscle length than to change of afterload. Our results clarify a long‐standing uncertainty in the field of cardiac muscle energetics.
    August 22, 2017   doi: 10.1113/JP274680   open full text
  • Differential calcium sensitivity in NaV1.5 mixed syndrome mutants.
    Mena Abdelsayed, Alban‐Elouen Baruteau, Karen Gibbs, Shubhayan Sanatani, Andrew D. Krahn, Vincent Probst, Peter C. Ruben.
    The Journal of Physiology. August 20, 2017
    Key points SCN5a mutations may express gain‐of‐function (Long QT Syndrome‐3), loss‐of‐function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium‐sensitive regions. Elevated cytosolic calcium attenuates gain‐of‐function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain‐of‐function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium‐to‐calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. Abstract Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage‐gated sodium channel, NaV1.5. Mutants in NaV1.5 result in Brugada Syndrome (BrS1), Long‐QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome NaV1.5 mutants. We used whole‐cell patch clamp to assess the biophysical properties of NaV1.5 wild‐type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the NaV1.5 α subunit (WT or mutants), β1 subunit and enhanced green fluorescent protein. Voltage‐dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500 nm. In silico, action potential (AP) model simulations were performed using a modified O'Hara Rudy model. Elevated cytosolic calcium attenuates the late sodium current in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Elevated cytosolic calcium restores steady‐state slow inactivation (SSSI) to the WT‐form in Q1909R, but depolarized SSSI in E1784K. Our AP simulations showed a frequency‐dependent reduction of AP duration in ∆KPQ, 1795insD and Q1909R carriers. In E1784K, AP duration is relatively prolonged at both low and high heart rates, resulting in a sodium overload. Cellular perturbations during exercise may affect BrS1/LQT3 patients differently depending on their individual genetic signature. Thus, exercise may be therapeutic or may be an arrhythmogenic trigger in some SCN5a patients.
    August 20, 2017   doi: 10.1113/JP274536   open full text
  • Paired motor cortex and cervical epidural electrical stimulation timed to converge in the spinal cord promotes lasting increases in motor responses.
    Asht M. Mishra, Ajay Pal, Disha Gupta, Jason B. Carmel.
    The Journal of Physiology. August 20, 2017
    Key points Pairing motor cortex stimulation and spinal cord epidural stimulation produced large augmentation in motor cortex evoked potentials if they were timed to converge in the spinal cord. The modulation of cortical evoked potentials by spinal cord stimulation was largest when the spinal electrodes were placed over the dorsal root entry zone. Repeated pairing of motor cortex and spinal cord stimulation caused lasting increases in evoked potentials from both sites, but only if the time between the stimuli was optimal. Both immediate and lasting effects of paired stimulation are likely mediated by convergence of descending motor circuits and large diameter afferents onto common interneurons in the cervical spinal cord. Abstract Convergent activity in neural circuits can generate changes at their intersection. The rules of paired electrical stimulation are best understood for protocols that stimulate input circuits and their targets. We took a different approach by targeting the interaction of descending motor pathways and large diameter afferents in the spinal cord. We hypothesized that pairing stimulation of motor cortex and cervical spinal cord would strengthen motor responses through their convergence. We placed epidural electrodes over motor cortex and the dorsal cervical spinal cord in rats; motor evoked potentials (MEPs) were measured from biceps. MEPs evoked from motor cortex were robustly augmented with spinal epidural stimulation delivered at an intensity below the threshold for provoking an MEP. Augmentation was critically dependent on the timing and position of spinal stimulation. When the spinal stimulation was timed to coincide with the descending volley from motor cortex stimulation, MEPs were more than doubled. We then tested the effect of repeated pairing of motor cortex and spinal stimulation. Repetitive pairing caused strong augmentation of cortical MEPs and spinal excitability that lasted up to an hour after just 5 min of pairing. Additional physiology experiments support the hypothesis that paired stimulation is mediated by convergence of descending motor circuits and large diameter afferents in the spinal cord. The large effect size of this protocol and the conservation of the circuits being manipulated between rats and humans makes it worth pursuing for recovery of sensorimotor function after injury to the central nervous system.
    August 20, 2017   doi: 10.1113/JP274663   open full text
  • Cortical contributions to sensory gating in the ipsilateral somatosensory cortex during voluntary activity.
    Yuming Lei, Monica A. Perez.
    The Journal of Physiology. August 18, 2017
    Key points It has long been known that the somatosensory cortex gates sensory inputs from the contralateral side of the body. Here, we examined the contribution of the ipsilateral somatosensory cortex (iS1) to sensory gating during index finger voluntary activity. The amplitude of the P25/N33, but not other somatosensory evoked potential (SSEP) components, was reduced during voluntary activity compared with rest. Interhemispheric inhibition between S1s and intracortical inhibition in the S1 modulated the amplitude of the P25/N33. Note that changes in interhemispheric inhibition between S1s correlated with changes in cortical circuits in the ipsilateral motor cortex. Our findings suggest that cortical circuits, probably from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans. Abstract An important principle in the organization of the somatosensory cortex is that it processes afferent information from the contralateral side of the body. The role of the ipsilateral somatosensory cortex (iS1) in sensory gating in humans remains largely unknown. Using electroencephalographic (EEG) recordings over the iS1 and electrical stimulation of the ulnar nerve at the wrist, we examined somatosensory evoked potentials (SSEPs; P14/N20, N20/P25 and P25/N33 components) and paired‐pulse SSEPs between S1s (interhemispheric inhibition) and within (intracortical inhibition) the iS1 at rest and during tonic index finger voluntary activity. We found that the amplitude of the P25/N33, but not other SSEP components, was reduced during voluntary activity compared with rest. Interhemispheric inhibition increased the amplitude of the P25/N33 and intracortical inhibition reduced the amplitude of the P25/N33, suggesting a cortical origin for this effect. The P25/N33 receives inputs from the motor cortex, so we also examined the contribution of distinct sets of cortical interneurons by testing the effect of ulnar nerve stimulation on motor‐evoked potentials (MEPs) elicited by transcranial magnetic stimulation over the ipsilateral motor cortex with the coil in the posterior–anterior (PA) and anterior–posterior (AP) orientation. Afferent input attenuated PA, but not AP, MEPs during voluntary activity compared with rest. Notably, changes in interhemispheric inhibition correlated with changes in PA MEPs. Our novel findings suggest that interhemispheric projections between S1s and intracortical circuits, probably from somatosensory and motor cortex, contribute to sensory gating in the iS1 during voluntary activity in humans.
    August 18, 2017   doi: 10.1113/JP274504   open full text
  • The role of T‐type calcium channels in the subiculum: to burst or not to burst?
    Srdjan M. Joksimovic, Pierce Eggan, Yukitoshi Izumi, Sonja Lj. Joksimovic, Vesna Tesic, Robert M. Dietz, James E. Orfila, Michael R. DiGruccio, Paco S. Herson, Vesna Jevtovic‐Todorovic, Charles F. Zorumski, Slobodan M. Todorovic.
    The Journal of Physiology. August 18, 2017
    Key points Pharmacological, molecular and genetic data indicate a prominent role of low‐voltage‐activated T‐type calcium channels (T‐channels) in the firing activity of both pyramidal and inhibitory interneurons in the subiculum. Pharmacological inhibition of T‐channels switched burst firing with lower depolarizing stimuli to regular spiking, and fully abolished hyperpolarization‐induced burst firing. Our molecular studies showed that CaV3.1 is the most abundantly expressed isoform of T‐channels in the rat subiculum. Consistent with this finding, both regular‐spiking and burst firing patterns were profoundly depressed in the mouse with global deletion of CaV3.1 isoform of T‐channels. Selective inhibition of T‐channels and global deletion of CaV3.1 channels completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway. Abstract Several studies suggest that voltage‐gated calcium currents are involved in generating high frequency burst firing in the subiculum, but the exact nature of these currents remains unknown. Here, we used selective pharmacology, molecular and genetic approaches to implicate Cav3.1‐containing T‐channels in subicular burst firing, in contrast to several previous reports discounting T‐channels as major contributors to subicular neuron physiology. Furthermore, pharmacological antagonism of T‐channels, as well as global deletion of CaV3.1 isoform, completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway. Our results indicate that excitability and synaptic plasticity of subicular neurons relies heavily on T‐channels. Hence, T‐channels may be a promising new drug target for different cognitive deficits.
    August 18, 2017   doi: 10.1113/JP274565   open full text
  • Decline in cellular function of aged mouse c‐kit+ cardiac progenitor cells.
    Alessandra Castaldi, Ramsinh Mansinh Dodia, Amabel M. Orogo, Cristina M. Zambrano, Rita H. Najor, Åsa B. Gustafsson, Joan Heller Brown, Nicole H. Purcell.
    The Journal of Physiology. August 18, 2017
    Key points While autologous stem cell‐based therapies are currently being tested on elderly patients, there are limited data on the function of aged stem cells and in particular c‐kit+ cardiac progenitor cells (CPCs). We isolated c‐kit+ cells from young (3 months) and aged (24 months) C57BL/6 mice to compare their biological properties. Aged CPCs have increased senescence, decreased stemness and reduced capacity to proliferate or to differentiate following dexamethasone (Dex) treatment in vitro, as evidenced by lack of cardiac lineage gene upregulation. Aged CPCs fail to activate mitochondrial biogenesis and increase proteins involved in mitochondrial oxidative phosphorylation in response to Dex. Aged CPCs fail to upregulate paracrine factors that are potentially important for proliferation, survival and angiogenesis in response to Dex. The results highlight marked differences between young and aged CPCs, which may impact future design of autologous stem cell‐based therapies. Abstract Therapeutic use of c‐kit+ cardiac progenitor cells (CPCs) is being evaluated for regenerative therapy in older patients with ischaemic heart failure. Our understanding of the biology of these CPCs has, however, largely come from studies of young cells and animal models. In the present study we examined characteristics of CPCs isolated from young (3 months) and aged (24 months) mice that could underlie the diverse outcomes reported for CPC‐based therapeutics. We observed morphological differences and altered senescence indicated by increased senescence‐associated markers β‐galactosidase and p16 mRNA in aged CPCs. The aged CPCs also proliferated more slowly than their young counterparts and expressed lower levels of the stemness marker LIN28. We subsequently treated the cells with dexamethasone (Dex), routinely used to induce commitment in CPCs, for 7 days and analysed expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs also failed to increase mitochondrial biogenesis and expression of the mitochondrial proteins Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. Dex‐treated aged CPCs also had impaired ability to upregulate expression of paracrine factor genes and the conditioned media from these cells had reduced ability to induce angiogenesis in vitro. These findings could impact the design of future CPC‐based therapeutic approaches for the treatment of older patients suffering from cardiac injury.
    August 18, 2017   doi: 10.1113/JP274775   open full text
  • Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle.
    Edward S. A. Humphries, Tomoko Kamishima, John M. Quayle, Caroline Dart.
    The Journal of Physiology. August 14, 2017
    Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain. In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII. The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown. In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII. To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Abstract Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.
    August 14, 2017   doi: 10.1113/JP274754   open full text
  • Post‐translational palmitoylation controls the voltage gating and lipid raft association of the CALHM1 channel.
    Akiyuki Taruno, Hongxin Sun, Koichi Nakajo, Tatsuro Murakami, Yasuyoshi Ohsaki, Mizuho A. Kido, Fumihito Ono, Yoshinori Marunaka.
    The Journal of Physiology. August 14, 2017
    Key points Calcium homeostasis modulator 1 (CALHM1), a new voltage‐gated ATP‐ and Ca2+‐permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post‐translationally regulated through the process of protein S‐palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine‐tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. Abstract Emerging roles of CALHM1, a recently discovered voltage‐gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post‐translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S‐palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance–voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent‐resistant membranes. Our results comprehensively uncover a post‐translational regulation of the voltage‐dependent gating of CALHM1 by palmitoylation.
    August 14, 2017   doi: 10.1113/JP274164   open full text
  • Depletion of Pax7+ satellite cells does not affect diaphragm adaptations to running in young or aged mice.
    Kevin A. Murach, Amy L. Confides, Angel Ho, Janna R. Jackson, Lina S. Ghazala, Charlotte A. Peterson, Esther E. Dupont‐Versteegden.
    The Journal of Physiology. August 14, 2017
    Key points Satellite cell depletion does not affect diaphragm adaptations to voluntary wheel running in young or aged mice. Satellite cell depletion early in life (4 months of age) has minimal effect on diaphragm phenotype by old age (24 months). Prolonged satellite cell depletion in the diaphragm does not result in excessive extracellular matrix accumulation, in contrast to what has been reported in hind limb muscles. Up‐regulation of Pax3 mRNA+ cells after satellite cell depletion in young and aged mice suggests that Pax3+ cells may compensate for a loss of Pax7+ satellite cells in the diaphragm. Future investigations should focus on the role of Pax3+ cells in the diaphragm during adaptation to exercise and ageing. Abstract Satellite cell contribution to unstressed diaphragm is higher compared to hind limb muscles, which is probably attributable to constant activation of this muscle to drive ventilation. Whether satellite cell depletion negatively impacts diaphragm quantitative and qualitative characteristics under stressed conditions in young and aged mice is unknown. We therefore challenged the diaphragm with prolonged running activity in the presence and absence of Pax7+ satellite cells in young and aged mice using an inducible Pax7CreER‐R26RDTA model. Mice were vehicle (Veh, satellite cell‐replete) or tamoxifen (Tam, satellite cell‐depleted) treated at 4 months of age and were then allowed to run voluntarily at 6 months (young) and 22 months (aged). Age‐matched, cage‐dwelling, Veh‐ and Tam‐treated mice without wheel access served as activity controls. Diaphragm muscles were analysed from young (8 months) and aged (24 months) mice. Satellite cell depletion did not alter diaphragm mean fibre cross‐sectional area, fibre type distribution or extracellular matrix content in young or aged mice, regardless of running activity. Resting in vivo diaphragm function was also unaffected by satellite cell depletion. Myonuclear density was maintained in young satellite cell‐depleted mice regardless of running, although it was modestly reduced in aged sedentary (–7%) and running (–19%) mice without satellite cells (P < 0.05). Using fluorescence in situ hybridization, we detected higher Pax3 mRNA+ cell density in both young and aged satellite cell‐depleted diaphragm muscle (P < 0.05), which may compensate for the loss of Pax7+ satellite cells.
    August 14, 2017   doi: 10.1113/JP274611   open full text
  • ATP and astrocytes play a prominent role in the control of the respiratory pattern generator in the lamprey.
    Elenia Cinelli, Ludovica Iovino, Donatella Mutolo.
    The Journal of Physiology. August 08, 2017
    Key points The paratrigeminal respiratory group (pTRG) is responsible for the respiratory pattern generation in the lamprey. The role of ATP and astrocytes, known to control respiratory activity in mammals, was investigated in the lamprey respiratory network. ATP microinjected into the pTRG induces a biphasic response consisting of marked increases in respiratory frequency mediated by P2X receptors followed by a decrease in the respiratory motor output due to the ATP metabolite adenosine. We provide evidence that astrocytes are involved in the genesis of the normal respiratory pattern, ATP‐induced responses and acidification‐induced increases of the respiratory activity. The function of astrocytes in rhythmic networks appears to be phylogenetically conserved. Abstract The role of ATP and astrocytes in respiratory rhythm modulation has been recently investigated in neonatal rodents. However, no information on the role of ATP and astrocytes within the respiratory network of the lamprey is available, particularly within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator. To address these issues, the present study was carried out on isolated brainstems of the adult lamprey. Bath application of ATP caused marked increases in respiratory frequency followed by decreases in the respiratory motor output, mediated by the ATP metabolite adenosine at the level of the pTRG. Bath applications and microinjections of agonists and antagonists of purinergic receptors showed that ATP increased respiratory activity through an action on pTRG P2X receptors. To disclose the respiratory role of astrocytes, we used bath application of the gliotoxin aminoadipic acid, which dramatically depressed the respiratory motor output that, however, promptly recovered following glutamine application. Furthermore, the excitatory responses to ATP‐γ‐S (a non‐hydrolysable ATP analogue), but not to substance P, microinjected into the pTRG, were abolished. Finally, we also demonstrated that acidification‐induced increases in respiratory activity were ATP‐independent, but mediated by the astrocytes’ glutamate–glutamine cycle. The results show for the first time that ATP and especially astrocytes strongly contribute to the modulation of the lamprey respiratory pattern. Their role in the modulation or maintenance of rhythmic neuronal activities appears to be phylogenetically conserved.
    August 08, 2017   doi: 10.1113/JP274749   open full text
  • Distinct temporal filters in mitral cells and external tufted cells of the olfactory bulb.
    Christopher E. Vaaga, Gary L. Westbrook.
    The Journal of Physiology. August 08, 2017
    Short‐term synaptic plasticity is a critical regulator of neural circuits, and largely determines how information is temporally processed. In the olfactory bulb, afferent olfactory receptor neurons respond to increasing concentrations of odorants with barrages of action potentials, and their terminals have an extraordinarily high release probability (Sicard, 1986; Murphy et al. 2004). These features suggest that during naturalistic stimuli, afferent input to the olfactory bulb is subject to strong synaptic depression, presumably truncating the postsynaptic response to afferent stimuli. To examine this issue, we used single glomerular stimulation in mouse olfactory bulb slices to measure the synaptic dynamics of afferent‐evoked input at physiological stimulus frequencies. In cell‐attached recordings, mitral cells responded to high frequency stimulation with sustained responses, whereas external tufted cells responded transiently. Consistent with previous reports (Murphy et al. 2004), olfactory nerve terminals onto both cell types had a high release probability (0.7), from a single pool of slowly recycling vesicles, indicating that the distinct responses of mitral and external tufted cells to high frequency stimulation did not originate presyaptically. Rather, distinct temporal response profiles in mitral cells and external tufted cells could be attributed to slow dendrodendritic responses in mitral cells, as blocking this slow current in mitral cells converted mitral cell responses to a transient response profile, typical of external tufted cells. Our results suggest that despite strong axodendritic synaptic depression, the balance of axodendritic and dendrodendritic circuitry in external tufted cells and mitral cells, respectively, tunes the postsynaptic responses to high frequency, naturalistic stimulation. This article is protected by copyright. All rights reserved
    August 08, 2017   doi: 10.1113/JP274608   open full text
  • Gene expression analyses reveal metabolic specifications in acute O2‐sensing chemoreceptor cells.
    Lin Gao, Victoria Bonilla‐Henao, Paula García‐Flores, Ignacio Arias‐Mayenco, Patricia Ortega‐Sáenz, José López‐Barneo.
    The Journal of Physiology. August 08, 2017
    Key points Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2‐sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2‐sensitive, with superior cervical ganglion neurons, which are practically O2‐insensitive. In O2‐sensitive cells, we found a characteristic prolyl hydroxylase 3 down‐regulation and hypoxia inducible factor 2α up‐regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a ‘signature metabolic profile’. Abstract Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2‐sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2‐sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells, which are O2‐sensitive (glomus cells > chromaffin cells), and superior cervical ganglion neurons, which are practically O2‐insensitive. In the O2‐sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up‐regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a ‘signature metabolic profile’ in chemoreceptor cells.
    August 08, 2017   doi: 10.1113/JP274684   open full text
  • NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.
    Brian J. Kinsman, Kirsteen N. Browning, Sean D. Stocker.
    The Journal of Physiology. August 02, 2017
    Key points Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi‐osmotic mannitol/sorbitol did not alter any variable. In vitro whole‐cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi‐osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi‐osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl‐ versus osmo‐sensing. Abstract Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi‐osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague‐Dawley rats. First, intracerebroventricular (icv) infusion (5 μl/10 min) of 1.0 m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi‐osmotic sorbitol (2.0 osmol l−1). Second, OVLT microinjection (20 nl) of 1.0 m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi‐osmotic sorbitol did not alter any variable. Third, in vitro whole‐cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5 mm) and mannitol (+15 mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single‐unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration‐dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi‐osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5 m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi‐osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.
    August 02, 2017   doi: 10.1113/JP274537   open full text
  • A reduction in SK channels contributes to increased activity of hypothalamic magnocellular neurons during heart failure.
    Hildebrando C. Ferreira‐Neto, Vinicia C. Biancardi, Javier E. Stern.
    The Journal of Physiology. August 02, 2017
    Key points Small conductance Ca2+‐activated K+ (SK) channels play an important role in regulating the excitability of magnocellular neurosecretory cells (MNCs). Although an increased SK channel function contributes to adaptive physiological responses, it remains unknown whether changes in SK channel function/expression contribute to exacerbated MNC activity under disease conditions. We show that the input–output function of MNCs in heart failure (HF) rats is enhanced. Moreover, the SK channel blocker apamin enhanced the input–output function in sham, although not in HF rats. We found that both the after‐hyperpolarizing potential magnitude and the underlying apamin‐sensitive IAHP are blunted in MNCs from HF rats. The magnitude of spike‐induced increases in intracellular Ca2+ levels was not affected in MNCs of HF rats. We found a diminished expression of SK2/SK3 channel subunit mRNA expression in the supraoptic nucleus of HF rats. Our studies suggest that a reduction in SK channel expression, but not changes in Ca2+‐mediated activation of SK channels, contributes to exacerbated MNC activity in HF rats. Abstract Small conductance Ca2+‐activated K+ channels (SK) play an important role in regulating the activity of magnocellular neurosecretory cells (MNCs) and hormone release from the posterior pituitary. Moreover, enhanced SK activity contributes to the adaptive responses of MNCs to physiological challenge, such as lactation. Nevertheless, whether changes in SK function/expression contribute to exacerbated MNC activity during diseases such as heart failure (HF) remains unknown. In the present study, we used a combination of patch clamp electrophysiology, confocal Ca2+ imaging and molecular biology in a rat model of ischaemic HF. We found that the input–output function of MNCs was enhanced in HF compared to sham rats. Moreover, although the SK blocker apamin (200 nm) strengthened the input–output function in sham rats, it failed to have an effect in HF rats. The magnitude of the after‐hyperpolarizing potential (AHP) following a train of spikes and the underlying apamin‐sensitive IAHP were blunted in MNCs from HF rats. However, spike‐induced increases in intracellular Ca2+ were not affected in the MNCs of HF rats. Real‐time PCR measurements of SK channel subunits mRNA in supraoptic nucleus punches revealed a diminished expression of SK2/SK3 subunits in HF compared to sham rats. Together, our studies demonstrate that MNCs from HF rats exhibit increased membrane excitability and an enhanced input–output function, and also that a reduction in SK channel‐mediated, apamin‐sensitive AHP is a critical contributing mechanism. Moreover, our results suggest that the reduced AHP is related to a down‐regulation of SK2/SK3 channel subunit expression but not the result of a blunted activity‐dependent intracellular Ca2+ increase following a burst of action potentials.
    August 02, 2017   doi: 10.1113/JP274730   open full text
  • Release of ATP by pre‐Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+‐dependent P2Y1 receptor mechanism.
    Vishaal Rajani, Yong Zhang, Venkatesh Jalubula, Vladimir Rancic, Shahriar SheikhBahaei, Jennifer D. Zwicker, Silvia Pagliardini, Clayton T. Dickson, Klaus Ballanyi, Sergey Kasparov, Alexander V. Gourine, Gregory D. Funk.
    The Journal of Physiology. July 27, 2017
    Key points The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre‐Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor‐mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. Abstract The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady‐state phase II) that can be life‐threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre‐Bötzinger Complex (preBötC) inspiratory rhythm‐generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia‐induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor‐mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+]i) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.
    July 27, 2017   doi: 10.1113/JP274727   open full text
  • In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury.
    Andreas Patsalos, Attila Pap, Tamas Varga, Gyorgy Trencsenyi, Gerardo Alvarado Contreras, Ildiko Garai, Zoltan Papp, Balazs Dezso, Eva Pintye, Laszlo Nagy.
    The Journal of Physiology. July 17, 2017
    Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for necrotic debris clearance and for coordination of tissue regeneration. This highly dynamic process is characterized by an in‐situ transition of infiltrating monocytes from an inflammatory (Ly6Chigh) to a repair (Ly6Clow) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross‐talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging we show that preserved muscle integrity and cell composition prior to the injury is necessary for repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross‐talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions are shaping the macrophages in‐situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics. This article is protected by copyright. All rights reserved
    July 17, 2017   doi: 10.1113/JP274361   open full text
  • Effect of dietary salt intake on Epithelial Na+ Channels (ENaC) in vasopressin magnocellular neurosecretory neurons in the rat supraoptic nucleus.
    Kaustubh Sharma, Masudul Haque, Richard Guidry, Yoichi Ueta, Ryoichi Teruyama.
    The Journal of Physiology. July 17, 2017
    All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) were located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na+ leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β‐ and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch‐clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high‐salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high‐salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaC which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs. This article is protected by copyright. All rights reserved
    July 17, 2017   doi: 10.1113/JP274856   open full text
  • Acetylcholine‐dependent upregulation of TASK‐1 channels in thalamic interneurons by a smooth muscle‐like signalling pathway.
    Michael Leist, Susanne Rinné, Maia Datunashvili, Ania Aissaoui, Hans‐Christian Pape, Niels Decher, Sven G. Meuth, Thomas Budde.
    The Journal of Physiology. July 17, 2017
    The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state‐dependent transmission of visual information. Non‐retinal inputs from the ascending arousal system and inhibition provided by γ‐aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay (TC) neurons by inhibiting two‐pore domain potassium (K2P) channels. Conversely, ACh also hyperpolarizes INs via an as‐yet‐unknown mechanism. By using whole cell patch‐clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors (M2AChRs) induces IN hyperpolarization by recruiting the G beta‐gamma complex (Gβγ), class‐1A phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K), and cellular and sarcoma (c‐Src) tyrosine kinase (TK), leading to activation of two‐pore domain weakly inwardly rectifying K+ channel (TWIK)‐related acid‐sensitive K+ (TASK)‐1 channels. The latter was confirmed by the use of TASK‐1 deficient mice. Furthermore inhibition of phospholipase Cβ (PLCβ) as well as an increase in the intracellular level of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c‐Src TK in regulating IN function in the brain and identified a novel mechanism by which TASK‐1 channels are activated in neurons. This article is protected by copyright. All rights reserved
    July 17, 2017   doi: 10.1113/JP274527   open full text
  • N‐glycan content modulates kainate receptor functional properties.
    Claire G. Vernon, Bryan A. Copits, Jacob R. Stolz, Yomayra F. Guzmán, Geoffrey T. Swanson.
    The Journal of Physiology. July 17, 2017
    Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between 4 and 12 consensus sites for N‐glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post‐translational modification. N‐glycosylation is required for proper folding of iGluRs in mammalian cells, but the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged, and mediate many protein‐protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. We show here that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide HNK‐1. Glycosylation patterns likely differ between cell types, across development, or with pathologies, and thus our findings reveal a potential mechanism for context‐specific fine‐tuning of KAR function through diversity in glycan structure. This article is protected by copyright. All rights reserved
    July 17, 2017   doi: 10.1113/JP274790   open full text
  • Ivermectin activates GIRK channels in a PIP2‐dependent, Gβγ‐independent manner and an amino acid residue at the slide helix governs the activation.
    I‐Shan Chen, Michihiro Tateyama, Yuko Fukata, Motonari Uesugi, Yoshihiro Kubo.
    The Journal of Physiology. July 17, 2017
    Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate‐gated Cl− channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand‐gated channels, such as Cys‐loop receptors and P2X receptors. In this study, we found that the G‐protein‐gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. By electrophysiological recordings in Xenopus oocytes, we observed that IVM activates GIRK channel in a phosphatidylinositol‐4,5‐biphosphate (PIP2)‐dependent manner, and that the IVM‐mediated GIRK activation is independent of Gβγ. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. By chimeric and mutagenesis analyses, we identified a unique amino acid residue of GIRK2 among GIRK family, Ile82, located in the slide helix between the TM1 and the N‐terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM‐CTD interface in GIRK channel, rather than the TMs, governs IVM‐mediated activation. These findings provide us with novel insights on the action mode of IVM in ion channels, and information toward identification of new pharmacophores which activate GIRK channel. This article is protected by copyright. All rights reserved
    July 17, 2017   doi: 10.1113/JP274871   open full text
  • Muscle carnitine availability plays a central role in regulating fuel metabolism in the rodent.
    Craig Porter, Dumitru Constantin‐Teodosiu, Despina Constantin, Brendan Leighton, Simon M. Poucher, Paul L. Greenhaff.
    The Journal of Physiology. July 16, 2017
    Key points Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium‐mediated muscle carnitine depletion on whole‐body fuel selection, and muscle fuel metabolism and its molecular regulation is under‐investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole‐body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole‐body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low‐density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. Abstract The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3‐(2,2,2‐trimethylhydrazinium)‐propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole‐body fuel selection, muscle fuel metabolism and its molecular regulation is under‐investigated. Male lean Zucker rats received water (control, n = 8) or meldonium‐supplemented water (meldonium, n = 8) for 10 days [1.6 g kg−1 body mass (BM) day−1 days 1–2, 0.8 g kg−1 BM day−1 thereafter]. From days 7–10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole‐body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium‐supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo.
    July 16, 2017   doi: 10.1113/JP274415   open full text
  • Pronounced limb and fibre type differences in subcellular lipid droplet content and distribution in elite skiers before and after exhaustive exercise.
    Han‐Chow E. Koh, Joachim Nielsen, Bengt Saltin, Hans‐Christer Holmberg, Niels Ørtenblad.
    The Journal of Physiology. July 16, 2017
    Key points Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle – one beneath the sarcolemma and the other between myofibrils. At rest, well‐trained leg muscles of cross‐country skiers contain 4‐ to 6‐fold more lipid droplets than equally well‐trained arm muscles, with a 3‐fold higher content in type 1 than in type 2 fibres. During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres. These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres. Abstract Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85–97% of the total volume fraction, while the subsarcolemmal pool made up 3–15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4‐ to 6‐fold higher in leg than in arm muscles (P < 0.001). Furthermore, the volume fraction of intermyofibrillar lipid droplets was 3‐fold higher in type 1 than in type 2 fibres (P < 0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise, intermyofibrillar lipid droplet volume fraction was 53% lower (P = 0.0082) in both fibre types in arm, but not leg muscles. This reduction was positively associated with the corresponding volume fraction prior to exercise (R2 = 0.84, P < 0.0001). No exercise‐induced change in the subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular distribution of lipid droplets in the type 1 and 2 fibres of well‐trained arm and leg muscles, as well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise. Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.
    July 16, 2017   doi: 10.1113/JP274462   open full text
  • Calcium signalling in medial intercalated cell dendrites and spines.
    Cornelia Strobel, Robert K. P. Sullivan, Peter Stratton, Pankaj Sah.
    The Journal of Physiology. July 16, 2017
    Key points Dendritic and spine calcium imaging in combination with electrophysiology in acute slices revealed that in medial intercalated cells of the amygdala: Action potentials back‐propagate into the dendritic tree, but due to the presence of voltage‐dependent potassium channels, probably Kv4.2 channels, attenuate over distance. A mixed population of AMPA receptors with rectifying and linear I–V relations are present at individual spines of a single neuron. Decay kinetics and pharmacology suggest tri‐heteromeric NMDA receptors at basolateral–intercalated cell synapses. NMDA receptors are the main contributors to spine calcium entry in response to synaptic stimulation. Calcium signals in response to low‐ and high‐frequency stimulation, and in combination with spontaneous action potentials are locally restricted to the vicinity of active spines. Together, these data show that calcium signalling in these GABAergic neurons is tightly controlled and acts as a local signal. Abstract The amygdala plays a central role in fear conditioning and extinction. The medial intercalated (mITC) neurons are GABAergic cell clusters interspaced between the basolateral (BLA) and central amygdala (CeA). These neurons are thought to play a key role in fear and extinction, controlling the output of the CeA by feed‐forward inhibition. BLA to mITC cell inputs are thought to undergo synaptic plasticity, a mechanism underlying learning, which is mediated by NMDA receptor‐dependent mechanisms that require changes in cytosolic calcium. Here, we studied the electrical and calcium signalling properties of mITC neurons in GAD67‐eGFP mice using whole‐cell patch clamp recordings and two‐photon calcium imaging. We show that action potentials back‐propagate (bAP) into dendrites, and evoke calcium transients in both the shaft and the dendritic spine. However, bAP‐mediated calcium rises in the dendrites attenuate with distance due to shunting by voltage‐gated potassium channels. Glutamatergic inputs make dual component synapses on spines. At these synapses, postsynaptic AMPA receptors can have linear or rectifying I–V relationships, indicating that some synapses express GluA2‐lacking AMPA receptors. Synaptic NMDA receptors had intermediate decay kinetics, and were only partly blocked by GuN2B selective blockers, indicating these receptors are GluN1/GluN2A/GluN2B trimers. Low‐ or high‐frequency synaptic stimulation raised spine calcium, mediated by calcium influx via NMDA receptors, was locally restricted and did not invade neighbouring spines. Our results show that in mITC neurons, postsynaptic calcium is tightly controlled, and acts as a local signal.
    July 16, 2017   doi: 10.1113/JP274261   open full text
  • Lack of linear correlation between dynamic and steady‐state cerebral autoregulation.
    Daan L. K. Jong, Takashi Tarumi, Jie Liu, Rong Zhang, Jurgen A. H. R. Claassen.
    The Journal of Physiology. July 14, 2017
    Key points For correct application and interpretation of cerebral autoregulation (CA) measurements in research and in clinical care, it is essential to understand differences and similarities between dynamic and steady‐state CA. The present study found no correlation between dynamic and steady‐state CA indices in healthy older adults. There was variability between individuals in all (steady‐state and dynamic) autoregulatory indices, ranging from low (almost absent) to highly efficient CA in this healthy population. These findings challenge the assumption that assessment of a single CA parameter or a single set of parameters can be generalized to overall CA functioning. Therefore, depending on specific research purposes, the choice for either steady‐state or dynamic measures or both should be weighed carefully. Abstract The present study aimed to investigate the relationship between dynamic (dCA) and steady‐state cerebral autoregulation (sCA). In 28 healthy older adults, sCA was quantified by a linear regression slope of proportionate (%) changes in cerebrovascular resistance (CVR) in response to proportionate (%) changes in mean blood pressure (BP) induced by stepwise sodium nitroprusside (SNP) and phenylephrine (PhE) infusion. Cerebral blood flow (CBF) was measured at the internal carotid artery (ICA) and vertebral artery (VA) and CBF velocity at the middle cerebral artery (MCA). With CVR = BP/CBF, Slope‐CVRICA, Slope‐CVRVA and Slope‐CVRiMCA were derived. dCA was assessed (i) in supine rest, analysed with transfer function analysis (gain and phase) and autoregulatory index (ARI) fit from spontaneous oscillations (ARIBaseline), and (ii) with transient changes in BP using a bolus injection of SNP (ARISNP) and PhE (ARIPhE). Comparison of sCA and dCA parameters (using Pearson's r for continuous and Spearman's ρ for ordinal parameters) demonstrated a lack of linear correlations between sCA and dCA measures. However, comparisons of parameters within dCA and within sCA were correlated. For sCA slope‐CVRVA with Slope‐CVRiMCA (r = 0.45, P < 0.03); for dCA ARISNP with ARIPhE (ρ = 0.50, P = 0.03), ARIBaseline (ρ = 0.57, P = 0.03) and PhaseLF (ρ = 0.48, P = 0.03); and for GainVLF with GainLF (r = 0.51, P = 0.01). By contrast to the commonly held assumption based on an earlier study, there were no linear correlations between sCA and dCA. As an additional observation, there was strong inter‐individual variability, both in dCA and sCA, in this healthy group of elderly, in a range from low to high CA efficiency.
    July 14, 2017   doi: 10.1113/JP274304   open full text
  • EEA1 restores homeostatic synaptic plasticity in hippocampal neurons from Rett syndrome mice.
    Xin Xu, Lucas Pozzo‐Miller.
    The Journal of Physiology. July 12, 2017
    Key points Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Mecp2 deletion in mice results in an imbalance of excitation and inhibition in hippocampal neurons, which affects ‘Hebbian’ synaptic plasticity. We show that Mecp2‐deficient neurons also lack homeostatic synaptic plasticity, likely due to reduced levels of EEA1, a protein involved in AMPA receptor endocytosis. Expression of EEA1 restored homeostatic synaptic plasticity in Mecp2‐deficient neurons, providing novel targets of intervention in Rett syndrome. Abstract Rett syndrome is a neurodevelopmental disorder caused by loss‐of‐function mutations in MECP2, the gene encoding the transcriptional regulator methyl‐CpG‐binding protein 2 (MeCP2). Deletion of Mecp2 in mice results in an imbalance of synaptic excitation and inhibition in hippocampal pyramidal neurons, which affects ‘Hebbian’ long‐term synaptic plasticity. Since the excitatory–inhibitory balance is maintained by homeostatic mechanisms, we examined the role of MeCP2 in homeostatic synaptic plasticity (HSP) at excitatory synapses. Negative feedback HSP, also known as synaptic scaling, maintains the global synaptic strength of individual neurons in response to sustained alterations in neuronal activity. Hippocampal neurons from Mecp2 knockout (KO) mice do not show the characteristic homeostatic scaling up of the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and of synaptic levels of the GluA1 subunit of AMPA‐type glutamate receptors after 48 h silencing with the Na+ channel blocker tetrodotoxin. This deficit in HSP is bidirectional because Mecp2 KO neurons also failed to scale down mEPSC amplitudes and GluA1 synaptic levels after 48 h blockade of type A GABA receptor (GABAAR)‐mediated inhibition with bicuculline. Consistent with the role of synaptic trafficking of AMPA‐type of glutamate receptors in HSP, Mecp2 KO neurons have lower levels of early endosome antigen 1 (EEA1), a protein involved in AMPA‐type glutamate receptor endocytosis. In addition, expression of EEA1 in Mecp2 KO neurons reduced mEPSC amplitudes to wild‐type levels, and restored synaptic scaling down of mEPSC amplitudes after 48 h blockade of GABAAR‐mediated inhibition with bicuculline. The identification of a molecular deficit in HSP in Mecp2 KO neurons provides potentially novel targets of intervention for improving hippocampal function in Rett syndrome individuals.
    July 12, 2017   doi: 10.1113/JP274450   open full text
  • Heterotypic endosomal fusion as an initial trigger for insulin‐induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.
    Hiroyasu Hatakeyama, Makoto Kanzaki.
    The Journal of Physiology. July 10, 2017
    Key points Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot‐based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving ‘static retention’ and ‘stimulus‐dependent liberation’. Vital imaging analyses and super‐resolution microscopy‐based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4‐containing vesicles in skeletal myofibres. Prior exposure to exercise‐mimetic stimuli potentiated this insulin‐responsive endomembrane fusion event involving GLUT4‐containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Abstract Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin‐induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4‐containing vesicles with a subset of transferrin receptor‐containing endosomes. Importantly, pretreatment with exercise‐mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe‐like localization close to sarcomeric α‐actinin and that insulin induced a reduction of the stripe‐like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.
    July 10, 2017   doi: 10.1113/JP273985   open full text
  • Mechanisms underlying vestibulo‐cerebellar motor learning in mice depend on movement direction.
    Kai Voges, Bin Wu, Laura Post, Martijn Schonewille, Chris I. Zeeuw.
    The Journal of Physiology. July 10, 2017
    Key points Directionality, inherent to movements, has behavioural and neuronal correlates. Direction of vestibular stimulation determines motor learning efficiency. Vestibulo‐ocular reflex gain–increase correlates with Purkinje cell simple spike potentiation. The locus of neural correlates for vestibulo‐ocular reflex adaptation is paradigm specific. Abstract Compensatory eye movements elicited by head rotation, also known as vestibulo‐ocular reflex (VOR), can be adapted with the use of visual feedback. The cerebellum is essential for this type of movement adaptation, although its neuronal correlates remain to be clarified. In the present study, we show that the direction of vestibular input determines the magnitude of eye movement adaptation induced by mismatched visual input in mice, with larger changes during contraversive head rotation. Moreover, the location of the neural correlate of this changed behaviour depends on the type of paradigm. Gain–increase paradigms induce increased simple spike (SS) activity in ipsilateral cerebellar Purkinje cells (PC), which is in line with eye movements triggered by optogenetic PC activation. By contrast, gain–decrease paradigms do not induce changes in SS activity, indicating that the murine vestibulo‐cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements.
    July 10, 2017   doi: 10.1113/JP274346   open full text
  • Ecto‐5′‐nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia.
    Andrew P. Holmes, Clare J. Ray, Selina A. Pearson, Andrew M. Coney, Prem Kumar.
    The Journal of Physiology. July 09, 2017
    Key points Carotid body dysfunction is recognized as a cause of hypertension in a number of cardiorespiratory diseases states and has therefore been identified as a potential therapeutic target. Purinergic transmission is an important element of the carotid body chemotransduction pathway. We show that inhibition of ecto‐5′‐nucleotidase (CD73) in vitro reduces carotid body basal discharge and responses to hypoxia and mitochondrial inhibition. Additionally, inhibition of CD73 in vivo decreased the hypoxic ventilatory response, reduced the hypoxia‐induced heart rate elevation and exaggerated the blood pressure decrease in response to hypoxia. Our data show CD73 to be a novel regulator of carotid body sensory function and therefore suggest that this enzyme may offer a new target for reducing carotid body activity in selected cardiovascular diseases. Abstract Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies, including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The present study investigated whether ecto‐5′‐nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of CD73 by α,β‐methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8‐(p‐sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (ΔV̇E control 74 ± 6%, ΔV̇E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as indicated by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify CD73 as a novel regulator of CB sensory activity. Future investigations are warranted to clarify whether inhibition of CD73 can effectively reduce CB activity in CB‐mediated cardiovascular pathology.
    July 09, 2017   doi: 10.1113/JP274498   open full text
  • Sympatholytic effect of intravascular ATP is independent of nitric oxide, prostaglandins, Na+/K+‐ATPase and KIR channels in humans.
    Christopher M. Hearon, Jennifer C. Richards, Mathew L. Racine, Gary J. Luckasen, Dennis G. Larson, Michael J. Joyner, Frank A. Dinenno.
    The Journal of Physiology. July 09, 2017
    Key points Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia. Similar to exercise, ATP‐mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR), and synthesis of nitric oxide (NO) and prostaglandins (PG). However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1‐adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR, NO, PGs and Na+/K+‐ATPase activity. Blockade of KIR channels alone or in combination with NO, PGs and Na+/K+‐ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1‐adrenergic vasoconstriction. This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans. Abstract Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α‐adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+/K+‐ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+/K+‐ATPase would not alter the ability of ATP to blunt α1‐adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n = 7; Protocol 1); NO (l‐NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+/K+‐ATPase (ouabain) and KIR channel inhibition (n = 6; Protocol 2). ATP attenuated PE‐mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE‐mediated ΔFVC: ATP: −16 ± 2; ADO: −38 ± 6; SNP: −59 ± 6%; P < 0.05 vs. ADO and SNP). Blockade of KIR channels alone or combined with NO, PGs and Na+/K+‐ATPase, attenuated ATP‐mediated vasodilatation (∼35 and ∼60% respectively; P < 0.05 vs. control). However, ATP maintained the ability to blunt PE‐mediated vasoconstriction (PE‐mediated ΔFVC: KIR blockade alone: −6 ± 5%; combined blockade:−4 ± 14%; P > 0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1‐adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+/K+‐ATPase in humans, consistent with a role for endothelium‐derived hyperpolarization in functional sympatholysis.
    July 09, 2017   doi: 10.1113/JP274532   open full text
  • Transcriptomic analysis identifies a role of PI3K/Akt signalling in the responses of skeletal muscle to acute hypoxia in vivo.
    Zhuohui Gan, Frank L. Powell, Alexander C. Zambon, Kyle S. Buchholz, Zhenxing Fu, Karen Ocorr, Rolf Bodmer, Esteban A. Moya, Jennifer C. Stowe, Gabriel G. Haddad, Andrew D. McCulloch.
    The Journal of Physiology. July 08, 2017
    The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia‐tolerant tissues like skeletal muscles. We used RNA‐seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down‐regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down‐regulated genes had mRNA half‐lives longer than 2 h, suggesting a role for post‐transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K/Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K/Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K/Akt targets, 75% could be explained by a deactivation of ARE‐binding protein BRF1, a target of PI3K/Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K/Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the de‐phosphorylation of ARE‐binding protein BRF1. This article is protected by copyright. All rights reserved
    July 08, 2017   doi: 10.1113/JP274556   open full text
  • Sensory feedback from the urethra evokes state‐dependent lower urinary tract reflexes in rat.
    Zachary C. Danziger, Warren M. Grill.
    The Journal of Physiology. July 07, 2017
    Key points The lower urinary tract is regulated by reflexes responsible for maintaining continence and producing efficient voiding. It is unclear how sensory information from the bladder and urethra engages differential, state‐dependent reflexes to either maintain continence or promote voiding. Using a new in vivo experimental approach, we quantified how sensory information from the bladder and urethra are integrated to switch reflex responses to urethral sensory feedback from maintaining continence to producing voiding. The results demonstrate how sensory information regulates state‐dependent reflexes in the lower urinary tract and contribute to our understanding of the pathophysiology of urinary retention and incontinence where sensory feedback may engage these reflexes inappropriately. Abstract Lower urinary tract reflexes are mediated by peripheral afferents from the bladder (primarily in the pelvic nerve) and the urethra (in the pudendal and pelvic nerves) to maintain continence or initiate micturition. If fluid enters the urethra at low bladder volumes, reflexes relax the bladder and evoke external urethral sphincter (EUS) contraction (guarding reflex) to maintain continence. Conversely, urethral flow at high bladder volumes, excites the bladder (micturition reflex) and relaxes the EUS (augmenting reflex). We conducted measurements in a urethane‐anaesthetized in vivo rat preparation to characterize systematically the reflexes evoked by fluid flow through the urethra. We used a novel preparation to manipulate sensory feedback from the bladder and urethra independently by controlling bladder volume and urethral flow. We found a distinct bladder volume threshold (74% of bladder capacity) above which flow‐evoked bladder contractions were 252% larger and evoked phasic EUS activation 2.6 times as often as responses below threshold, clearly demonstrating a discrete transition between continence (guarding) and micturition (augmenting) reflexes. Below this threshold urethral flow evoked tonic EUS activity, indicative of the guarding reflex, that was proportional to the urethral flow rate. These results demonstrate the complementary roles of sensory feedback from the bladder and urethra in regulating reflexes in the lower urinary tract that depend on the state of the bladder. Understanding the neural control of functional reflexes and how they are mediated by sensory information in the bladder and urethra will open new opportunities, especially in neuromodulation, to treat pathologies of the lower urinary tract.
    July 07, 2017   doi: 10.1113/JP274191   open full text
  • Impact of ageing on postsynaptic neuronal nicotinic neurotransmission in auditory thalamus.
    Sarah Y. Sottile, Lynne Ling, Brandon C. Cox, Donald M. Caspary.
    The Journal of Physiology. July 07, 2017
    Key points Neuronal nicotinic acetylcholine receptors (nAChRs) play a fundamental role in the attentional circuitry throughout the mammalian CNS. In the present study, we report a novel finding that ageing negatively impacts nAChR efficacy in auditory thalamus, and this is probably the result of a loss of nAChR density (Bmax) and changes in the subunit composition of nAChRs. Our data support the hypothesis that age‐related maladaptive changes involving nAChRs within thalamocortical circuits partially underpin the difficulty that elderly adults experience with respect to attending to speech and other salient acoustic signals. Abstract The flow of auditory information through the medial geniculate body (MGB) is regulated, in part, by cholinergic projections from the pontomesencephalic tegmentum. The functional significance of these projections is not fully established, although they have been strongly implicated in the allocation of auditory attention. Using in vitro slice recordings, we have analysed postsynaptic function and pharmacology of neuronal nicotinic ACh receptors (nAChRs) in young adult and the aged rat MGB. We find that ACh produces significant excitatory postsynaptic actions on young MGB neurons, probably mediated by β2‐containing heteromeric nAChRs. Radioligand binding studies show a significant age‐related loss of heteromeric nAChR receptor number, which supports patch clamp data showing an age‐related loss in ACh efficacy in evoking postsynaptic responses. Use of the β2‐selective nAChR antagonist, dihydro‐β‐erythroidine, suggests that loss of cholinergic efficacy may also be the result of an age‐related subunit switch from high affinity β2‐containing nAChRs to low affinity β4‐containing nAChRs, in addition to the loss of total nAChR number. This age‐related nAChR dysfunction may partially underpin the attentional deficits that contribute to the loss of speech understanding in the elderly.
    July 07, 2017   doi: 10.1113/JP274467   open full text
  • Promotion of endocytosis efficiency through an ATP‐independent mechanism at rat calyx of Held terminals.
    Hai‐Yuan Yue, Erhard Bieberich, Jianhua Xu.
    The Journal of Physiology. July 05, 2017
    Key points At rat calyx of Held terminals, ATP was required not only for slow endocytosis, but also for rapid phase of compensatory endocytosis. An ATP‐independent form of endocytosis was recruited to accelerate membrane retrieval at increased activity and temperature. ATP‐independent endocytosis primarily involved retrieval of pre‐existing membrane, which depended on Ca2+ and the activity of neutral sphingomyelinase but not clathrin‐coated pit maturation. ATP‐independent endocytosis represents a non‐canonical mechanism that can efficiently retrieve membrane at physiological conditions without competing for the limited ATP at elevated neuronal activity. Abstract Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP‐dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP‐independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at rat calyx of Held terminals by whole‐cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis but only partially inhibited excess endocytosis following intense stimulation. The ATP‐independent endocytosis occurred at calyces from postnatal days 8–15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin‐coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre‐existing at the surface, and was impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP‐independent endocytosis became more significant at 34–35°C, and recovered membrane by an amount that, on average, was close to exocytosis. The results of the present study suggest that activity and temperature recruit ATP‐independent endocytosis of pre‐existing membrane (in addition to ATP‐dependent endocytosis) to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non‐canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.
    July 05, 2017   doi: 10.1113/JP274275   open full text
  • Chronic electromyograms in treadmill running SOD1 mice reveal early changes in muscle activation.
    Katharina A. Quinlan, Elma Kajtaz, Jody D. Ciolino, Rebecca D. Imhoff‐Manuel, Matthew C. Tresch, Charles J. Heckman, Vicki M. Tysseling.
    The Journal of Physiology. July 05, 2017
    Key points The present study demonstrates that electromyograms (EMGs) obtained during locomotor activity in mice were effective for identification of early physiological markers of amyotrophic lateral sclerosis (ALS). These measures could be used to evaluate therapeutic intervention strategies in animal models of ALS. Several parameters of locomotor activity were shifted early in the disease time course in SOD1G93A mice, especially when the treadmill was inclined, including intermuscular phase, burst skew and amplitude of the locomotor bursts. The results of the present study indicate that early compensatory changes may be taking place within the neural network controlling locomotor activity, including spinal interneurons. Locomotor EMGs could have potential use as a clinical diagnostic tool. Abstract To improve our understanding of early disease mechanisms and to identify reliable biomarkers of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, we measured electromyogram (EMG) activity in hind limb muscles of SOD1G93A mice. By contrast to clinical diagnostic measures using EMGs, which are performed on quiescent patients, we monitored activity during treadmill running aiming to detect presymptomatic changes in motor patterning. Chronic EMG electrodes were implanted into vastus lateralis, biceps femoris posterior, lateral gastrocnemius and tibialis anterior in mice from postnatal day 55 to 100 and the results obtained were assessed using linear mixed models. We evaluated differences in parameters related to EMG amplitude (peak and area) and timing (phase and skew, a measure of burst shape) when animals ran on level and inclined treadmills. There were significant changes in both the timing of activity and the amplitude of EMG bursts in SOD1G93A mice. Significant differences between wild‐type and SOD1G93A mice were mainly observed when animals locomoted on inclined treadmills. All muscles had significant effects of mutation that were independent of age. These novel results indicate (i) locomotor EMG activity might be an early measure of disease onset; (ii) alterations in locomotor patterning may reflect changes in neuronal drive and compensation at the network level including altered activity of spinal interneurons; and (iii) the increased power output necessary on an inclined treadmill was important in revealing altered activity in SOD1G93A mice.
    July 05, 2017   doi: 10.1113/JP274170   open full text
  • Altered post‐capillary and collecting venular reactivity in skeletal muscle with metabolic syndrome.
    Kent A. Lemaster, Zahra Farid, Robert W. Brock, Carl D. Shrader, Daniel Goldman, Dwayne N. Jackson, Jefferson C. Frisbee.
    The Journal of Physiology. July 05, 2017
    Key points With the development of the metabolic syndrome, both post‐capillary and collecting venular dilator reactivity within the skeletal muscle of obese Zucker rats (OZR) is impaired. The impaired dilator reactivity in OZR reflects a loss in venular nitric oxide and PGI2 bioavailability, associated with the chronic elevation in oxidant stress. Additionally, with the impaired dilator responses, a modest increase in adrenergic constriction combined with an elevated thromboxane A2 production may contribute to impaired functional dilator and hyperaemic responses at the venular level. For the shift in skeletal muscle venular function with development of the metabolic syndrome, issues such as aggregate microvascular perfusion resistance, mass transport and exchange within with capillary networks, and fluid handling across the microcirculation are compelling avenues for future investigation. Abstract While research into vascular outcomes of the metabolic syndrome has focused on arterial/arteriolar and capillary levels, investigation into venular function and how this impacts responses has received little attention. Using the in situ cremaster muscle of obese Zucker rats (OZR; with lean Zucker rats (LZR) as controls), we determined indices of venular function. At ∼17 weeks of age, skeletal muscle post‐capillary venular density was reduced by ∼20% in LZR vs. OZR, although there was no evidence of remodelling of the venular wall. Venular tone at ∼25 μm (post‐capillary) and ∼75 μm (collecting) diameter was elevated in OZR vs. LZR. Venular dilatation to acetylcholine was blunted in OZR vs. LZR due to increased oxidant stress‐based loss of nitric oxide bioavailability (post‐capillary) and increased α1‐ (and α2‐) mediated constrictor tone (collecting). Venular constrictor responses in OZR were comparable to LZR for most stimuli, although constriction to α1‐adrenoreceptor stimulation was elevated. In response to field stimulation of the cremaster muscle (0.5, 1, 3 Hz), venular dilator and hyperaemic responses to lower frequencies were blunted in OZR, but responses at 3 Hz were similar between strains. Venous production of TxA2 was higher in OZR than LZR and significantly higher than PGI2 production in either following arachidonic acid challenge. These results suggest that multi‐faceted alterations to skeletal muscle venular function in OZR may contribute to alterations in upstream capillary pressure profiles and the transcapillary exchange of solutes and water under conditions of metabolic syndrome.
    July 05, 2017   doi: 10.1113/JP274291   open full text
  • Glial EAAT2 regulation of extracellular nTS glutamate critically controls neuronal activity and cardiorespiratory reflexes.
    Michael P. Matott, David D. Kline, Eileen M. Hasser.
    The Journal of Physiology. July 05, 2017
    Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, their influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2 specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK‐induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex‐mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function. This article is protected by copyright. All rights reserved
    July 05, 2017   doi: 10.1113/JP274620   open full text
  • Inhibitory modulation of medial prefrontal cortical activation on lateral orbitofrontal cortex‐amygdala information flow.
    Chun‐hui Chang, Ta‐wen Ho.
    The Journal of Physiology. July 05, 2017
    Several neocortical projections converge onto the basolateral complex of the amygdala (BLA), including the lateral orbitofrontal cortex (lOFC) and medial prefrontal cortex (mPFC). Lateral orbitofrontal input to BLA is important for cue‐outcome contingencies, while medial prefrontal input is essential for emotion control. In this study, we examined how mPFC, specifically the infralimbic (IL) division of mPFC, modulates the lOFC‐BLA information flow, using combined in vivo extracellular single‐unit recordings and pharmacological manipulations in anesthetized rats. We found that the majority (over 95%) of BLA neurons that responded to lOFC stimulation also responded to mPFC stimulation. Compared to basal condition, pharmacological (N‐Methyl‐D‐aspartate, NMDA) or electrical activation of the mPFC exerted an inhibitory modulation of the lOFC‐BLA pathway, which was reversed with intra‐amygdala blockade of GABAergic receptors with combined GABAA and GABAB antagonists (bicuculline and saclofen). Moreover, mPFC tetanus potentiated the lOFC‐BLA pathway, but mPFC tetanus or low‐frequency stimulation (LFS) did not alter its inhibitory modulatory gating on the lOFC‐BLA pathway. These results show that the mPFC potently inhibits lOFC drive of BLA neurons in a GABA‐dependent manner. Our result is informative in understanding the normal and potential pathophysiological state of emotion and contingency associations regulating behaviour. This article is protected by copyright. All rights reserved
    July 05, 2017   doi: 10.1113/JP274568   open full text
  • Kir2.1 and K2P1 channels reconstitute two levels of resting membrane potential in cardiomyocytes.
    Dongchuan Zuo, Kuihao Chen, Min Zhou, Zheng Liu, Haijun Chen.
    The Journal of Physiology. July 04, 2017
    Key points Outward and inward background currents across the cell membrane balance, determining resting membrane potential. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the resting membrane potential of cardiomyocytes. Human cardiomyocytes exhibit two levels of resting membrane potential at subphysiological extracellular K+ concentrations or pathological hypokalaemia, however, the underlying mechanism is unclear. In the present study, we show that human cardiomyocytes derived from induced pluripotent stem cells with enhanced expression of isoform 1 of Kir2 (Kir2.1) channels and mouse HL‐1 cardiomyocytes with ectopic expression of two pore‐domain K+ channel isoform 1 (K2P1) recapitulate two levels of resting membrane potential, indicating the contributions of Kir2.1 and K2P1 channels to the phenomenon. In Chinese hamster ovary cells that express the channels, Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced K2P1 leak cation currents, reconstituting two levels of resting membrane potential. These findings support the hypothesis that Kir2 currents non‐linearly counterbalance inward background cation currents, such as K2P1 currents, accounting for two levels of resting membrane potential in human cardiomyocytes and demonstrating a novel mechanism that regulates excitability. Abstract Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the normal resting membrane potential of cardiomyocytes. At subphysiological extracellular K+ concentrations or pathological hypokalaemia, human cardiomyocytes show both hyperpolarized and depolarized resting membrane potentials; these depolarized potentials cause cardiac arrhythmia; however, the underlying mechanism is unknown. In the present study, we show that inward rectifier K+ channel subfamily 2 isoform 1 (Kir2.1) currents non‐linearly counterbalance hypokalaemia‐induced two pore‐domain K+ channel isoform 1 (K2P1) leak cation currents, reconstituting two levels of resting membrane potential in cardiomyocytes. Under hypokalaemic conditions, both human cardiomyocytes derived from induced pluripotent stem cells with enhanced Kir2.1 expression and mouse HL‐1 cardiomyocytes with ectopic expression of K2P1 channels recapitulate two levels of resting membrane potential. These cardiomyocytes display N‐shaped current–voltage relationships that cross the voltage axis three times and the first and third zero‐current potentials match the two levels of resting membrane potential. Inhibition of K2P1 expression eliminates the phenomenon, indicating contributions of Kir2.1 and K2P1 channels to two levels of resting membrane potential. Second, in Chinese hamster ovary cells that heterologously express the channels, Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced K2P1 leak cation currents, yielding the N‐shaped current–voltage relationships, causing the resting membrane potential to spontaneously jump from hyperpolarization at the first zero‐current potential to depolarization at the third zero‐current potential, again recapitulating two levels of resting membrane potential. These findings reveal ionic mechanisms of the two levels of resting membrane potential, demonstrating a previously unknown mechanism for the regulation of excitability, and support the hypothesis that Kir2 currents non‐linearly balance inward background cation currents, accounting for two levels of resting membrane potential of human cardiomyocytes.
    July 04, 2017   doi: 10.1113/JP274268   open full text
  • A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions.
    Gregory K. Potts, Rachel M. McNally, Rocky Blanco, Jae‐Sung You, Alexander S. Hebert, Michael S. Westphall, Joshua J. Coon, Troy A. Hornberger.
    The Journal of Physiology. July 04, 2017
    Key points Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill‐defined. Here we report a mass spectrometry‐based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal‐intensity contractions (MICs). Our dataset provides the first comprehensive map of the MIC‐regulated phosphoproteome. Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well‐known MIC‐regulated signalling pathways, as well as to a plethora of novel MIC‐regulated events. We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field. Abstract The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal‐intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal‐intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC‐regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation‐based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase‐substrate predictions indicated that the MIC‐regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z‐disc, with over 74% of the Z‐disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z‐disc kinases (striated muscle‐specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.
    July 04, 2017   doi: 10.1113/JP273904   open full text
  • VEGF‐A165b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF‐A splice isoforms from the kidney.
    Megan Stevens, Christopher R. Neal, Andrew H. J. Salmon, David O. Bates, Steven J. Harper, Sebastian Oltean.
    The Journal of Physiology. July 03, 2017
    Key points Progressive depletion of all vascular endothelial growth factor A (VEGF‐A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over‐expression of VEGF‐A165b only. VEGF‐A165b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub‐podocyte space coverage, produced by VEGF‐A depletion. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF‐A165b has opposite effects to VEGF‐A165 on the expression of genes involved in endothelial cell migration and proliferation. Abstract Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF‐A). However, little is known about the contribution of VEGF‐A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF‐A165b (resulting from alternative usage of a 3′ splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad‐transgenic model, that over‐expression of VEGF‐A165b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF‐A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub‐podocyte space coverage is reduced when VEGF‐A is depleted, all of which are rescued in VEGF‐A165b over‐expressors. VEGF‐A165b restores the expression of platelet endothelial cell adhesion molecule‐1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down‐regulates genes involved in migration and proliferation of endothelial cells, otherwise up‐regulated by the canonical isoform VEGF‐A165. The results of the present study indicate that manipulation of VEGF‐A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.
    July 03, 2017   doi: 10.1113/JP274481   open full text
  • Impaired activity of adherens junctions contributes to endothelial dilator dysfunction in ageing rat arteries.
    Fumin Chang, Sheila Flavahan, Nicholas A. Flavahan.
    The Journal of Physiology. June 30, 2017
    Key points Ageing‐induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE‐cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium‐dependent dilatation. Ageing increased internalization and degradation of VE‐cadherin, resulting in impaired activity of adherens junctions. Inhibition of VE‐cadherin clustering at adherens junctions (function‐blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries. Src tyrosine kinase activity and tyrosine phosphorylation of VE‐cadherin were increased in old arteries. Src inhibition increased VE‐cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE‐cadherin FBA. Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing. Abstract Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3–4 months) and old (22–24 months) F344 rats. VE‐cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE‐cadherin species was present on the cell surface and in TTX‐insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE‐cadherin were decreased, whereas levels of a TTX‐soluble 115 kDa VE‐cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium‐dependent dilatation that was decreased in old compared to young arteries. Disruption of VE‐cadherin clustering at AJs (function‐blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE‐cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE‐cadherin in the TTX‐insoluble fraction, (ii) VE‐cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src‐induced phosphorylation, internalization and degradation of VE‐cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing.
    June 30, 2017   doi: 10.1113/JP274189   open full text
  • Enhancement of synchronized activity between hippocampal CA1 neurons during initial storage of associative fear memory.
    Yu‐Zhang Liu, Yao Wang, Weida Shen, Zhiru Wang.
    The Journal of Physiology. June 30, 2017
    Key points Learning and memory storage requires neuronal plasticity induced in the hippocampus and other related brain areas, and this process is thought to rely on synchronized activity in neural networks. We used paired whole‐cell recording in vivo to examine the synchronized activity that was induced in hippocampal CA1 neurons by associative fear learning. We found that both membrane potential synchronization and spike synchronization of CA1 neurons could be transiently enhanced after task learning, as observed on day 1 but not day 5. On day 1 after learning, CA1 neurons showed a decrease in firing threshold and rise times of suprathreshold membrane potential changes as well as an increase in spontaneous firing rates, possibly contributing to the enhancement of spike synchronization. The transient enhancement of CA1 neuronal synchronization may play important roles in the induction of neuronal plasticity for initial storage and consolidation of associative memory. Abstract The hippocampus is critical for memory acquisition and consolidation. This function requires activity‐ and experience‐induced neuronal plasticity. It is known that neuronal plasticity is largely dependent on synchronized activity. As has been well characterized, repetitive correlated activity of presynaptic and postsynaptic neurons can lead to long‐term modifications at their synapses. Studies on network activity have also suggested that memory processing in the hippocampus may involve learning‐induced changes of neuronal synchronization, as observed in vivo between hippocampal CA3 and CA1 networks as well as between the rhinal cortex and the hippocampus. However, further investigation of learning‐induced synchronized activity in the hippocampus is needed for a full understanding of hippocampal memory processing. In this study, by performing paired whole‐cell recording in vivo on CA1 pyramidal cells (PCs) in anaesthetized adult rats, we examined CA1 neuronal synchronization before and after associative fear learning. We first found in naive animals that there was a low level of membrane potential (MP) synchronization and spike synchronization of CA1 PCs. In conditioned animals, we found a significant enhancement of both MP synchronization and spike synchronization, as observed on day 1 after learning, and this enhancement was transient and not observed on day 5. Accompanying learning‐induced synchronized activity was a decreased firing threshold and rise time of suprathreshold MP changes as well as an increased spontaneous firing rate, possibly contributing to the enhanced spike synchronization. The transiently enhanced CA1 neuronal synchronization may have important roles in generating neuronal plasticity for hippocampal storage and consolidation of associative memory traces.
    June 30, 2017   doi: 10.1113/JP274212   open full text
  • Threshold position control of anticipation in humans: a possible role of corticospinal influences.
    Lei Zhang, Nicolas A. Turpin, Anatol G. Feldman.
    The Journal of Physiology. June 28, 2017
    Key points Sudden unloading of preloaded wrist muscles elicits motion to a new wrist position. Such motion is prevented if subjects unload muscles using the contralateral arm (self‐unloading). Corticospinal influences originated from the primary motor cortex maintain tonic influences on motoneurons of wrist muscles before sudden unloading but modify these influences prior to the onset and until the end of self‐unloading. Results are interpreted based on the previous finding that intentional actions are caused by central, particularly corticospinal, shifts in the spatial thresholds at which wrist motoneurons are activated, thus predetermining the attractor point at which the neuromuscular periphery achieves mechanical balance with environment forces. By maintaining or shifting the thresholds, descending systems let body segments go to the equilibrium position in the respective unloading tasks without the pre‐programming of kinematics or muscle activation patterns. The study advances the understanding of how motor actions in general, and anticipation in particular, are controlled. Abstract The role of corticospinal (CS) pathways in anticipatory motor actions was evaluated using transcranial magnetic stimulation (TMS) of the primary motor cortex projecting to motoneurons (MNs) of wrist muscles. Preloaded wrist flexors were suddenly unloaded by the experimenter or by the subject using the other hand (self‐unloading). After sudden unloading, the wrist joint involuntarily flexed to a new position. In contrast, during self‐unloading the wrist remained almost motionless, implying that an anticipatory postural adjustment occurred. In the self‐unloading task, anticipation was manifested by a decrease in descending facilitation of pre‐activated flexor MNs starting ∼72 ms before changes in the background EMG activity. Descending facilitation of extensor MNs began to increase ∼61 ms later. Conversely, these influences remained unchanged before sudden unloading, implying the absence of anticipation. We also tested TMS responses during EMG silent periods produced by brief muscle shortening, transiently resulting in similar EMG levels before the onset and after the end of self‐unloading. We found reduced descending facilitation of flexor MNs after self‐unloading. To explain why the wrist excursion was minimized in self‐unloading due to these changes in descending influences, we relied on previous demonstrations that descending systems pre‐set the threshold positions of body segments at which muscles begin to be activated, thus predetermining the equilibrium point to which the system is attracted. Based on this notion, a more consistent explanation of the kinematic, EMG and descending patterns in the two types of unloading is proposed compared to the alternative notion of direct pre‐programming of kinematic and/or EMG patterns.
    June 28, 2017   doi: 10.1113/JP274309   open full text
  • Skeletal myofiber vascular endothelial growth factor is required for the exercise training‐induced increase in dentate gyrus neuronal precursor cells.
    Benjamin Rich, Miriam Scadeng, Masahiro Yamaguchi, Peter D. Wagner, Ellen C. Breen.
    The Journal of Physiology. June 28, 2017
    Key points Peripheral vascular endothelial growth factor (VEGF) is necessary for exercise to stimulate hippocampal neurogenesis. Here we report that skeletal myofiber VEGF directly or indirectly regulates exercise‐signalled proliferation of neuronal precursor cells. Our results found skeletal myofiber VEGF to be necessary for maintaining blood flow through hippocampal regions independent of exercise training state. This study demonstrates that skeletal myofiber VEGF is required for the hippocampal VEGF response to acute exercise. These results help to establish the mechanisms by which exercise, through skeletal myofiber VEGF, affects the hippocampus. Abstract Exercise signals neurogenesis in the dentate gyrus of the hippocampus. This phenomenon requires vascular endothelial growth factor (VEGF) originating from outside the blood–brain barrier, but no cellular source has been identified. Thus, we hypothesized that VEGF produced by skeletal myofibers plays a role in regulating hippocampal neuronal precursor cell proliferation following exercise training. This was tested in adult conditional skeletal myofiber‐specific VEGF gene‐ablated mice (VEGFHSA−/−) by providing VEGFHSA−/− and non‐ablated (VEGFf/f) littermates with running wheels for 14 days. Following this training period, hippocampal cerebral blood flow (CBF) was measured by functional magnetic resonance imaging (fMRI), and neuronal precursor cells (BrdU+/Nestin+) were detected by immunofluorescence. The VEGFf/f trained group showed improvements in both speed and endurance capacity in acute treadmill running tests (P < 0.05). The VEGFHSA−/− group did not. The number of proliferating neuronal precursor cells was increased with training in VEGFf/f (P < 0.05) but not in VEGFHSA−/− mice. Endothelial cell (CD31+) number did not change in this region with exercise training or skeletal myofiber VEGF gene deletion. However, resting blood flow through the hippocampal region was lower in VEGFHSA−/− mice, both untrained and trained, than untrained VEGFf/f mice (P < 0.05). An acute hypoxic challenge decreased CBF (P < 0.05) in untrained VEGFf/f, untrained VEGFHSA−/− and trained VEGFHSA−/− mice, but not trained VEGFf/f mice. VEGFf/f, but not VEGFHSA−/−, mice were able to acutely run on a treadmill at an intensity sufficient to increase hippocampus VEGF levels. These data suggest that VEGF expressed by skeletal myofibers may directly or indirectly regulate both hippocampal blood flow and neurogenesis.
    June 28, 2017   doi: 10.1113/JP273994   open full text
  • Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome.
    Chih‐Jen Cheng, Yi‐Fen Lo, Jen‐Chi Chen, Chou‐Long Huang, Shih‐Hua Lin.
    The Journal of Physiology. June 27, 2017
    Key points The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype–phenotype association has not been established. Low‐level expression of the human ClC‐Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC‐Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C‐terminal in‐frame green fluorescent protein fusion may slow down the proteasome‐mediated proteolysis. Barttin co‐expression necessarily improves the stability, membrane trafficking and gating of ClC‐Kb. CLCNKB mutations in barttin‐binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC‐Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. Abstract Mutations in the CLCNKB gene encoding the human voltage‐gated chloride ClC‐Kb (hClC‐Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC‐Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype–phenotype association has not been established. In this study, we found that hClC‐Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In‐frame fusion of green fluorescent protein (GFP) to the C‐terminus of the channel may ameliorate proteasome degradation. Co‐expression of barttin increased protein abundance and membrane trafficking of hClC‐Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC‐Kb‐GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well‐described functional consequences and clinical presentations for genotype–phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC‐Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C‐terminus of hClC‐Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.
    June 27, 2017   doi: 10.1113/JP274344   open full text
  • Training alters the distribution of perilipin proteins in muscle following acute free fatty acid exposure.
    S. O. Shepherd, J. A. Strauss, Q. Wang, J. J. Dube, B. Goodpaster, D. G. Mashek, L. S. Chow.
    The Journal of Physiology. June 27, 2017
    Key points The lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown. We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only. We conclude that trained individuals are able to redistribute the pre‐existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG. Abstract Because the lipid droplet (LD)‐associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic–euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type‐specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P < 0.05) but did not affect PLIN protein content. At baseline, PLIN2 (+65%), PLIN3 (+105%) and PLIN5 (+53%; all P < 0.05) protein content was higher in trained compared to sedentary individuals. In trained individuals, lipid infusion increased the number of LDs associated with PLIN2 (+27%), PLIN3 (+73%) and PLIN5 (+40%; all P < 0.05) in type I fibres. By contrast, in sedentary individuals, lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute free fatty acid elevation therefore induces a redistribution of PLIN proteins to an expanded LD pool in trained individuals only and this may be part of the mechanism that enables fatty acids to be stored in IMTG.
    June 27, 2017   doi: 10.1113/JP274374   open full text
  • Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis.
    E. Matthew Morris, Colin S. McCoin, Julie A. Allen, Michelle L. Gastecki, Lauren G. Koch, Steven L. Britton, Justin A. Fletcher, Xiarong Fu, Wen‐Xing Ding, Shawn C. Burgess, R. Scott Rector, John P. Thyfault.
    The Journal of Physiology. June 27, 2017
    Key points Low intrinsic aerobic capacity is associated with increased all‐cause and liver‐related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high‐fat/high‐sucrose diet‐induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high‐fat/high‐sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non‐alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. Abstract Low aerobic capacity increases risk for non‐alcoholic fatty liver disease and liver‐related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet‐induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16‐week ‘western diet’ (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD‐fed low‐fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP‐1, TLR4, TLR2 and IL‐1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD‐induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD‐induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.
    June 27, 2017   doi: 10.1113/JP274281   open full text
  • Spike threshold dynamics in spinal motoneurons during scratching and swimming.
    Ramunas Grigonis, Aidas Alaburda.
    The Journal of Physiology. June 27, 2017
    During functional spinal neural network activity motoneurons receive intense synaptic input, and this could modulate the threshold for action potential generation, providing the ability to dynamically adjust the excitability and recruitment order for functional needs. In the present study we investigated the dynamics of action potential threshold during motor network activity. Intracellular recordings from spinal motoneurons in an ex vivo carapace‐spinal cord preparation from adult turtles were performed during two distinct types of motor behaviour – fictive scratching and fictive swimming. We found that the threshold of the first spike in episodes of scratching and swimming was the lowest. The threshold potential depolarizes by about 10 mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Depolarization of the threshold potential results in decreased excitability of motoneurons. Synaptic inputs do not modulate the threshold of the first action potential during episodes of scratching or of swimming. There is no correlation between changes in spike threshold and interspike intervals within bursts. Slow synaptic integration that results in a wave of membrane potential depolarization rather than fast synaptic events preceding each spike is the factor influencing the threshold potential within firing bursts during motor behaviours. This article is protected by copyright. All rights reserved
    June 27, 2017   doi: 10.1113/JP274434   open full text
  • Heterogeneity of Purkinje cell simple spike–complex spike interactions: zebrin‐ and non‐zebrin‐related variations.
    Tianyu Tang, Jianqiang Xiao, Colleen Y. Suh, Amelia Burroughs, Nadia L. Cerminara, Linjia Jia, Sarah P. Marshall, Andrew K. Wise, Richard Apps, Izumi Sugihara, Eric J. Lang.
    The Journal of Physiology. June 26, 2017
    Key points Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist. Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike–complex spike interactions both within and across zebrin populations. Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre‐ and post‐complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions. Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs. The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions. Abstract Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS‐associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre‐CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z−) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre‐CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the amplitudes of the SS modulation phases were generally weak. Division of spikelets into likely axonally propagated and non‐propagated groups (based on their interspikelet interval) showed that the correlation of spikelet number with SS firing rate primarily reflected a relationship with non‐propagated spikelets. In sum, the results show both zebrin‐related and non‐zebrin‐related physiological heterogeneity in SS–CS interactions among PCs, which suggests that the cerebellar cortex is more functionally diverse than is assumed by standard theories of cerebellar function.
    June 26, 2017   doi: 10.1113/JP274252   open full text
  • Coupling of excitation to Ca2+ release is modulated by dysferlin.
    Valeriy Lukyanenko, Joaquin M. Muriel, Robert J. Bloch.
    The Journal of Physiology. June 26, 2017
    Key points Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage‐induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin‐null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L‐type Ca2+ channels protect voltage‐induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+‐free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury‐induced SR Ca2+ leak. Abstract Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage‐induced Ca2+ transients elicited in dysferlin‐null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus‐dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L‐type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI‐induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1–FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus‐dysferlin prevented OSI‐induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1‐mediated Ca2+ leak from the SR into the cytoplasm.
    June 26, 2017   doi: 10.1113/JP274515   open full text
  • Blood flow restricted training leads to myocellular macrophage infiltration and upregulation of heat shock proteins, but no apparent muscle damage.
    Jakob L. Nielsen, Per Aagaard, Tatyana A. Prokhorova, Tobias Nygaard, Rune D. Bech, Charlotte Suetta, Ulrik Frandsen.
    The Journal of Physiology. June 23, 2017
    Key points Muscular contractions performed using a combination of low external loads and partial restriction of limb blood flow appear to induce substantial gains in muscle strength and muscle mass. This exercise regime may initially induce muscular stress and damage; however, the effects of a period of blood flow restricted training on these parameters remain largely unknown. The present study shows that short‐term, high‐frequency, low‐load muscle training performed with partial blood flow restriction does not induce significant muscular damage. However, signs of myocellular stress and inflammation that were observed in the early phase of training and after the training intervention, respectively, may be facilitating the previously reported gains in myogenic satellite cell content and muscle hypertrophy. The present results improve our current knowledge about the physiological effects of low‐load muscular contractions performed under blood flow restriction and may provide important information of relevance for future therapeutic treatment of muscular atrophy. Abstract Previous studies indicate that low‐load muscle contractions performed under local blood flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study protocols were conducted, covering 3 weeks (3 wk) or one week (1 wk). Subjects performed BFR exercise (100 mmHg, 20% 1RM) to concentric failure (BFRE) (3 wk/1 wk), while controls performed work‐matched (LLE) (3 wk) or high‐load (HLE; 70% 1RM) (1 wk) free‐flow exercise. Muscle biopsies (3 wk) were obtained at baseline (Pre), 8 days into the intervention (Mid8), and 3 and 10 days after training cessation (Post3, Post10) to examine macrophage (M1/M2) content as well as heat shock protein (HSP27/70) and tenascin‐C expression. Blood samples (1 wk) were collected before and after (0.1–24 h) the first and last training session to examine markers of muscle damage (creatine kinase), oxidative stress (total antibody capacity, glutathione) and inflammation (monocyte chemotactic protein‐1, interleukin‐6, tumour necrosis factor α). M1‐macrophage content increased 108–165% with BFRE and LLE at Post3 (P < 0.05), while M2‐macrophages increased (163%) with BFRE only (P < 0.01). Membrane and intracellular HSP27 expression increased 60–132% at Mid8 with BFRE (P < 0.05–0.01). No or only minor changes were observed in circulating markers of muscle damage, oxidative stress and inflammation. The amplitude, timing and localization of the above changes indicate that only limited muscle damage was evoked with BFRE. This study is the first to show that a period of high‐frequency, low‐load BFR training does not appear to induce general myocellular damage. However, signs of tissue inflammation and focal myocellular membrane stress and/or reorganization were observed that may be involved in the adaptation processes evoked by BFR muscle exercise.
    June 23, 2017   doi: 10.1113/JP273907   open full text
  • Presence of ethanol‐sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens.
    B. Förstera, B. Muñoz, M. K. Lobo, R. Chandra, D. M. Lovinger, L. G. Aguayo.
    The Journal of Physiology. June 23, 2017
    Key points The nucleus accumbens (nAc) is involved in addiction‐related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse. Abstract Alcohol abuse causes major social, economic and health‐related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)‐positive MSNs. Interestingly, the glycine‐evoked currents in dissociated DRD1‐positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR‐mediated tonic current by ethanol suggests that they modulate the excitability of DRD1‐positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction‐related and compulsive behaviours.
    June 23, 2017   doi: 10.1113/JP273767   open full text
  • Cerebral haemodynamic response to somatosensory stimulation in neonatal lambs.
    Shinji Nakamura, David W. Walker, Flora Y. Wong.
    The Journal of Physiology. June 23, 2017
    The neurovascular coupling response has been defined for the adult brain but in the neonate non‐invasive measurement of local cerebral perfusion using NIRS or BOLD fMRI have yielded variable and inconsistent results, including negative responses suggesting decreased perfusion and localised tissue tissue hypoxia. Also, the impact of permissive hypercapnia (PaCO2 > 50 mmHg) in the management of neonates on cerebrovascular responses to somatosensory input is unknown. Using NIRS to measure changes in cerebral oxy‐ and deoxy‐haemoglobin (ΔoxyHb, ΔdeoxyHb) in 8 anaesthetised newborn lambs, we studied the cerebral haemodynamic functional response to left median nerve stimulation using stimulus trains of 1.8, 4.8 and 7.8 s. Stimulation always produced a somatosensory evoked response, and superficial cortical perfusion measured by Laser Doppler Flowmetry predominantly increased following median nerve stimulation. However, with 1.8 s stimulation, oxyHb responses in the contralateral hemisphere were either positive (i.e. increased oxyHb), negative, or absent; and with 4.8 and 7.8 s stimulations, both positive and negative responses were observed. Hypercapnia increased baseline oxyHb and total Hb consistent with cerebral vasodilatation, and 6 of 7 lambs tested showed increased Δtotal Hb responses after the 7.8 s stimulation; among which 4 lambs also showed increased ΔoxyHb responses. In 2 of 3 lambs, the negative ΔoxyHb response became a positive pattern during hypercapnia. These results show that instead of functional hyperaemia, somatosensory stimulation can evoke negative (decreased oxyHb, total Hb) functional responses in the neonatal brain suggestive of decreased local perfusion and vasoconstriction, and that hypercapnia produces both baseline hyperperfusion and increased functional hyperaemia. This article is protected by copyright. All rights reserved
    June 23, 2017   doi: 10.1113/JP274244   open full text
  • Lack of adaptation during prolonged split‐belt locomotion in the intact and spinal cat.
    Victoria Kuczynski, Alessandro Telonio, Yann Thibaudier, Marie‐France Hurteau, Charline Dambreville, Etienne Desrochers, Adam Doelman, Tayler Ross, Alain Frigon.
    The Journal of Physiology. June 23, 2017
    In humans, gait adapts to prolonged walking on a split‐belt treadmill, where one leg steps faster than the other, by gradually restoring the symmetry of interlimb kinematic variables, such as double support periods and step lengths, and by reducing muscle activity (EMG, electromyography). The adaptation is also characterized by reversing the asymmetry of interlimb variables observed during the early split‐belt period when returning to tied‐belt locomotion, termed an after‐effect. To determine if cats adapt to prolonged split‐belt locomotion and to assess if spinal locomotor circuits participate in the adaptation, we measured interlimb variables and EMG in intact and spinal‐transected cats before, during and after 10 min of split‐belt locomotion. In spinal cats only the hindlimbs performed stepping with the forelimbs stationary. In intact and spinal cats, step lengths and double support periods were, on average, symmetric, during tied‐belt locomotion. They became asymmetric during split‐belt locomotion and remained asymmetric throughout the split‐belt period. Upon returning to tied‐belt locomotion, symmetry was immediately restored. In intact cats, the mean EMG amplitude of hindlimb extensors increased during split‐belt locomotion and remained increased throughout the split‐belt period, whereas in spinal cats, EMG amplitude did not change. Therefore, the results indicate that the locomotor pattern of cats does not adapt to prolonged split‐belt locomotion, suggesting an important physiological difference in the control of locomotion between cats and humans. We propose that restoring left‐right symmetry is not required to maintain balance during prolonged asymmetric locomotion in the cat, a quadruped, as opposed to human bipedal locomotion. This article is protected by copyright. All rights reserved
    June 23, 2017   doi: 10.1113/JP274518   open full text
  • Intraglomerular gap junctions enhance interglomerular synchrony in a sparsely‐connected olfactory bulb network.
    Frederic Pouille, Thomas S. McTavish, Lawrence E. Hunter, Diego Restrepo, Nathan E. Schoppa.
    The Journal of Physiology. June 22, 2017
    A dominant feature of the olfactory bulb response to odour is fast synchronized oscillations at beta (15–40 Hz) or gamma (40–90 Hz) frequencies, thought to be involved in integration of olfactory signals. Mechanistically, the bulb presents an interesting case study for understanding how beta/gamma oscillations arise. Fast oscillatory synchrony in the activity of output mitral cells (MCs) appears to result from interactions with GABAergic granule cells (GCs), yet the incidence of MC‐GC connections is very low, around 4%. Here, we combined computational and experimental approaches to examine how oscillatory synchrony can nevertheless arise, focusing mainly on activity between “non‐sister” MCs affiliated with different glomeruli (interglomerular synchrony). In a sparsely connected model of MCs and GCs, we found first that interglomerular synchrony was generally quite low, but could be increased by a factor of 4 by physiological‐levels of gap junctional coupling between sister MCs at the same glomerulus. This effect was due to enhanced mutually synchronizing interactions between MC and GC populations. The potent role of gap junctions was confirmed in patch‐clamp recordings in bulb slices from wild‐type and connexin 36‐knockout (KO) mice. KO reduced both beta/gamma local field potential oscillations as well as synchrony of inhibitory signals in pairs of non‐sister MCs. These effects were independent of potential KO‐actions on network excitation. Divergent synaptic connections did not contribute directly to the vast majority of synchronized signals. Thus, in a sparsely connected network, gap junctions between a small subset of cells can, through population‐effects, greatly amplify oscillatory synchrony amongst unconnected cells. This article is protected by copyright. All rights reserved
    June 22, 2017   doi: 10.1113/JP274408   open full text
  • Exercise‐induced quadriceps muscle fatigue in men and women: effects of arterial oxygen content and respiratory muscle work.
    Paolo B. Dominelli, Yannick Molgat‐Seon, Donald E. G. Griesdale, Carli M. Peters, Jean‐Sébastien Blouin, Mypinder Sekhon, Giulio S. Dominelli, William R. Henderson, Glen E. Foster, Lee M. Romer, Michael S. Koehle, A. William Sheel.
    The Journal of Physiology. June 19, 2017
    Key points High work of breathing and exercise‐induced arterial hypoxaemia (EIAH) can decrease O2 delivery and exacerbate exercise‐induced quadriceps fatigue in healthy men. Women have a higher work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles and develop EIAH. Despite a greater reduction in men's work of breathing, the attenuation of quadriceps fatigue was similar between the sexes. The degree of EIAH was similar between sexes, and regardless of sex, those who developed the greatest hypoxaemia during exercise demonstrated the most attenuation of quadriceps fatigue. Based on our previous finding that women have a greater relative oxygen cost of breathing, women appear to be especially susceptible to work of breathing‐related changes in quadriceps muscle fatigue. Abstract Reducing the work of breathing or eliminating exercise‐induced arterial hypoxaemia (EIAH) during exercise decreases the severity of quadriceps fatigue in men. Women have a greater work of breathing during exercise, dedicate a greater fraction of whole‐body V̇O2 towards their respiratory muscles, and demonstrate EIAH, suggesting women may be especially susceptible to quadriceps fatigue. Healthy subjects (8 male, 8 female) completed three constant load exercise tests over 4 days. During the first (control) test, subjects exercised at ∼85% of maximum while arterial blood gases and work of breathing were assessed. Subsequent constant load exercise tests were iso‐time and iso‐work rate, but with EIAH prevented by inspiring hyperoxic gas or work of breathing reduced via a proportional assist ventilator (PAV). Quadriceps fatigue was assessed by measuring force in response to femoral nerve stimulation. For both sexes, quadriceps force was equally reduced after the control trial (−27 ± 2% baseline) and was attenuated with hyperoxia and PAV (−18 ± 1 and −17 ± 2% baseline, P < 0.01, respectively), with no sex difference. EIAH was similar between the sexes, and regardless of sex, subjects with the lowest oxyhaemoglobin saturation during the control test had the greatest quadriceps fatigue attenuation with hyperoxia (r2 = 0.79, P < 0.0001). For the PAV trial, despite reducing the work of breathing to a greater degree in men (men: 60 ± 5, women: 75 ± 6% control, P < 0.05), the attenuation of quadriceps fatigue was similar between the sexes (36 ± 4 vs. 37 ± 7%). Owing to a greater relative V̇O2 of the respiratory muscles in women, less of a change in work of breathing is needed to reduce quadriceps fatigue.
    June 19, 2017   doi: 10.1113/JP274068   open full text
  • Functional expression of calcium‐permeable Canonical Transient Receptor Potential 4‐containing channels promotes migration of medulloblastoma cells.
    Wei‐Chun Wei, Wan‐Chen Huang, Yu‐Ping Lin, Esther B. E. Becker, Olaf Ansorge, Veit Flockerzi, Daniele Conti, Giovanna Cenacchi, Maike D. Glitsch.
    The Journal of Physiology. June 19, 2017
    Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilised in response to activation of cell surface receptors. OGR1 (Ovarian cancer G protein coupled Receptor 1, aka GPR68) is a proton‐sensing Gq‐coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We find that nine distinct human MB samples all express OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promotes expression of the proton‐potentiated member of the Canonical Transient Receptor Potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we find that all MB samples also express TRPC4. In DAOY cells, activation of TRPC4‐containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration was observed. Our results suggest that OGR1‐dependent increases in TRPC4 expression may favour formation of highly Ca2+‐permeable TRPC4‐containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB. This article is protected by copyright. All rights reserved
    June 19, 2017   doi: 10.1113/JP274659   open full text
  • Chemosensitive Phox2b‐expressing neurons are crucial for hypercapnic ventilatory response in the nucleus tractus solitarius.
    Congrui Fu, Jinyu Xue, Ri Wang, Jinting Chen, Lan Ma, Yixian Liu, Xuejiao Wang, Fang Guo, Yi Zhang, Xiangjian Zhang, Sheng Wang.
    The Journal of Physiology. June 16, 2017
    Key points Central hypercapnic hypoventilation is highly prevalent in children suffering from congenital central hypoventilation syndrome (CCHS). Mutations of the gene for paired‐like homeobox 2b (Phox2b) are aetiologically associated with CCHS and Phox2b is present in central components of respiratory chemoreflex, such as the nucleus tractus solitarius (NTS). Injection of the neurotoxin substance P‐saporin into NTS destroys Phox2b‐expressing neurons. Impaired hypercapnic ventilatory response caused by this neurotoxin is attributable to a loss of CO2‐sensitive Phox2b‐expressing NTS neurons. A subgroup of Phox2b‐expressing neurons exhibits intrinsic chemosensitivity. A background K+ channel‐like current is partially responsible for such chemosensitivity in Phox2b‐expressing neurons. The present study helps us better understand the mechanism of respiratory deficits in CCHS and potentially locates a brainstem site for development of precise clinical intervention. Abstract The nucleus tractus solitarius (NTS) neurons have been considered to function as central respiratory chemoreceptors. However, the common molecular marker defined for these neurons remains unknown. The present study investigated whether paired‐like homeobox 2b (Phox2b)‐expressing NTS neurons are recruited in hypercapnic ventilatory response (HCVR) and whether these neurons exhibit intrinsic chemosensitivity. HCVR was assessed using whole body plethysmography and neuronal chemosensitivity was examined by patch clamp recordings in brainstem slices or dissociated neurons from Phox2b‐EGFP transgenic mice. Injection of the neurotoxin substance P‐saporin (SSP‐SAP) into NTS destroyed Phox2b‐expressing neurons. Minute ventilation and tidal volume were both reduced by 13% during exposure to 8% CO2 in inspired air when ∼13% of the Phox2b‐expressing neurons were eliminated. However, a loss of ∼18% of these neurons was associated with considerable decreases in minute ventilation by ≥18% and in tidal volume by≥22% when challenged by ≥4% CO2. In both cases, breathing frequency was unaffected. Most CO2‐activated neurons were immunoreactive to Phox2b. In brainstem slices, ∼43% of Phox2b‐expressing neurons from Phox2b‐EGFP mice displayed a sustained or transient increase in firing rate during physiological acidification (pH 7.0 or 8% CO2). Such a response was also present in dissociated neurons in favour of an intrinsic property. In voltage clamp recordings, a background K+ channel‐like current was found in a subgroup of Phox2b‐expressing neurons. Thus, the respiratory deficits caused by injection of SSP‐SAP into the NTS are attributable to proportional lesions of CO2/H+‐sensitive Phox2b‐expressing neurons.
    June 16, 2017   doi: 10.1113/JP274437   open full text
  • An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.
    Karla Droguett, Mariana Rios, Daniela V. Carreño, Camilo Navarrete, Christian Fuentes, Manuel Villalón, Nelson P. Barrera.
    The Journal of Physiology. June 15, 2017
    Key points Extracellular ATP, in association with [Ca2+]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Abstract Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+]i‐dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml−1) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1–100 μm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.
    June 15, 2017   doi: 10.1113/JP273996   open full text
  • Systolic [Ca2+]i regulates diastolic levels in rat ventricular myocytes.
    R. Sankaranarayanan, K. Kistamas, D. J. Greensmith, L. A. Venetucci, D. A. Eisner.
    The Journal of Physiology. June 15, 2017
    [Ca2+]i must be low enough in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the RyR leaky (with caffeine or ryanodine) or by decreasing SERCA activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Diastolic and time‐averaged [Ca2+]i were decreased by decreasing the L‐type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres which decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+]i. This identifies a novel mechanism whereby changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i. This article is protected by copyright. All rights reserved
    June 15, 2017   doi: 10.1113/JP274366   open full text
  • In vivo analysis of synaptic activity in cerebellar nuclei neurons unravels the efficacy of excitatory inputs.
    Yasmin Yarden‐Rabinowitz, Yosef Yarom.
    The Journal of Physiology. June 15, 2017
    It is commonly agreed that the main function of the cerebellar system is to provide well‐timed signals used for the execution of motor commands or prediction of sensory inputs. This function is manifested as a temporal sequence of spiking that should be expressed in the cerebellar nuclei projection neurons (CN). Whether spiking activity is generated by excitation or release from inhibition is still a hotly debated issue. In an attempt to resolve this debate, we recorded intracellularly from CN neurons in anaesthetized mice and performed an analysis of synaptic activity that yielded a number of important observations. First, we demonstrate that CN neurons can be classified into four groups. Second, shape‐index plots of the excitatory events suggest that they are distributed all over the dendritic tree. Third, the rise time of excitatory events is linearly related to the amplitude, suggesting that all excitatory events contribute equally to the generation of action potentials. Fourth, we identified a temporal pattern of spontaneous excitatory events that represent climbing fibre inputs and confirm the results by direct stimulation and analysis on harmaline evoked activity. Finally, we demonstrate that the probability of excitatory inputs to generate an AP is 0.1 yet half of the APs are generated by excitatory events. Moreover, the probability of a presumably spontaneous climbing fibre input to generate an AP is higher, reaching mean population value of 0.15. In view of these results, the mode of synaptic integration at the level of the CN should be re‐considered. This article is protected by copyright. All rights reserved
    June 15, 2017   doi: 10.1113/JP274115   open full text
  • Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects.
    Lasse Gliemann, Nicolai Rytter, Mads Lindskrog, Martina H. Lundberg Slingsby, Thorbjörn Åkerström, Lykke Sylow, Erik A. Richter, Ylva Hellsten.
    The Journal of Physiology. June 15, 2017
    Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor‐ and vascular function in humans, twelve young healthy male subjects supplemented their diet with 3 × 75 g sucrose day−1 for 14 days in a randomized cross‐over design. Before and after the intervention period, the hyperemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2 %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM‐1), endothelial nitric oxide synthase, NADPH oxidase and the Rho family GTPase Rac1 protein expression in the muscle tissue as well as an increased basal phosphorylation status of Vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM‐1. The phosphorylation status was not acutely altered with passive leg movement. These findings indicate that regular intake of high levels of sucrose can impair vascular mechanotransduction, increase the oxidative stress potential and suggest that dietary excessive sugar intake may contribute to the development of vascular disease. This article is protected by copyright. All rights reserved
    June 15, 2017   doi: 10.1113/JP274623   open full text
  • Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating.
    A. Liavas, G. Lignani, S. Schorge.
    The Journal of Physiology. June 15, 2017
    Voltage‐gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well‐tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals. This article is protected by copyright. All rights reserved
    June 15, 2017   doi: 10.1113/JP274693   open full text
  • A multiscale computational modelling approach predicts mechanisms of female sex risk in the setting of arousal‐induced arrhythmias.
    Pei‐Chi Yang, Laura L. Perissinotti, Fernando López‐Redondo, Yibo Wang, Kevin R. DeMarco, Mao‐Tsuen Jeng, Igor Vorobyov, Robert D. Harvey, Junko Kurokawa, Sergei Y. Noskov, Colleen E. Clancy.
    The Journal of Physiology. June 14, 2017
    Key points This study represents a first step toward predicting mechanisms of sex‐based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo‐EM structure for the Eag‐family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. Abstract Female sex is a risk factor for inherited and acquired long‐QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long‐QT syndrome patients. We used a combined experimental and computational approach to predict ‘the perfect storm’ of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long‐QT. More specifically, we developed mathematical models of acquired and inherited long‐QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These ‘male’ and ‘female’ model myocytes and tissues then were used to predict how various sex‐based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long‐QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP‐related blockers in the intra‐cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal‐induced arrhythmias.
    June 14, 2017   doi: 10.1113/JP273142   open full text
  • Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with post‐traumatic stress disorder.
    Jeanie Park, Paul J. Marvar, Peizhou Liao, Melanie L. Kankam, Seth D. Norrholm, Ryan M. Downey, S. Ashley McCullough, Ngoc‐Anh Le, Barbara O. Rothbaum.
    The Journal of Physiology. June 14, 2017
    Key points Patients with post‐traumatic stress disorder (PTSD) are at a significantly higher risk of developing hypertension and cardiovascular disease. The mechanisms underlying this increased risk are not known. Studies have suggested that PTSD patients have an overactive sympathetic nervous system (SNS) that could contribute to cardiovascular risk; however, sympathetic function has not previously been rigorously evaluated in PTSD patients. Using direct measurements of sympathetic nerve activity and pharmacological manipulation of blood pressure, we show that veterans with PTSD have augmented SNS and haemodynamic reactivity during both combat‐related and non‐combat related mental stress, impaired sympathetic and cardiovagal baroreflex sensitivity, and increased inflammation. Identifying the mechanisms contributing to increased cardiovascular (CV) risk in PTSD will pave the way for developing interventions to improve sympathetic function and reduce CV risk in these patients. Abstract Post‐traumatic stress disorder (PTSD) is associated with increased cardiovascular (CV) risk. We tested the hypothesis that PTSD patients have augmented sympathetic nervous system (SNS) and haemodynamic reactivity during mental stress, as well as impaired arterial baroreflex sensitivity (BRS). Fourteen otherwise healthy Veterans with combat‐related PTSD were compared with 14 matched Controls without PTSD.  Muscle sympathetic nerve activity (MSNA), continuous blood pressure (BP) and electrocardiography were measured at baseline, as well as during two types of mental stress:  combat‐related mental stress using virtual reality combat exposure (VRCE) and non‐combat related stress using mental arithmetic (MA). A cold pressor test (CPT) was administered for comparison. BRS was tested using pharmacological manipulation of BP via the Modified Oxford technique at rest and during VRCE. Blood samples were analysed for inflammatory biomarkers. Baseline characteristics, MSNA and haemodynamics were similar between the groups. In PTSD vs. Controls, MSNA (+8.2 ± 1.0 vs. +1.2 ± 1.3 bursts min–1, P < 0.001) and heart rate responses (+3.2 ± 1.1 vs. −2.3 ± 1.0 beats min–1, P = 0.003) were significantly augmented during VRCE.  Similarly, in PTSD vs. Controls, MSNA (+21.0 ± 2.6 vs. +6.7 ± 1.5 bursts min–1, P < 0.001) and diastolic BP responses (+6.3 ± 1.0 vs. +3.5 ± 1.0 mmHg, P = 0.011) were significantly augmented during MA but not during CPT (P = not significant). In the PTSD group, sympathetic BRS (–1.2 ± 0.2 vs. –2.0 ± 0.3 burst incidence mmHg−1, P = 0.026) and cardiovagal BRS (9.5 ± 1.4 vs. 23.6 ± 4.3 ms mmHg−1, P = 0.008) were significantly blunted at rest. PTSD patients had significantly higher highly sensitive‐C‐reactive protein levels compared to Controls (2.1 ± 0.4 vs. 1.0 ± 0.3 mg L−1, P = 0.047). Augmented SNS and haemodynamic responses to mental stress, blunted BRS and inflammation may contribute to an increased CV risk in PTSD.
    June 14, 2017   doi: 10.1113/JP274269   open full text
  • Facilitation of mossy fibre‐driven spiking in the cerebellar nuclei by the synchrony of inhibition.
    Yeechan Wu, Indira M. Raman.
    The Journal of Physiology. June 11, 2017
    Key points Large premotor neurons of the cerebellar nuclei (CbN cells) integrate synaptic inhibition from Purkinje neurons and synaptic excitation from mossy fibres to generate cerebellar output. We find that mossy fibre inputs to CbN cells generate unitary AMPA receptor EPSCs of ∼1 nS that decay in ∼1 ms and mildly voltage‐dependent NMDA receptor EPSCs of ∼0.6 nS that decay in ∼7 ms. A few hundred mossy fibres active at a few tens of spikes s−1 must converge on CbN cells to generate physiological CbN spike rates (∼60 spikes s−1) during convergent inhibition from spontaneously active Purkinje cells. Dynamic clamp studies in cerebellar slices from weanling mice demonstrate that synaptic excitation from mossy fibres becomes more effective at increasing the rate of CbN cell spiking when the coherence (synchrony) of convergent inhibition is increased. Abstract Large projection neurons of the cerebellar nuclei (CbN cells), whose activity generates movement, are inhibited by Purkinje cells and excited by mossy fibres. The high convergence, firing rates and strength of Purkinje inputs predict powerful suppression of CbN cell spiking, raising the question of what activity patterns favour excitation over inhibition. Recording from CbN cells at near‐physiological temperatures in cerebellar slices from weanling mice, we measured the amplitude, kinetics, voltage dependence and short‐term plasticity of mossy fibre‐mediated EPSCs. Unitary EPSCs were small and brief (AMPA receptor, ∼1 nS, ∼1 ms; NMDA receptor, ∼0.6 nS, ∼7 ms) and depressed moderately. Using these experimentally measured parameters, we applied combinations of excitation and inhibition to CbN cells with dynamic clamp. Because Purkinje cells can fire coincident simple spikes during cerebellar behaviours, we varied the proportion (0–20 of 40) and precision (0–4 ms jitter) of synchrony of inhibitory inputs, along with the rates (0–100 spikes s−1) and number (0–800) of excitatory inputs. Even with inhibition constant, when inhibitory synchrony was higher, excitation increased CbN cell firing rates more effectively. Partial inhibitory synchrony also dictated CbN cell spike timing, even with physiological rates of excitation. These effects were present with ≥10 inhibitory inputs active within 2–4 ms of each other. Conversely, spiking was most effectively suppressed when inhibition was maximally asynchronous. Thus, the rate and relative timing of Purkinje‐mediated inhibition set the rate and timing of cerebellar output. The results suggest that increased coherence of Purkinje cell activity can facilitate mossy fibre‐driven spiking by CbN cells, in turn driving movements.
    June 11, 2017   doi: 10.1113/JP274321   open full text
  • Large‐conductance Ca2+‐activated K+ channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium.
    Nathan A. Zaidman, Angela Panoskaltsis‐Mortari, Scott M. O'Grady.
    The Journal of Physiology. June 11, 2017
    Key points Hydrocortisone (HC) is required for activation of large‐conductance Ca2+‐activated K+ current (BK) by purinergic receptor agonists. HC reduces insertion of the stress‐regulated exon (STREX) in the KCNMA1 gene, permitting protein kinase C (PKC)‐dependent channel activation. Overlapping and unique purinergic signalling regions exist at the apical border of differentiated surface cells. BK channels localize in the cilia of surface cells. Abstract In the present study we investigated the role of hydrocortisone (HC) on uridine‐5ʹ‐triphosphate (UTP)‐stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. The presence of a UTP‐stimulated, paxilline‐sensitive large‐conductance Ca2+‐activated K+ (BK) current was demonstrated in control epithelia but was not stimulated in epithelia differentiated in the absence of HC (HC0). Addition of the BK channel opener NS11021 directly activated channels in control epithelia; however, under HC0 conditions, activation only occurred when UTP was added after NS11021. The PKC inhibitors GF109203x and Gö6983 blocked BK activation by UTP in control epithelia, suggesting that PKC‐mediated phosphorylation plays a permissive role in purinoceptor‐stimulated BK activation. Moreover, HC0 epithelia expressed significantly more KCNMA1 containing the stress‐regulated exon (STREX), a splice‐variant of the α‐subunit that displays altered channel regulation by phosphorylation, compared to control epithelia. Furthermore, BK channels as well as purinergic receptors were shown to localize in unique and overlapping domains at the apical membrane of ciliated surface cells. These results establish a previously unrecognized role for glucocorticoids in regulation of BK channels in airway epithelial cells.
    June 11, 2017   doi: 10.1113/JP274200   open full text
  • Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice.
    Maximilian Kleinert, Benjamin L. Parker, Andreas M. Fritzen, Jonas R. Knudsen, Thomas E. Jensen, Rasmus Kjøbsted, Lykke Sylow, Markus Ruegg, David E. James, Erik A. Richter.
    The Journal of Physiology. June 11, 2017
    Key points Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle. Abstract Exercise increases glucose uptake into insulin‐resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin‐controlled glucose uptake, has been reported to interact with ras‐related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise‐induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running‐induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild‐type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5′ AMP‐activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N‐myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise‐dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion‐transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.
    June 11, 2017   doi: 10.1113/JP274203   open full text
  • Evolved changes in the intracellular distribution and physiology of muscle mitochondria in high‐altitude native deer mice.
    Sajeni Mahalingam, Grant B. McClelland, Graham R. Scott.
    The Journal of Physiology. June 07, 2017
    Key points Mitochondrial function changes over time at high altitudes, but the potential benefits of these changes for hypoxia resistance remains unclear. We used high‐altitude‐adapted populations of deer mice, which exhibit enhanced aerobic performance in hypoxia, to examine whether changes in mitochondrial physiology or intracellular distribution in the muscle contribute to hypoxia resistance. Permeabilized muscle fibres from the gastrocnemius muscle had higher respiratory capacities in high‐altitude mice than in low‐altitude mice. Highlanders also had higher mitochondrial volume densities, due entirely to an enriched abundance of subsarcolemmal mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. There were several effects of hypoxia acclimation on mitochondrial function, some of which were population specific, but they differed from the evolved changes in high‐altitude natives, which probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes. Abstract High‐altitude natives that have evolved to live in hypoxic environments provide a compelling system to understand how animals can overcome impairments in oxygen availability. We examined whether these include changes in mitochondrial physiology or intracellular distribution that contribute to hypoxia resistance in high‐altitude deer mice (Peromyscus maniculatus). Mice from populations native to high and low altitudes were born and raised in captivity, and as adults were acclimated to normoxia or hypobaric hypoxia (equivalent to 4300 m elevation). We found that highlanders had higher respiratory capacities in the gastrocnemius (but not soleus) muscle than lowlanders (assessed using permeabilized fibres with single or multiple inputs to the electron transport system), due in large part to higher mitochondrial volume densities in the gastrocnemius. The latter was attributed to an increased abundance of subsarcolemmal (but not intermyofibrillar) mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. Hypoxia acclimation had no significant effect on these population differences, but it did increase mitochondrial cristae surface densities of mitochondria in both populations. Hypoxia acclimation also altered the physiology of isolated mitochondria by affecting respiratory capacities and cytochrome c oxidase activities in population‐specific manners. Chronic hypoxia decreased the release of reactive oxygen species by isolated mitochondria in both populations. There were subtle differences in O2 kinetics between populations, with highlanders exhibiting increased mitochondrial O2 affinity or catalytic efficiency in some conditions. Our results suggest that evolved changes in mitochondrial physiology in high‐altitude natives are distinct from the effects of hypoxia acclimation, and probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes.
    June 07, 2017   doi: 10.1113/JP274130   open full text
  • Structure and function of human muscle fibres and muscle proteome in physically active older men.
    Lorenza Brocca, Jamie S. McPhee, Emanuela Longa, Monica Canepari, Olivier Seynnes, Giuseppe Vito, Maria Antonietta Pellegrino, Marco Narici, Roberto Bottinelli.
    The Journal of Physiology. June 05, 2017
    Key points Loss of muscle mass and strength in the growing population of elderly people is a major health concern for modern societies. This condition, termed sarcopenia, is a major cause of falls and of the subsequent increase in morbidity and mortality. Despite numerous studies on the impact of ageing on individual muscle fibres, the contribution of single muscle fibre adaptations to ageing‐induced atrophy and functional impairment is still unsettled. The level of physical function and disuse is often associated with ageing. We studied relatively healthy older adults in order to understand the effects of ageing per se without the confounding impact of impaired physical function. We found that in healthy ageing, structural and functional alterations of muscle fibres occur. Protein post‐translational modifications, oxidation and phosphorylation contribute to such alterations more than loss of myosin and other muscle protein content. Abstract Contradictory results have been reported on the impact of ageing on structure and functions of skeletal muscle fibres, likely to be due to a complex interplay between ageing and other phenomena such as disuse and diseases. Here we recruited healthy, physically and socially active young (YO) and elderly (EL) men in order to study ageing per se without the confounding effects of impaired physical function. In vivo analyses of quadriceps and in vitro analyses of vastus lateralis muscle biopsies were performed. In EL subjects, our results show that (i) quadriceps volume, maximum voluntary contraction isometric torque and patellar tendon force were significantly lower; (ii) muscle fibres went through significant atrophy and impairment of specific force (isometric force/cross‐sectional area) and unloaded shortening velocity; (iii) myosin/actin ratio and myosin content in individual muscle fibres were not altered; (iv) the muscle proteome went through quantitative adaptations, namely an up‐regulation of the content of several groups of proteins among which were myofibrillar proteins and antioxidant defence systems; (v) the muscle proteome went through qualitative adaptations, namely phosphorylation of several proteins, including myosin light chain‐2 slow and troponin T and carbonylation of myosin heavy chains. The present results indicate that impairment of individual muscle fibre structure and function is a major feature of ageing per se and that qualitative adaptations of muscle proteome are likely to be more involved than quantitative adaptations in determining such a phenomenon.
    June 05, 2017   doi: 10.1113/JP274148   open full text
  • Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol‐induced proliferation of ovine uterine artery endothelial cells.
    Rosalina Villalon Landeros, Sheikh O. Jobe, Gabrielle Aranda‐Pino, Gladys E. Lopez, Jing Zheng, Ronald R. Magness.
    The Journal of Physiology. June 05, 2017
    Key points The catechol metabolites of 17β‐oestradiol (E2β), 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate proliferation of pregnancy‐derived ovine uterine artery endothelial cells (P‐UAECs) through β‐adrenoceptors (β‐ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2‐OHE2‐ and 4‐OHE2‐induced P‐UAEC proliferation, as well as proliferation induced by the parent hormone E2β and other β‐AR signalling hormones (i.e. catecholamines). Conversely, although 2‐OHE2 and 4‐OHE2 rapidly activate phosphatidylinositol 3‐kinase (PI3K), its activation is not involved in catecholoestradiol‐induced P‐UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol‐induced P‐UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2β‐ and catecholamine‐induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. Abstract Previously we demonstrated that the biologically active metabolites of 17β‐oestradiol, 2‐hydroxyoestradiol (2‐OHE2) and 4‐hydroxyoestradiol (4‐OHE2), stimulate pregnancy‐specific proliferation of uterine artery endothelial cells derived from pregnant (P‐UAECs), but not non‐pregnant ewes. However, unlike 17β‐oestradiol, which induces proliferation via oestrogen receptor‐β (ER‐β), the catecholoestradiols mediate P‐UAEC proliferation via β‐adrenoceptors (β‐AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P‐UAECs. P‐UAECs were treated with 2‐OHE2 and 4‐OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3‐kinase (PI3K)–AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist‐induced P‐UAEC proliferation. 2‐OHE2 and 4‐OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol‐induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β‐oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P‐UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol‐, 17β‐oestradiol‐ and catecholamine‐induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.
    June 05, 2017   doi: 10.1113/JP274119   open full text
  • The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.
    Paulo W. Pires, Eun‐A. Ko, Harry A.T. Pritchard, Michael Rudokas, Evan Yamasaki, Scott Earley.
    The Journal of Physiology. June 01, 2017
    Key points The angiotensin II receptor type 1b (AT1Rb) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane‐stretch induced stimulation of AT1Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT1Ra or AT1Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT1Rb mRNA is ∼30‐fold higher than AT1Ra in whole cerebral arteries and ∼45‐fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT1Rb for pressure‐induced vasoconstriction. Abstract Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT1R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1R – AT1Ra and AT1Rb – and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1Ra knockout animals compared with controls. In patch‐clamp experiments using native cerebral arterial myocytes, membrane stretch‐induced cation currents were blocked by the TRPM4 inhibitor 9‐phenanthrol in both groups. Further, the AT1R blocker losartan (1 μm) diminished myogenic tone and blocked stretch‐induced cation currents in cerebral arteries from both groups. Activation of AT1R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1Rb mRNA was ∼30‐fold greater than AT1Ra in cerebral arteries, and knockdown of AT1Rb selectively diminished myogenic constriction. We conclude that AT1Rb, acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.
    June 01, 2017   doi: 10.1113/JP274310   open full text
  • Does the intercept of the heat–stress relation provide an accurate estimate of cardiac activation heat?
    Toan Pham, Kenneth Tran, Kimberley M Mellor, Anthony Hickey, Amelia Power, Marie‐Louise Ward, Andrew Taberner, June‐Chiew Han, Denis Loiselle.
    The Journal of Physiology. June 01, 2017
    Key points The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15–20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. Abstract Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+‐ATPase and Ca2+ translocation by the Na+/Ca2+ exchanger coupled to the Na+,K+‐ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l−1), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat–stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat–force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long‐standing disputes in the literature.
    June 01, 2017   doi: 10.1113/JP274174   open full text
  • Genotype‐specific pathogenic effects in human dilated cardiomyopathy.
    Ilse A. E. Bollen, Maike Schuldt, Magdalena Harakalova, Aryan Vink, Folkert W. Asselbergs, Jose R. Pinto, Martina Krüger, Diederik W. D. Kuster, Jolanda Velden.
    The Journal of Physiology. June 01, 2017
    Key points Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+‐sensitivity and reduced length‐dependent activation. TNNT2p.K217del caused increased passive tension. A mutation in the gene encoding Lamin A/C (LMNAp.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy. Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. Abstract Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.
    June 01, 2017   doi: 10.1113/JP274145   open full text
  • Light adaptation and the evolution of vertebrate photoreceptors.
    Ala Morshedian, Gordon L. Fain.
    The Journal of Physiology. June 01, 2017
    Key Points Lamprey are cyclostomes, a group of vertebrates that diverged from lines leading to jawed vertebrates (including mammals) in the late Cambrian, 500 million years ago. It may therefore be possible to infer properties of photoreceptors in early vertebrate progenitors by comparing lamprey to other vertebrates. We show that lamprey rods and cones respond to light much like rods and cones in amphibians and mammals. They operate over a similar range of light intensities and adapt to backgrounds and bleaches nearly identically. These correspondences are pervasive and detailed; they argue for the presence of rods and cones very early in the evolution of vertebrates with properties much like those of rods and cones in existing vertebrate species. Abstract The earliest vertebrates were agnathans – fish‐like organisms without jaws, which first appeared near the end of the Cambrian radiation. One group of agnathans became cyclostomes, which include lamprey and hagfish. Other agnathans gave rise to jawed vertebrates or gnathostomes, the group including all other existing vertebrate species. Because cyclostomes diverged from other vertebrates 500 million years ago, it may be possible to infer some of the properties of the retina of early vertebrate progenitors by comparing lamprey to other vertebrates. We have previously shown that rods and cones in lamprey respond to light much like photoreceptors in other vertebrates and have a similar sensitivity. We now show that these affinities are even closer. Both rods and cones adapt to background light and to bleaches in a manner almost identical to other vertebrate photoreceptors. The operating range in darkness is nearly the same in lamprey and in amphibian or mammalian rods and cones; moreover background light shifts response–intensity curves downward and to the right over a similar range of ambient intensities. Rods show increment saturation at about the same intensity as mammalian rods, and cones never saturate. Bleaches decrease sensitivity in part by loss of quantum catch and in part by opsin activation of transduction. These correspondences are so numerous and pervasive that they are unlikely to result from convergent evolution but argue instead that early vertebrate progenitors of both cyclostomes and mammals had photoreceptors much like our own.
    June 01, 2017   doi: 10.1113/JP274211   open full text
  • Regionalization of the stretch reflex in the human vastus medialis.
    Alessio Gallina, Jean‐Sébastien Blouin, Tanya D. Ivanova, S. Jayne Garland.
    The Journal of Physiology. June 01, 2017
    Key points Regionalization of the stretch reflex, i.e. the notion that the activation of 1a afferents from a muscle region influences only the activation of motor units in the same region, has been demonstrated previously in animals but not in humans. Mechanical stretches applied to regions of vastus medialis as close as 10 mm apart resulted in recruitment of motor units localized topographically with respect to the location of the mechanical stretch. Stretch reflexes are regionalized in the human vastus medialis. The human spinal cord has the neuromuscular circuitry to preferentially activate motoneurones innervating muscle fibres located in different regions of the vastus medialis. Abstract The localization of motor unit territories provides an anatomical basis to suggest that the CNS may have more independence in motor unit recruitment and control strategies than what was previously thought. In this study, we investigated whether the human spinal cord has the neuromuscular circuitry to independently activate motor units located in different regions of the vastus medialis. Mechanical taps were applied to multiple locations in the vastus medialis (VM) in nine healthy individuals. Regional responses within the muscle were observed using a grid of 5 × 13 surface EMG electrodes. The EMG amplitude was quantified for each channel, and a cluster of channels showing the largest activation was identified. The spatial location of the EMG response was quantified as the position of the channels in the cluster. In a subset of three participants, intramuscular recordings were performed simultaneously with the surface EMG recordings. Mechanical taps resulted in localized, discrete responses for each participant. The spatial location of the elicited responses was dependent on the location of the tap (P < 0.001). Recordings with intramuscular electrodes confirmed the regional activation of the VM for different tap locations. Selective stimulation of 1a afferents localized in a region of the VM results in reflex recruitment of motor units in the same region. These findings suggest that the human spinal cord has the neuromuscular circuitry to modulate spatially the motoneuronal output to vastus medialis regions, which is a neuroanatomical prerequisite for regional activation.
    June 01, 2017   doi: 10.1113/JP274458   open full text
  • Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.
    Sergej Pirkmajer, Henriette Kirchner, Leonidas S. Lundell, Pavel V. Zelenin, Juleen R. Zierath, Kira S. Makarova, Yuri I. Wolf, Alexander V. Chibalin.
    The Journal of Physiology. May 29, 2017
    Key points Small transmembrane proteins such as FXYDs, which interact with Na+,K+‐ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+‐ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny‐based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Abstract Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1–12), which regulate Na+,K+‐ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence‐based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.
    May 29, 2017   doi: 10.1113/JP274254   open full text
  • Long‐term plasticity of corticostriatal synapses is modulated by pathway‐specific co‐release of opioids through κ‐opioid receptors.
    Sarah L. Hawes, Armando G. Salinas, David M. Lovinger, Kim T. Blackwell.
    The Journal of Physiology. May 26, 2017
    Key points Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal‐directed actions, and can also modify long‐term plasticity of corticostriatal synapses. Striatal projection neurons of the direct pathway co‐release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ‐opioid receptors. Theta‐burst stimulation of corticostriatal fibres produces long‐term potentiation (LTP) in striatal projection neurons when measured using whole‐cell patch recording. Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release. Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse. Abstract Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction‐related striatal plasticity. Though the role of opioid receptors in long‐term depression in striatum has been characterized, their effect on long‐term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor‐containing; D1R‐) spiny projection neurons (SPNs) co‐release the opioid neuropeptide dynorphin, which acts at presynaptic κ‐opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co‐released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R‐SPNs in brain slice while using whole‐cell patch recording to measure changes in the synaptic response of SPNs following theta‐burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R‐SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R‐SPNs reduces stimulation‐evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R‐SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R‐SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation.
    May 26, 2017   doi: 10.1113/JP274190   open full text
  • Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.
    Mitsue Takeya, Hikaru Hashitani, Tokumasa Hayashi, Ryuhei Higashi, Kei‐ichiro Nakamura, Makoto Takano.
    The Journal of Physiology. May 25, 2017
    Key points The mucosa may have neuron‐like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco‐endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco‐endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. Abstract The role of the mucosa in generating the spontaneous activity of guinea‐pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi‐fluorescence Ca2+ imaging, respectively. Mucosa‐intact but not mucosa‐denuded SV preparations generated TTX‐ (1 μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μm). Consistently, SMCs developed mucosa‐dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μm), a sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor. DIDS (300 μm) and niflumic acid (100 μm), blockers for Ca2+‐activated Cl− channels (CACCs), or low Cl− solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L‐type voltage‐dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco‐endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.
    May 25, 2017   doi: 10.1113/JP273872   open full text
  • α‐Linolenic acid and exercise training independently, and additively, decrease blood pressure and prevent diastolic dysfunction in obese Zucker rats.
    Pierre‐Andre Barbeau, Tanya M. Holloway, Jamie Whitfield, Brittany L. Baechler, Joe Quadrilatero, Luc J. C. Loon, Adrian Chabowski, Graham P. Holloway.
    The Journal of Physiology. May 24, 2017
    Key points α‐linolenic acid (ALA) and exercise training both attenuate hyperlipidaemia‐related cardiovascular derangements, however, there is a paucity of information pertaining to their mechanisms of action when combined. We investigated both the independent and combined effects of exercise training and ALA consumption in obese Zucker rats, aiming to determine the potential for additive improvements in cardiovascular function. ALA and exercise training independently improved cardiac output, end‐diastolic volume, left ventricular fibrosis and mean blood pressure following a 4 week intervention. Combining ALA and endurance exercise yielded greater improvements in these parameters, independent of changes in markers of oxidative stress or endogenous anti‐oxidants. We postulate that divergent mechanisms of action may explain these changes: ALA increases peripheral vasodilation, and exercise training stimulates angiogenesis. Abstract Although α‐linolenic acid (ALA) and endurance exercise training independently attenuate hyperlipidaemia‐related cardiovascular derangements, there is a paucity of information pertaining to their mechanisms of action and efficacy when combined as a preventative therapeutic approach. Therefore, we used obese Zucker rats to investigate the independent and combined effects of these interventions on cardiovascular disease. Specifically, animals were randomly assigned to one of the following groups: control diet‐sedentary, ALA supplemented‐sedentary, control diet‐exercise trained or ALA supplemented‐exercise trained. Following a 4 week intervention, although the independent and combined effects of ALA and exercise reduced (P < 0.05) the serum free/esterified cholesterol ratio, only the ALA supplemented‐exercise trained animals displayed a reduction in the content of both serum free and esterified cholesterol. Moreover, although ALA and endurance training individually increased cardiac output, stroke volume and end‐diastolic volume, as well as reduced left ventricle fibrosis, mean blood pressure and total peripheral resistance, these responses were all greater following the combined intervention (ALA supplemented‐exercise trained). These effects occurred independent of changes in oxidative phosphorylation proteins, markers of oxidative stress or endogenous anti‐oxidant capacity. We propose that the beneficial effects of a combined intervention occur as a result of divergent mechanisms of action elicited by ALA and endurance exercise because only exercise training increased the capillary content in the left ventricle and skeletal muscle, and tended to decrease protein carbonylation in the left ventricle (P = 0.06). Taken together, our data indicate that combining ALA and endurance exercise provides additional improvements in cardiovascular disease risk reduction compared to singular interventions in the obese Zucker rat.
    May 24, 2017   doi: 10.1113/JP274036   open full text
  • Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC‐A.
    Sam El‐Ajouz, Elisa Venturi, Katja Witschas, Matthew Beech, Abigail D. Wilson, Chris Lindsay, David Eberhardt, Fiona O'Brien, Tsunaki Iida, Miyuki Nishi, Hiroshi Takeshima, Rebecca Sitsapesan.
    The Journal of Physiology. May 23, 2017
    Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1). We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+. In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice. Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. Abstract The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.
    May 23, 2017   doi: 10.1113/JP273550   open full text
  • Increased transient Na+ conductance and action potential output in layer 2/3 prefrontal cortex neurons of the fmr1−/y mouse.
    Brandy N. Routh, Rahul K. Rathour, Michael E. Baumgardner, Brian E. Kalmbach, Daniel Johnston, Darrin H. Brager.
    The Journal of Physiology. May 23, 2017
    Key points Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1−/y mice. In fmr1−/y L2/3 neurons, action potentials are taller, faster and narrower. Outside‐out patch clamp recordings revealed that the maximum Na+ conductance density is higher in fmr1−/y L2/3 neurons. Measurements of three biophysically distinct K+ currents revealed a depolarizing shift in the activation of a rapidly inactivating (A‐type) K+ conductance. Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype. Abstract Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1−/y mouse is significantly altered due to changes in several voltage‐gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole‐cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1−/y mouse fired more action potentials for a given stimulus compared with wild‐type neurons. In addition, action potentials in fmr1−/y neurons were significantly larger, faster and narrower. Voltage clamp of outside‐out patches from L2/3 neurons revealed that the transient Na+ current was significantly larger in fmr1−/y neurons. Furthermore, the activation curve of somatic A‐type K+ current was depolarized. Realistic conductance‐based simulations revealed that these biophysical changes in Na+ and K+ channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1−/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome.
    May 23, 2017   doi: 10.1113/JP274258   open full text
  • Exercise training reverses age‐induced diastolic dysfunction and restores coronary microvascular function.
    Kazuki Hotta, Bei Chen, Bradley J. Behnke, Payal Ghosh, John N. Stabley, Jeremy A. Bramy, Jaime L. Sepulveda, Michael D. Delp, Judy M. Muller‐Delp.
    The Journal of Physiology. May 23, 2017
    Key points In a rat model of ageing that is free of atherosclerosis or hypertension, E/A, a diagnostic measure of diastolic filling, decreases, and isovolumic relaxation time increases, indicating that both active and passive ventricular relaxation are impaired with advancing age. Resting coronary blood flow and coronary functional hyperaemia are reduced with age, and endothelium‐dependent vasodilatation declines with age in coronary resistance arterioles. Exercise training reverses age‐induced declines in diastolic and coronary microvascular function. Thus, microvascular dysfunction and inadequate coronary perfusion are likely mechanisms of diastolic dysfunction in aged rats. Exercise training, initiated at an advanced age, reverses age‐related diastolic and microvascular dysfunction; these data suggest that late‐life exercise training can be implemented to improve coronary perfusion and diastolic function in the elderly. Abstract The risk for diastolic dysfunction increases with advancing age. Regular exercise training ameliorates age‐related diastolic dysfunction; however, the underlying mechanisms have not been identified. We investigated whether (1) microvascular dysfunction contributes to the development of age‐related diastolic dysfunction, and (2) initiation of late‐life exercise training reverses age‐related diastolic and microvascular dysfunction. Young and old rats underwent 10 weeks of exercise training or remained as sedentary, cage‐controls. Isovolumic relaxation time (IVRT), early diastolic filling (E/A), myocardial performance index (MPI) and aortic stiffness (pulse wave velocity; PWV) were evaluated before and after exercise training or cage confinement. Coronary blood flow and vasodilatory responses of coronary arterioles were evaluated in all groups at the end of training. In aged sedentary rats, compared to young sedentary rats, a 42% increase in IVRT, a 64% decrease in E/A, and increased aortic stiffness (PWV: 6.36 ± 0.47 vs.4.89 ± 0.41, OSED vs. YSED, P < 0.05) was accompanied by impaired coronary blood flow at rest and during exercise. Endothelium‐dependent vasodilatation was impaired in coronary arterioles from aged rats (maximal relaxation to bradykinin: 56.4 ± 5.1% vs. 75.3 ± 5.2%, OSED vs. YSED, P < 0.05). After exercise training, IVRT, a measure of active ventricular relaxation, did not differ between old and young rats. In old rats, exercise training reversed the reduction in E/A, reduced aortic stiffness, and eliminated impairment of coronary blood flow responses and endothelium‐dependent vasodilatation. Thus, age‐related diastolic and microvascular dysfunction are reversed by late‐life exercise training. The restorative effect of exercise training on coronary microvascular function may result from improved endothelial function.
    May 23, 2017   doi: 10.1113/JP274172   open full text
  • Beneficial effects of leptin treatment in a setting of cardiac dysfunction induced by transverse aortic constriction in mouse.
    Nieves Gómez‐Hurtado, Alejandro Domínguez‐Rodríguez, Philippe Mateo, María Fernández‐Velasco, Almudena Val‐Blasco, Rafael Aizpún, Jessica Sabourin, Ana María Gómez, Jean‐Pierre Benitah, Carmen Delgado.
    The Journal of Physiology. May 23, 2017
    Key points Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non‐hypertensive dose of leptin on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters. Abstract Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg–1 day–1) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin‐treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+/calmodulin‐dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.
    May 23, 2017   doi: 10.1113/JP274030   open full text
  • An increased extrasynaptic NMDA tone inhibits A‐type K+ current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.
    Meng Zhang, Vinicia C. Biancardi, Javier E. Stern.
    The Journal of Physiology. May 23, 2017
    Key points A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR–IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA, contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats. Abstract We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A‐type K+ current (IA) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR–IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch‐clamp electrophysiology and real‐time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1‐NR2A–D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.
    May 23, 2017   doi: 10.1113/JP274327   open full text
  • Hippocampal electrical stimulation disrupts associative learning when targeted at dentate spikes.
    Miriam S. Nokia, Irina Gureviciene, Tomi Waselius, Heikki Tanila, Markku Penttonen.
    The Journal of Physiology. May 23, 2017
    Key points Dentate spikes are fast fluctuations of hilar local‐field potentials that take place during rest and are thought to reflect input arriving from the entorhinal cortex to the hippocampus. During dentate spikes, neuronal firing in hippocampal input (dentate gyrus) and output (CA1/CA3) regions is uncoupled. To date, the behavioural significance of dentate spikes is unknown. Here, we provide evidence that disrupting the dentate spike‐related uncoupling of the dentate gyrus and the CA1/CA3 subregions for 1 h after training retards associative learning. We suggest dentate spikes play a significant role in memory consolidation. Abstract Hippocampal electrophysiological oscillations, namely theta and ripples, have been implicated in encoding and consolidation of new memories, respectively. According to existing literature, hippocampal dentate spikes are prominent, short‐duration (<30 ms), large‐amplitude (∼2–4 mV) fluctuations in hilar local‐field potentials that take place during awake immobility and sleep. Interestingly, previous studies indicate that during dentate spikes dentate gyrus granule cells increase their firing while firing of CA1 pyramidal cells are suppressed, thus resulting in momentary uncoupling of the two hippocampal subregions. To date, the behavioural significance of dentate spikes is unknown. Here, to study the possible role of dentate spikes in learning, we trained adult male Sprague–Dawley rats in trace eyeblink classical conditioning. For 1 h immediately following each conditioning session, one group of animals received hippocampal stimulation via the ventral hippocampal commissure (vHC) contingent on dentate spikes to disrupt the uncoupling between the dentate gyrus and the CA1 subregions. A yoked control group was stimulated during immobility, irrespective of brain state, and another control group was not stimulated at all. As a result, learning was impaired only in the group where vHC stimulation was administered contingent on dentate spikes. Our results suggest dentate spikes and/or the associated uncoupling of the dentate gyrus and the CA1 play a significant role in memory consolidation. Dentate spikes could possibly reflect reactivation and refinement of a memory trace within the dentate gyrus triggered by input from the entorhinal cortex.
    May 23, 2017   doi: 10.1113/JP274023   open full text
  • Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.
    Abigail L. Mackey, Mélanie Magnan, Bénédicte Chazaud, Michael Kjaer.
    The Journal of Physiology. May 23, 2017
    Key points Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross‐talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell‐culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell–cell cross‐talk during physiological and pathological muscle remodelling. Abstract Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross‐sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co‐cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross‐sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.
    May 23, 2017   doi: 10.1113/JP273997   open full text
  • Mechanical tuning and amplification within the apex of the guinea pig cochlea.
    Alberto Recio‐Spinoso, John S. Oghalai.
    The Journal of Physiology. May 21, 2017
    Key points A popular conception of mammalian cochlear physiology is that tuned mechanical vibration of the basilar membrane defines the frequency response of the innervating auditory nerve fibres However, the data supporting these concepts come from vibratory measurements at cochlear locations tuned to high frequencies (>7 kHz). Here, we measured the travelling wave in regions of the guinea pig cochlea that respond to low frequencies (<2 kHz) and found that mechanical tuning was broad and did not match auditory nerve tuning characteristics. Non‐linear amplification of the travelling wave functioned over a broad frequency range and did not substantially sharpen frequency tuning. Thus, the neural encoding of low‐frequency sounds, which includes most of the information conveyed by human speech, is not principally determined by basilar membrane mechanics. Abstract The popular notion of mammalian cochlear function is that auditory nerves are tuned to respond best to different sound frequencies because basilar membrane vibration is mechanically tuned to different frequencies along its length. However, this concept has only been demonstrated in regions of the cochlea tuned to frequencies >7 kHz, not in regions sensitive to lower frequencies where human speech is encoded. Here, we overcame historical technical limitations and non‐invasively measured sound‐induced vibrations at four locations distributed over the apical two turns of the guinea pig cochlea. In turn 3, the responses demonstrated low‐pass filter characteristics. In turn 2, the responses were low‐pass‐like, in that they occasionally did have a slight peak near the corner frequency. The corner frequencies of the responses were tonotopically tuned and ranged from 384 to 668 Hz. Non‐linear gain, or amplification of the vibrations in response to low‐intensity stimuli, was found both below and above the corner frequencies. Post mortem, cochlear gain disappeared. The non‐linear gain was typically 10–30 dB and was broad‐band rather than sharply tuned. However, the gain did reach nearly 50 dB in turn 2 for higher stimulus frequencies, nearly the amount of gain found in basal cochlear regions. Thus, our data prove that mechanical responses do not match neural responses and that cochlear amplification does not appreciably sharpen frequency tuning for cochlear regions that respond to frequencies <2 kHz. These data indicate that the non‐linear processing of sound performed by the guinea pig cochlea varies substantially between the cochlear apex and base.
    May 21, 2017   doi: 10.1113/JP273881   open full text
  • Compensatory and decompensatory alterations in cardiomyocyte Ca2+ dynamics in hearts with diastolic dysfunction following aortic banding.
    Sara Gattoni, Åsmund Treu Røe, Jan Magnus Aronsen, Ivar Sjaastad, William E. Louch, Nicolas P. Smith, Steven A. Niederer.
    The Journal of Physiology. May 21, 2017
    Key points At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics. Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality. Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic‐banded rats exhibiting diastolic dysfunction. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively. Results show that the ability to dynamically change systolic Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction. Abstract Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic‐banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel (LCC) and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) as the principal regulators of systolic and diastolic Ca2+, respectively. In the aortic banding model, the sensitivity of systolic Ca2+ to LCC density and diastolic Ca2+ to SERCA density decreased by 16‐fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca2+ is moderately increased.
    May 21, 2017   doi: 10.1113/JP273879   open full text
  • Maternal nutrient restriction during pregnancy and lactation leads to impaired right ventricular function in young adult baboons.
    Anderson H. Kuo, Cun Li, Hillary F. Huber, Matthias Schwab, Peter W. Nathanielsz, Geoffrey D. Clarke.
    The Journal of Physiology. May 18, 2017
    Key points Maternal nutrient restriction induces intrauterine growth restriction (IUGR) and leads to heightened cardiovascular risks later in life. We report right ventricular (RV) filling and ejection abnormalities in IUGR young adult baboons using cardiac magnetic resonance imaging. Both functional and morphological indicators of poor RV function were seen, many of which were similar to effects of ageing, but also with a few key differences. We observed more pronounced RV changes compared to our previous report of the left ventricle, suggesting there is likely to be a component of isolated RV abnormality in addition to expected haemodynamic sequelae from left ventricular dysfunction. In particular, our findings raise the suspicion of pulmonary hypertension after IUGR. This study establishes that IUGR also leads to impairment of the right ventricle in addition to the left ventricle classically studied. Abstract Maternal nutrient restriction induces intrauterine growth restriction (IUGR), increasing later life chronic disease including cardiovascular dysfunction. Our left ventricular (LV) CMRI studies in IUGR baboons (8 M, 8 F, 5.7 years – human equivalent approximately 25 years), control offspring (8 M, 8 F, 5.6 years), and normal elderly (OLD) baboons (6 M, 6 F, mean 15.9 years) revealed long‐term LV abnormalities in IUGR offspring. Although it is known that right ventricular (RV) function is dependent on LV health, the IUGR right ventricle remains poorly studied. We examined the right ventricle with cardiac magnetic resonance imaging in the same cohorts. We observed decreased ejection fraction (49 ± 2 vs. 33 ± 3%, P < 0.001), cardiac index (2.73 ± 0.27 vs. 1.89 ± 0.20 l min−1 m−2, P < 0.05), early filling rate/body surface area (BSA) (109.2 ± 7.8 vs. 44.6 ± 7.3 ml s−1 m−2, P < 0.001), wall thickening (61 ± 3 vs. 44 ± 5%, P < 0.05), and longitudinal shortening (26 ± 3 vs. 15 ± 2%, P < 0.01) in IUGR animals with increased chamber volumes. Many, but not all, of these changes share similarities to normal older animals. Our findings suggest IUGR‐induced pulmonary hypertension should be further investigated and that atrial volume, pulmonic outflow and interventricular septal motion may provide valuable insights into IUGR cardiovascular physiology. Overall, our findings reaffirm that gestational and neonatal challenges can result in long‐term programming of poor offspring cardiovascular health. To our knowledge, this is the first study reporting IUGR‐induced programmed adult RV dysfunction in an experimental primate model.
    May 18, 2017   doi: 10.1113/JP273928   open full text
  • Phosphatidylinositol 4,5‐bisphosphate (PIP2) modulates afterhyperpolarizations in oxytocin neurons of the supraoptic nucleus.
    Matthew K. Kirchner, Robert C. Foehring, Lie Wang, Giri Kumar Chandaka, Joseph C. Callaway, William E. Armstrong.
    The Journal of Physiology. May 15, 2017
    Key points Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2’s control of these AHPs. We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+]i, and whole cell Ca2+ currents. Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3‐triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2. PIP2 depletion reduced spike‐evoked Ca2+ entry and voltage‐gated Ca2+ currents. PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking. Abstract Oxytocin (OT)‐ and vasopressin (VP)‐secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium‐dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5‐bisphosphate (PIP2) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP) of OT, but not VP neurons with high affinity. We further tested this by introducing a water‐soluble PIP2 analogue (diC8‐PIP2) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP. Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐triggered Ca2+‐store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+. Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage‐gated Ca2+ channels opened during spike trains.
    May 15, 2017   doi: 10.1113/JP274219   open full text
  • Refuting the myth of non‐response to exercise training: ‘non‐responders’ do respond to higher dose of training.
    David Montero, Carsten Lundby.
    The Journal of Physiology. May 14, 2017
    Key points The prevalence of cardiorespiratory fitness (CRF) non‐response gradually declines in healthy individuals exercising 60, 120, 180, 240 or 300 min per week for 6 weeks. Following a successive identical 6‐week training period but comprising 120 min of additional exercise per week, CRF non‐response is universally abolished. The magnitude of CRF improvement is primarily attributed to changes in haemoglobin mass. The potential for CRF improvement may be present and unveiled with appropriate exercise training stimuli in healthy individuals without exception. Abstract One in five adults following physical activity guidelines are reported to not demonstrate any improvement in cardiorespiratory fitness (CRF). Herein, we sought to establish whether CRF non‐response to exercise training is dose‐dependent, using a between‐ and within‐subject study design. Seventy‐eight healthy adults were divided into five groups (1–5) respectively comprising one, two, three, four and five 60 min exercise sessions per week but otherwise following an identical 6‐week endurance training (ET) programme. Non‐response was defined as any change in CRF, determined by maximal incremental exercise power output (Wmax), within the typical error of measurement (±3.96%). Participants classified as non‐responders after the ET intervention completed a successive 6‐week ET period including two additional exercise sessions per week. Maximal oxygen consumption (V̇O2 max ), haematology and muscle biopsies were assessed prior to and after each ET period. After the first ET period, Wmax increased (P < 0.05) in groups 2, 3, 4 and 5, but not 1. In groups 1, 2, 3, 4 and 5, 69%, 40%, 29%, 0% and 0% of individuals, respectively, were non‐responders. After the second ET period, non‐response was eliminated in all individuals. The change in V̇O2 max with exercise training independently determined Wmax response (partial correlation coefficient, rpartial ≥ 0.74, P < 0.001). In turn, total haemoglobin mass was the strongest independent determinant of V̇O2 max (rpartial = 0.49, P < 0.001). In conclusion, individual CRF non‐response to exercise training is abolished by increasing the dose of exercise and primarily a function of haematological adaptations in oxygen‐carrying capacity.
    May 14, 2017   doi: 10.1113/JP273480   open full text
  • When size matters: transient receptor potential vanilloid 4 channel as a volume‐sensor rather than an osmo‐sensor.
    Trine L. Toft‐Bertelsen, David Križaj, Nanna MacAulay.
    The Journal of Physiology. May 14, 2017
    Key points Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume‐sensing ion channel irrespective of the origin of the cell swelling. Abstract Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non‐selective cation channel permeable to Ca2+, it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+‐dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4–AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume‐sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co‐expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4‐mediated transmembrane currents. Similar TRPV4 activation was induced by co‐expression of a cognate channel, AQP1. The level of osmotically‐induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co‐expressed water‐translocating Na+/K+/2Cl− cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume‐dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume‐sensor, rather than as an osmo‐sensor.
    May 14, 2017   doi: 10.1113/JP274135   open full text
  • Sensitivity to ischaemia of single sympathetic nerve fibres innervating the dorsum of the human foot.
    W. J. Z'Graggen, R. Solà, N. E. Graf, J. Serra, H. Bostock.
    The Journal of Physiology. May 14, 2017
    Key points Changes in nerve conduction velocity following an impulse (i.e. velocity recovery cycles) reflect after‐potentials, and can provide an indication of altered nerve membrane properties. This study used microneurography to assess the effects of ischaemia on single human sympathetic fibres innervating the dorsum of the foot. It was found that velocity recovery cycles can distinguish whether a sympathetic nerve fibre is depolarized or not. The method may be used to detect membrane depolarization of sympathetic nerve fibres in human patients when autonomic neuropathy is suspected. Abstract The aim of this study was to determine whether velocity recovery cycles (VRCs) could detect the effects of ischaemia on sympathetic nerve fibres. VRCs of human sympathetic nerve fibres of the superficial peroneal nerve innervating the dorsum of the foot were recorded by microneurography in seven healthy volunteers. Sympathetic nerve fibres were identified by studying their response to manoeuvres increasing sympathetic outflow and by measuring activity‐dependent slowing at 2 Hz stimulation. VRCs were assessed at rest, during 30 min of induced limb ischaemia and during 20 min of recovery after ischaemia. From each VRC was measured the relative refractory period (RRP), the supernormality and the time to peak supernormality (SN@). During ischaemia, RRP increased from the baseline value of 37.4 ± 8.7 ms (mean ± SEM) to 67.1 ± 12.1 ms (P < 0.01) and SN@ increased from 68.6 ± 9.8 ms to 133.8 ± 11.0 ms (P < 0.005). The difference between SN@ and RRP separated ischaemic from non‐ischaemic sympathetic nerve fibres. It is concluded that these sympathetic nerve fibres are sensitive to ischaemia, and that VRCs provide a method to study changes of axonal membrane potential of human sympathetic nerve fibres in vivo.
    May 14, 2017   doi: 10.1113/JP274324   open full text
  • The impact of age and frailty on ventricular structure and function in C57BL/6J mice.
    H. A. Feridooni, A. E. Kane, O. Ayaz, A. Boroumandi, N. Polidovitch, R. G. Tsushima, R. A. Rose, S. E. Howlett.
    The Journal of Physiology. May 14, 2017
    Key points Heart size increases with age (called hypertrophy), and its ability to contract declines. However, these reflect average changes that may not be present, or present to the same extent, in all older individuals. That aging happens at different rates is well accepted clinically. People who are aging rapidly are frail and frailty is measured with a ‘frailty index’. We quantified frailty with a validated mouse frailty index tool and evaluated the impacts of age and frailty on cardiac hypertrophy and contractile dysfunction. Hypertrophy increased with age, while contractions, calcium currents and calcium transients declined; these changes were graded by frailty scores. Overall health status, quantified as frailty, may promote maladaptive changes associated with cardiac aging and facilitate the development of diseases such as heart failure. To understand age‐related changes in heart structure and function, it is essential to know both chronological age and the health status of the animal. Abstract On average, cardiac hypertrophy and contractile dysfunction increase with age. Still, individuals age at different rates and their health status varies from fit to frail. We investigated the influence of frailty on age‐dependent ventricular remodelling. Frailty was quantified as deficit accumulation in adult (≈7 months) and aged (≈27 months) C57BL/6J mice by adapting a validated frailty index (FI) tool. Hypertrophy and contractile function were evaluated in Langendorff‐perfused hearts; cellular correlates/mechanisms were investigated in ventricular myocytes. FI scores increased with age. Mean cardiac hypertrophy increased with age, but values in the adult and aged groups overlapped. When plotted as a function of frailty, hypertrophy was graded by FI score (r = 0.67–0.55, P < 0.0003). Myocyte area also correlated positively with FI (r = 0.34, P = 0.03). Left ventricular developed pressure (LVDP) plus rates of pressure development (+dP/dt) and decay (−dP/dt) declined with age and this was graded by frailty (r = −0.51, P = 0.0007; r = −0.48, P = 0.002; r = −0.56, P = 0.0002 for LVDP, +dP/dt and −dP/dt). Smaller, slower contractions graded by FI score were also seen in ventricular myocytes. Contractile dysfunction in cardiomyocytes isolated from frail mice was attributable to parallel changes in underlying Ca2+ transients. These changes were not due to reduced sarcoplasmic reticulum stores, but were graded by smaller Ca2+ currents (r = −0.40, P = 0.008), lower gain (r = −0.37, P = 0.02) and reduced expression of Cav1.2 protein (r = −0.68, P = 0.003). These results show that cardiac hypertrophy and contractile dysfunction in naturally aging mice are graded by overall health and suggest that frailty, in addition to chronological age, can help explain heterogeneity in cardiac aging.
    May 14, 2017   doi: 10.1113/JP274134   open full text
  • Endogenous nitric oxide formation in cardiac myocytes does not control respiration during β‐adrenergic stimulation.
    Michael Kohlhaas, Alexander G. Nickel, Stefanie Bergem, Barbara Casadei, Ulrich Laufs, Christoph Maack.
    The Journal of Physiology. May 14, 2017
    Key points In the heart, endothelial nitric oxide (NO) controls oxygen consumption in the working heart through paracrine mechanisms. While cardiac myocytes contain several isoforms of NO synthases, it is unclear whether these can control respiration in an intracrine fashion. A long‐standing controversy is whether a NOS exists within mitochondria. By combining fluorescence technologies with electrical field stimulation or the patch‐clamp technique in beating cardiac myocytes, we identified a neuronal NO synthase (nNOS) as the most relevant source of intracellular NO during β‐adrenergic stimulation, while no evidence for a mitochondria‐located NOS was obtained. The amounts of NO produced by non‐mitochondrial nNOS were insufficient to regulate respiration during β‐adrenergic stimulation, arguing against intracrine control of respiration by NO within cardiac myocytes. Abstract Endothelial nitric oxide (NO) controls cardiac oxygen (O2) consumption in a paracrine way by slowing respiration at the mitochondrial electron transport chain. While NO synthases (NOSs) are also expressed in cardiac myocytes, it is unclear whether they control respiration in an intracrine way. Furthermore, the existence of a mitochondrial NOS is controversial. Here, by combining fluorescence imaging with electrical field stimulation, the patch‐clamp method and knock‐out technology, we determined the sources and consequences of intracellular NO formation during workload transitions in isolated murine and guinea pig cardiac myocytes and mitochondria. Using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF) as a fluorescent NO‐sensor that locates to the cytosol and mitochondria, we observed that NO increased by ∼12% within 3 min of β‐adrenergic stimulation in beating cardiac myocytes. This NO stems from neuronal NOS (nNOS), but not endothelial (eNOS). After patch clamp‐mediated dialysis of cytosolic DAF, the remaining NO signals (mostly mitochondrial) were blocked by nNOS deletion, but not by inhibiting the mitochondrial Ca2+ uniporter with Ru360. While in isolated mitochondria exogenous NO inhibited respiration and reduced the NAD(P)H redox state, pyridine nucleotide redox states were unaffected by pharmacological or genetic disruption of endogenous nNOS or eNOS during workload transitions in cardiac myoctyes. We conclude that under physiological conditions, nNOS is the most relevant source for NO in cardiac myocytes, but this nNOS is not located in mitochondria and does not control respiration. Therefore, cardiac O2 consumption is controlled by endothelial NO in a paracrine, but not intracrine, fashion.
    May 14, 2017   doi: 10.1113/JP273750   open full text
  • Vasopressin casts light on the suprachiasmatic nucleus.
    Takahiro Tsuji, Andrew J. Allchorne, Meng Zhang, Chiharu Tsuji, Vicky A. Tobin, Rafael Pineda, Androniki Raftogianni, Javier E. Stern, Valery Grinevich, Gareth Leng, Mike Ludwig.
    The Journal of Physiology. May 14, 2017
    Key points A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light‐induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo‐entrainment of biological rhythms. Abstract In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light‐evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c‐fos in the SCN, which is involved in photic entrainment of circadian rhythms.
    May 14, 2017   doi: 10.1113/JP274025   open full text
  • Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial‐specific Na+/Ca2+ exchange knockout mice.
    Angelo G. Torrente, Rui Zhang, Heidi Wang, Audrey Zaini, Brian Kim, Xin Yue, Kenneth D. Philipson, Joshua I. Goldhaber.
    The Journal of Physiology. May 13, 2017
    Key points Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+. Abstract Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q‐PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+‐sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial‐specific Na+/Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+‐dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+. We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.
    May 13, 2017   doi: 10.1113/JP274249   open full text
  • Calcium‐activated BKCa channels govern dynamic membrane depolarizations of horizontal cells in rodent retina.
    Xiaoping Sun, Arlene A. Hirano, Nicholas C. Brecha, Steven Barnes.
    The Journal of Physiology. May 13, 2017
    Key points Large conductance, Ca2+‐activated K+ (BKCa) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells. BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 mm K+, single channels had a conductance close to 250 pS, within the range of all known BKCa channels. In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells. Abstract Large conductance, calcium‐activated potassium (BKCa) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between −30 and +60 mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole‐cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100 nm), iberiotoxin (10 μm), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30 μm). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS (n = 17; symmetrical 150 mm K+) with open probability being both voltage‐ and Ca2+‐dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells.
    May 13, 2017   doi: 10.1113/JP274132   open full text
  • Non‐muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.
    Wenwu Zhang, Susan J. Gunst.
    The Journal of Physiology. May 08, 2017
    Key points Non‐muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non‐phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh‐induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. Abstract The molecular function of non‐muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non‐phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh‐induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross‐bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA‐mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross‐bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.
    May 08, 2017   doi: 10.1113/JP273906   open full text
  • Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature.
    M. Nocella, G. Cecchi, B. Colombini.
    The Journal of Physiology. May 08, 2017
    Key points Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi] in the myoplasm. High [Pi] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low‐ or no‐force state and show that fibre length changes perturb the Pi‐sensitive force generation of the crossbridge cycle. Abstract Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi] in the myoplasm. Experiments in skinned fibres at low temperature (10–12°C) have shown that [Pi] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.
    May 08, 2017   doi: 10.1113/JP273672   open full text
  • Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.
    Erin V. McGillick, Sandra Orgeig, Beth J. Allison, Kirsty L. Brain, Youguo Niu, Nozomi Itani, Katie L. Skeffington, Andrew D. Kane, Emilio A. Herrera, Dino A. Giussani, Janna L. Morrison.
    The Journal of Physiology. May 07, 2017
    Key points Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air‐breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. Abstract Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2) or hypoxia (10% O2) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT‐PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti‐oxidant marker CAT and decreased expression of the pro‐oxidant marker NOX‐4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF‐3α, KDM3A, SLC2A1, EGLN‐3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B‐1, HSD11B‐2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1‐B, ATP1‐A1, ATP1‐B1) and water (AQP‐4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP‐B, SFTP‐D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air‐breathing at birth.
    May 07, 2017   doi: 10.1113/JP273842   open full text
  • Both standing and postural threat decrease Achilles’ tendon reflex inhibition from tendon electrical stimulation.
    Brian C. Horslen, J. Timothy Inglis, Jean‐Sébastien Blouin, Mark G. Carpenter.
    The Journal of Physiology. May 04, 2017
    Key points Golgi tendon organs (GTOs) and associated Ib reflexes contribute to standing balance, but the potential impacts of threats to standing balance on Ib reflexes are unknown. Tendon electrical stimulation to the Achilles’ tendon was used to probe changes in Ib inhibition in medial gastrocnemius with postural orientation (lying prone vs. upright standing; experiment 1) and height‐induced postural threat (standing at low and high surface heights; experiment 2). Ib inhibition was reduced while participants stood upright, compared to lying prone (42.2%); and further reduced when standing in the high, compared to low, threat condition (32.4%). These experiments will impact future research because they demonstrate that tendon electrical stimulation can be used to probe Ib reflexes in muscles engaged in standing balance. These results provide novel evidence that human short‐latency GTO‐Ib reflexes are dependent upon both task, as evidenced by changes with postural orientation, and context, such as height‐induced postural threat during standing. Abstract Golgi tendon organ Ib reflexes are thought to contribute to standing balance control, but it is unknown if they are modulated when people are exposed to a postural threat. We used a novel application of tendon electrical stimulation (TStim) to elicit Ib inhibitory reflexes in the medial gastrocnemius, while actively engaged in upright standing balance, to examine (a) how Ib reflexes to TStim are influenced by upright stance, and (b) the effects of height‐induced postural threat on Ib reflexes during standing. TStim evoked short‐latency (<47 ms) inhibition apparent in trigger‐averaged rectified EMG, which was quantified in terms of area, duration and mean amplitude of inhibition. In order to validate the use of TStim in a standing model, TStim‐Ib inhibition was compared from conditions where participants were lying prone vs. standing upright. TStim evoked Ib inhibition in both conditions; however, significant reductions in Ib inhibition area (42.2%) and duration (32.9%) were observed during stance. Postural threat, manipulated by having participants stand at LOW (0.8 m high, 0.6 m from edge) and HIGH (3.2 m, at edge) elevated surfaces, significantly reduced Ib inhibition area (32.4%), duration (16.4%) and amplitude (24.8%) in the HIGH, compared to LOW, threat condition. These results demonstrate TStim is a viable technique for investigating Ib reflexes in standing, and confirm Ib reflexes are modulated with postural orientation. The novel observation of reduced Ib inhibition with elevated postural threat reveals that human Ib reflexes are context dependent, and the human Ib reflex pathways are modulated by threat or emotional processing centres of the CNS.
    May 04, 2017   doi: 10.1113/JP273935   open full text
  • Mechanically sensitive Aδ nociceptors that innervate bone marrow respond to changes in intra‐osseous pressure.
    Sara Nencini, Jason Ivanusic.
    The Journal of Physiology. May 04, 2017
    Key points Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function. Here we use a novel in vivo bone–nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra‐osseous pressure. We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra‐osseous pressure stimulus and express the Piezo2 mechano‐transducer molecule. Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and that the Piezo2 mechano‐transducer may be involved. Abstract Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic–tonic units that appear to code for different intensities of intra‐osseous pressure, or phasic units that code for the rate of change in intra‐osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic–tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak‐to‐peak action potential amplitude. The majority of small‐diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano‐nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra‐osseous pressure, and imply that Piezo2 signalling may be involved in mechano‐transduction in these receptors.
    May 04, 2017   doi: 10.1113/JP273877   open full text
  • SIRT1 may play a crucial role in overload‐induced hypertrophy of skeletal muscle.
    Erika Koltai, Zoltán Bori, Clovis Chabert, Hervé Dubouchaud, Hisashi Naito, Shuichi Machida, Kelvin JA Davies, Zsolt Murlasits, Andrew C Fry, Istvan Boldogh, Zsolt Radak.
    The Journal of Physiology. April 28, 2017
    Key points Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload‐induced hypertrophy. SIRT1‐mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload‐induced changes in microRNA levels regulate SIRT1 and insulin‐like growth factor 1 signalling. Abstract Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P < 0.001). SIRT1‐regulated Akt, endothelial nitric oxide synthase and GLUT4 levels were also induced in hypertrophied muscles, and SIRT1 levels correlated with muscle mass, paired box protein 7 (Pax7), proliferating cell nuclear antigen (PCNA) and nicotinamide phosphoribosyltransferase (Nampt) levels. Alternatively, decreased forkhead box O 1 (FOXO1) and increased K48 polyubiquitination also suggest that SIRT1 could be involved in the catabolic process of hypertrophy. Furthermore, increased levels of K63 and muscle RING finger 2 (MuRF2) protein could also be important enhancers of muscle mass. We report here that the levels of miR1 and miR133a decrease in hypertrophy and negatively correlate with muscle mass, SIRT1 and Nampt levels. Our results reveal a strong correlation between SIRT1 levels and activity, SIRT1‐regulated pathways and overload‐induced hypertrophy. These findings, along with the well‐known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload‐induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing.
    April 28, 2017   doi: 10.1113/JP273774   open full text
  • Impact of perinatal exposure to sucrose or high fructose corn syrup (HFCS‐55) on adiposity and hepatic lipid composition in rat offspring.
    Carla R. Toop, Beverly S. Muhlhausler, Kerin O'Dea, Sheridan Gentili.
    The Journal of Physiology. April 26, 2017
    Perinatal exposure to excess maternal intake of added sugars, including fructose and sucrose, is associated with an increased risk of obesity and type 2 diabetes in adult life. However, it is unknown to what extent the type of sugar and the timing of exposure affect these outcomes. The aim of this study was to determine the impact of exposure to maternal consumption of a 10% w/v beverage containing sucrose or high fructose corn syrup‐55 (HFCS‐55) during the prenatal and/or suckling periods on offspring at 3 and 12 weeks, utilising a cross‐fostering approach in a rodent model. Perinatal sucrose exposure decreased plasma glucose concentrations in offspring at 3 weeks, but did not alter glucose tolerance. Increased adiposity was observed in 3‐week‐old offspring exposed to sucrose or HFCS‐55 during suckling, with increased hepatic fat content in HFCS‐55‐exposed offspring. In terms of specific fatty acids, hepatic monounsaturated (omega‐7 and ‐9) fatty acid content was elevated at weaning, and was most pronounced in sucrose offspring exposed during both the prenatal and suckling periods, and HFCS‐55 offspring exposed during suckling only. By 12 weeks, the effects on adiposity and hepatic lipid composition were largely normalised. However, exposure to either sucrose or HFCS‐55 during the prenatal period only was associated with elevated plasma free fatty acids at weaning, and this effect persisted until 12 weeks. This study suggests that the type of sugar and the timing of exposure (prenatal or suckling periods) are both important for determining the impact on metabolic health outcomes in the offspring. This article is protected by copyright. All rights reserved
    April 26, 2017   doi: 10.1113/JP274066   open full text
  • Functional impact of an oculopharyngeal muscular dystrophy mutation in PABPN1.
    Maricela García‐Castañeda, Ana Victoria Vega, Rocío Rodríguez, Maria Guadalupe Montiel‐Jaen, Bulmaro Cisneros, Angel Zarain‐Herzberg, Guillermo Avila.
    The Journal of Physiology. April 25, 2017
    Key points Mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1) result in oculopharyngeal muscular dystrophy (OPMD). This disease is of late‐onset, but the underlying mechanism is unclear. Ca2+ stimulates muscle growth and contraction and, because OPMD courses with muscle atrophy and weakness, we hypothesized that the homeostasis of Ca2+ is altered in this disorder. C2C12 myotubes were transfected with cDNAs encoding either PABPN1 or the PABPN1‐17A OPMD mutation. Subsequently, they were investigated concerning not only excitation–contraction coupling (ECC) and intracellular levels of Ca2+, but also differentiation stage and nuclear structure. PABPN1‐17A gave rise to: inhibition of Ca2+ release during ECC, depletion of sarcoplasmic reticulum Ca2+ content, reduced expression of ryanodine receptors, altered nuclear morphology and incapability to stimulate myoblast fusion. PABPN1‐17A failed to inhibit ECC in adult muscle fibres, suggesting that its effects are primarily related to muscle regeneration. Abstract Oculopharyngeal muscular dystrophy (OPMD) is linked to mutations in the gene encoding poly(A)‐binding protein nuclear 1 (PABPN1). OPMD mutations consist of an expansion of a tract that contains 10 alanines (to 12–17). This disease courses with muscle weakness that begins in adulthood, but the underlying mechanism is unclear. In the present study, we investigated the functional effects of PABPN1 and an OPMD mutation (PABPN1‐17A) using myotubes transfected with cDNAs encoding these proteins (GFP‐tagged). PABPN1 stimulated myoblast fusion (100%), whereas PABPN1‐17A failed to mimic this effect. Additionally, the OPMD mutation markedly altered nuclear morphology; specifically, it led to nuclei with a more convoluted and ovoid shape. Although PABPN1 and PABPN1‐17A modified the expression of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase and calsequestrin, the corresponding changes did not have a clear impact on [Ca2+]. Interestingly, neither L‐type Ca2+ channels, nor voltage‐gated sarcoplasmic reticulum (SR) Ca2+ release (VGCR) was altered by PABPN1. However, PABPN1‐17A produced a selective inhibition of VGCR (50%). This effect probably arises from both lower expression of RyR1 and depletion of SR Ca2+. The latter, however, was not related to inhibition of store‐operated Ca2+ entry. Both PABPN1 constructs promoted a moderated decrease in cytosolic [Ca2+], which apparently results from down‐regulation of excitation‐coupled Ca2+ entry. On the other hand, PABPN1‐17A did not alter ECC in muscle fibres, suggesting that adult muscle is less prone to developing deleterious effects. These results demonstrate that PABPN1 proteins regulate essential processes during myotube formation and support the notion that OPMD involves disruption of myogenesis, nuclear structure and homeostasis of Ca2+.
    April 25, 2017   doi: 10.1113/JP273948   open full text
  • In vitro characterization of cell‐level neurophysiological diversity in the rostral nucleus reuniens of adult mice.
    Darren A. Walsh, Jonathan T. Brown, Andrew D. Randall.
    The Journal of Physiology. April 25, 2017
    Key points The nucleus reuniens (Re), a nucleus of the midline thalamus, is part of a cognitive network including the hippocampus and the medial prefrontal cortex. To date, very few studies have examined the electrophysiological properties of Re neurons at a cellular level. The majority of Re neurons exhibit spontaneous action potential firing at rest. This is independent of classical amino‐acid mediated synaptic transmission. When driven by various forms of depolarizing current stimulus, Re neurons display considerable diversity in their firing patterns. As a result of the presence of a low threshold Ca2+ channel, spike output functions are strongly modulated by the prestimulus membrane potential. Finally, we describe a novel form of activity‐dependant intrinsic plasticity that eliminates the high‐frequency burst firing present in many Re neurons. These results provide a comprehensive summary of the intrinsic electrophysiological properties of Re neurons allowing us to better consider the role of the Re in cognitive processes. Abstract The nucleus reuniens (Re) is the largest of the midline thalamic nuclei. We have performed a detailed neurophysiological characterization of neurons in the rostral Re of brain slices prepared from adult male mice. At resting potential (−63.7 ± 0.6 mV), ∼90% of Re neurons fired action potentials, typically continuously at ∼8 Hz. Although Re neurons experience a significant spontaneous barrage of fast, amino‐acid‐mediate synaptic transmission, this was not predominantly responsible for spontaneous spiking because firing persisted in the presence of glutamate and GABA receptor antagonists. With resting potential preset to −80 mV, −20 pA current injections revealed a mean input resistance of 615 MΩ and a mean time constant of 38 ms. Following cessation of this stimulus, a significant rebound potential was seen that was sometimes sufficiently large to trigger a short burst of very high frequency (100–300 Hz) firing. In most cells, short (2 ms), strong (2 nA) current injections elicited a single spike followed by a large afterdepolarizing potential which, when suprathreshold, generated high‐frequency spiking. Similarly, in the majority of cells preset at −80 mV, 500 ms depolarizing current injections to cells led to a brief initial burst of very high‐frequency firing, although this was lost when cells were preset at −72 mV. Biophysical and pharmacological experiments indicate a prominent role for T‐type Ca2+ channels in the high‐frequency bursting of Re neurons. Finally, we describe a novel form of activity‐dependent intrinsic plasticity that persistently eliminates the burst firing potential of Re neurons.
    April 25, 2017   doi: 10.1113/JP273915   open full text
  • Evidence that 5‐HT stimulates intracellular Ca2+ signalling and activates pannexin‐1 currents in type II cells of the rat carotid body.
    Sindhubarathi Murali, Min Zhang, Colin A. Nurse.
    The Journal of Physiology. April 25, 2017
    Key points 5‐HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH). In the present study, we show that, in addition to type I cells, adjacent glial‐like type II cells express functional, ketanserin‐sensitive 5‐HT2 receptors, and their stimulation increases cytoplasmic Ca2+ derived from intracellular stores. In type II cells, 5‐HT activated a ketanserin‐sensitive inward current (I5‐HT) that was similar to that (IUTP) activated by the P2Y2R agonist, UTP. As previously shown for IUTP, I5‐HT was inhibited by BAPTA‐AM and carbenoxolone (5 μm), a putative blocker of ATP‐permeable pannexin (Panx)‐1 channels; IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide channel blocker, 10Panx peptide. Paracrine stimulation of type II cells by 5‐HT, leading to ATP release via Panx‐1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea). Abstract Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5‐HT and increased autocrine–paracrine 5‐HT2 receptor signalling contributes to sensory long‐term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial‐like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca2+]i) and activating carbenoxolone‐sensitive, ATP‐permeable, pannexin (Panx)‐1‐like channels. In the present study, using dissociated rat CB cultures, we found that 5‐HT induced Δ[Ca2+]i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5‐HT‐induced Ca2+ response in type II cells was dose‐dependent (EC50 ∼183 nm) and largely inhibited by the 5‐HT2A receptor blocker, ketanserin (1 μm), and also arose mainly from intracellular stores. 5‐HT also activated an inward current (I5‐HT) in type II cells (EC50 ∼200 nm) that was reversibly inhibited by ketanserin (1–10 nm), the Ca2+ chelator BAPTA‐AM (5 μm), and low concentrations of carbenoxolone (5 μm), a putative Panx‐1 channel blocker. I5‐HT reversed direction at approximately −11 mV and was indistinguishable from the UTP‐activated current (IUTP). Consistent with a role for Panx‐1 channels, IUTP was reversibly inhibited by the specific Panx‐1 mimetic peptide blocker 10Panx (100 μm), although not by its scrambled control peptide (scPanx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5‐HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx‐1 channels.
    April 25, 2017   doi: 10.1113/JP273473   open full text
  • Characterisation and functional mapping of surface potentials in the rat dorsal column nuclei.
    Alastair J. Loutit, Ted Maddess, Stephen J. Redmond, John W. Morley, Greg J. Stuart, Jason R. Potas.
    The Journal of Physiology. April 25, 2017
    Key points The brainstem dorsal column nuclei (DCN) process sensory information arising from the body before it reaches the brain and becomes conscious. Despite significant investigations into sensory coding in peripheral nerves and the somatosensory cortex, little is known about how sensory information arising from the periphery is represented in the DCN. Following stimulation of hind‐limb nerves, we mapped and characterised the evoked electrical signatures across the DCN surface. We show that evoked responses recorded from the DCN surface are highly reproducible and are unique to nerves carrying specific sensory information. Abstract The brainstem dorsal column nuclei (DCN) play a role in early processing of somatosensory information arising from a variety of functionally distinct peripheral structures, before being transmitted to the cortex via the thalamus. To improve our understanding of how sensory information is represented by the DCN, we characterised and mapped low‐ (<200 Hz) and high‐frequency (550–3300 Hz) components of nerve‐evoked DCN surface potentials. DCN surface potentials were evoked by electrical stimulation of the left and right nerves innervating cutaneous structures (sural nerve), or a mix of cutaneous and deep structures (peroneal nerve), in 8‐week‐old urethane‐anaesthetised male Wistar rats. Peroneal nerve‐evoked DCN responses demonstrated low‐frequency events with significantly longer durations, more high‐frequency events and larger magnitudes compared to responses evoked from sural nerve stimulation. Hotspots of low‐ and high‐frequency DCN activity were found ipsilateral to stimulated nerves but were not symmetrically organised. In conclusion, we find that sensory inputs from peripheral nerves evoke unique and characteristic DCN activity patterns that are highly reproducible both within and across animals.
    April 25, 2017   doi: 10.1113/JP273759   open full text
  • Early structural and functional signature of 3‐day human skeletal muscle disuse using the dry immersion model.
    Rémi Demangel, Loïc Treffel, Guillaume Py, Thomas Brioche, Allan F. Pagano, Marie‐Pierre Bareille, Arnaud Beck, Laurence Pessemesse, Robin Candau, Claude Gharib, Angèle Chopard, Catherine Millet.
    The Journal of Physiology. April 23, 2017
    Key points Our study contributes to the characterization of muscle loss and weakness processes induced by a sedentary life style, chronic hypoactivity, clinical bed rest, immobilization and microgravity. This study, by bringing together integrated and cellular evaluation of muscle structure and function, identifies the early functional markers and biomarkers of muscle deconditioning. Three days of muscle disuse in healthy adult subjects is sufficient to significantly decrease muscle mass, tone and force, and to induce changes in function relating to a weakness in aerobic metabolism and muscle fibre denervation. The outcomes of this study should be considered in the development of an early muscle loss prevention programme and/or the development of pre‐conditioning programmes required before clinical bed rest, immobilization and spaceflight travel. Abstract Microgravity and hypoactivity are associated with skeletal muscle deconditioning. The decrease of muscle mass follows an exponential decay, with major changes in the first days. The purpose of the study was to dissect out the effects of a short‐term 3‐day dry immersion (DI) on human quadriceps muscle function and structure. The DI model, by suppressing all support zones, accurately reproduces the effects of microgravity. Twelve healthy volunteers (32 ± 5 years) completed 3 days of DI. Muscle function was investigated through maximal voluntary contraction (MVC) tests and muscle viscoelasticity. Structural experiments were performed using MRI analysis and invasive experiments on muscle fibres. Our results indicated a significant 9.1% decrease of the normalized MVC constant (P = 0.048). Contraction and relaxation modelization kinetics reported modifications related to torque generation (kACT = −29%; P = 0.014) and to the relaxation phase (kREL = +34%; P = 0.040) after 3 days of DI. Muscle viscoelasticity was also altered. From day one, rectus femoris stiffness and tone decreased by, respectively, 7.3% (P = 0.002) and 10.2% (P = 0.002), and rectus femoris elasticity decreased by 31.5% (P = 0.004) after 3 days of DI. At the cellular level, 3 days of DI translated into a significant atrophy of type I muscle fibres (−10.6 ± 12.1%, P = 0.027) and an increased proportion of hybrid, type I/IIX fibre co‐expression. Finally, we report an increase (6‐fold; P = 0.002) in NCAM+ muscle fibres, showing an early denervation process. This study is the first to report experiments performed in Europe investigating human short‐term DI‐induced muscle adaptations, and contributes to deciphering the early changes and biomarkers of skeletal muscle deconditioning.
    April 23, 2017   doi: 10.1113/JP273895   open full text
  • Direct current stimulation boosts synaptic gain and cooperativity in vitro.
    Asif Rahman, Belen Lafon, Lucas C. Parra, Marom Bikson.
    The Journal of Physiology. April 23, 2017
    Key points Direct current stimulation (DCS) polarity specifically modulates synaptic efficacy during a continuous train of presynaptic inputs, despite synaptic depression. DCS polarizes afferent axons and postsynaptic neurons, boosting cooperativity between synaptic inputs. Polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during transcranial DCS (tDCS). A statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous in enhancing connectivity between co‐active brain regions. Abstract Transcranial direct current stimulation (tDCS) produces sustained and diffuse current flow in the brain with effects that are state dependent and outlast stimulation. A mechanistic explanation for tDCS should capture these spatiotemporal features. It remains unclear how sustained DCS affects ongoing synaptic dynamics and how modulation of afferent inputs by diffuse stimulation changes synaptic activity at the target brain region. We tested the effect of acute DCS (10–20 V m−1 for 3–5 s) on synaptic dynamics with constant rate (5–40 Hz) and Poisson‐distributed (4 Hz mean) trains of presynaptic inputs. Across tested frequencies, sustained synaptic activity was modulated by DCS with polarity‐specific effects. Synaptic depression attenuates the sensitivity to DCS from 1.1% per V m−1 to 0.55%. DCS applied during synaptic activity facilitates cumulative neuromodulation, potentially reversing endogenous synaptic depression. We establish these effects are mediated by both postsynaptic membrane polarization and afferent axon fibre polarization, which boosts cooperativity between synaptic inputs. This potentially extends the locus of neuromodulation from the nominal target to afferent brain regions. Based on these results we hypothesized the polarization of afferent neurons in upstream brain regions may modulate activity in the target brain region during tDCS. A multiscale model of transcranial electrical stimulation including a finite element model of brain current flow, numerical simulations of neuronal activity, and a statistical theory of coincident activity predicts that the diffuse and weak profile of current flow can be advantageous. Thus, we propose that specifically because tDCS is diffuse, weak and sustained it can boost connectivity between co‐active brain regions.
    April 23, 2017   doi: 10.1113/JP273005   open full text
  • Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high‐intensity exercise training.
    Victoria L. Wyckelsma, Itamar Levinger, Michael J. McKenna, Luke E. Formosa, Michael T. Ryan, Aaron C. Petersen, Mitchell J. Anderson, Robyn M. Murphy.
    The Journal of Physiology. April 23, 2017
    Key points Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high‐intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. Abstract Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High‐intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin‐2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70–100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults.
    April 23, 2017   doi: 10.1113/JP273950   open full text
  • mGluR1 enhances efferent inhibition of inner hair cells in the developing rat cochlea.
    Zhanlei Ye, Juan D. Goutman, Sonja J. Pyott, Elisabeth Glowatzki.
    The Journal of Physiology. April 21, 2017
    Key points Spontaneous activity of the sensory inner hair cells shapes maturation of the developing ascending (afferent) auditory system before hearing begins. Just before the onset of hearing, descending (efferent) input from cholinergic neurons originating in the brainstem inhibit inner hair cell spontaneous activity and may further refine maturation. We show that agonist activation of the group I metabotropic glutamate receptor mGluR1 increases the strength of this efferent inhibition by enhancing the presynaptic release of acetylcholine. We further show that the endogenous release of glutamate from the inner hair cells may increase the strength of efferent inhibition via the activation of group I metabotropic glutamate receptors. Thus, before the onset of hearing, metabotropic glutamate signalling establishes a local negative feedback loop that is positioned to regulate inner hair cell excitability and refine maturation of the auditory system. Abstract Just before the onset of hearing, the inner hair cells (IHCs) receive inhibitory efferent input from cholinergic medial olivocochlear (MOC) neurons originating in the brainstem. This input may serve a role in the maturation of the ascending (afferent) auditory system by inhibiting spontaneous activity of the IHCs. To investigate the molecular mechanisms regulating these IHC efferent synapses, we combined electrical stimulation of the efferent fibres with patch clamp recordings from the IHCs to measure efferent synaptic strength. By examining evoked responses, we show that activation of metabotropic glutamate receptors (mGluRs) by general and group I‐specific mGluR agonists enhances IHC efferent inhibition. This enhancement is blocked by application of a group I mGluR1‐specific antagonist, indicating that enhancement of IHC efferent inhibition is mediated by group I mGluRs and specifically by mGluR1s. By comparing spontaneous and evoked responses, we show that group I mGluR agonists act presynaptically to increase neurotransmitter release without affecting postsynaptic responsiveness. Moreover, endogenous glutamate released from the IHCs also enhances IHC efferent inhibition via the activation of group I mGluRs. Finally, immunofluorescence analysis indicates that the efferent terminals are sufficiently close to IHC glutamate release sites to allow activation of mGluRs on the efferent terminals by glutamate spillover. Together, these results suggest that glutamate released from the IHCs activates group I mGluRs (mGluR1s), probably present on the efferent terminals, which, in turn, enhances release of acetylcholine and inhibition of the IHCs. Thus, mGluRs establish a local negative feedback loop positioned to regulate IHC activity and maturation of the ascending auditory system in the developing cochlea.
    April 21, 2017   doi: 10.1113/JP272604   open full text
  • Vasopressin V1a receptors mediate the hypertensive effects of [Pyr1]apelin‐13 in the rat rostral ventrolateral medulla.
    Philip R. Griffiths, Stephen J. Lolait, Louise E. Harris, Julian F. R. Paton, Anne‐Marie O'Carroll.
    The Journal of Physiology. April 21, 2017
    Key points Dysfunctions in CNS regulation of arterial blood pressure lead to an increase in sympathetic nerve activity that participates in the pathogenesis of hypertension. The apelin‐apelin receptor system affects arterial blood pressure homeostasis; however, the central mechanisms underlying apelin‐mediated changes in sympathetic nerve activity and blood pressure have not been clarified. We explored the mechanisms involved in the regulation of [Pyr1]apelin‐13‐mediated cardiovascular control within the rostral ventrolateral medulla (RVLM) using selective receptor antagonists. We show that [Pyr1]apelin‐13 acts as a modulating neurotransmitter in the normotensive RVLM to affect vascular tone through interaction with the vasopressin V1a receptor but that [Pyr1]apelin‐13‐induced sympathoexcitation is independent of angiotensin II receptor type 1, oxytocin, ionotropic glutamate and GABAA receptors. Our data confirm a role for the apelin peptide system in cardiovascular regulation at the level of the RVLM and highlight that this system is a possible potential therapeutic target for the treatment of hypertension. Abstract Apelin is a ubiquitous peptide that can elevate arterial blood pressure (ABP) yet understanding of the mechanisms involved remain incomplete. Bilateral microinjection of [Pyr1]apelin‐13 into the rostral ventrolateral medulla (RVLM), a major source of sympathoexcitatory neurones, increases ABP and sympathetic nerve activity. We aimed to investigate the potential involvement of neurotransmitter systems through which the apelin pressor response may occur within the RVLM. Adult male Wistar rats were anaesthetized and ABP was monitored via a femoral arterial catheter. Bilateral RVLM microinjection of [Pyr1]apelin‐13 significantly increased ABP (9 ± 1 mmHg) compared to saline (−1 ± 2mmHg; P < 0.001), which was blocked by pretreatment with the apelin receptor antagonist, F13A (0 ± 1 mmHg; P < 0.01). The rise in ABP was associated with an increase in the low frequency spectra of systolic BP (13.9 ± 4.3% total power; P < 0.001), indicative of sympathetic vasomotor activation. The [Pyr1]apelin‐13‐mediated pressor response and the increased low frequency spectra of systolic BP response were fully maintained despite RVLM pretreatment with the angiotensin II type 1 receptor antagonist losartan, the oxytocin receptor antagonist desGly‐NH2, d(CH2)5[D‐Tyr2,Thr4]OVT, the ionotropic glutamate receptor antagonist kynurenate or the GABAA antagonist bicuculline (P > 0.05). By contrast, the [Pyr1]apelin‐13 induced pressor and sympathoexcitatory effects were abolished by pretreatment of the RVLM with the vasopressin V1a receptor antagonist, SR 49059 (−1 ± 1 mmHg; 1.1 ± 1.1% total power, respectively; P < 0.001). These findings suggest that the pressor action of [Pyr1]apelin‐13 in the RVLM of normotensive rats is not mediated via angiotensin II type 1 receptor, oxytocin, ionotropic glutamate or GABAA receptors but instead involves a close relationship with the neuropeptide modulator vasopressin.
    April 21, 2017   doi: 10.1113/JP274178   open full text
  • Angiotensin II activates CaV1.2 Ca2+ channels through β‐arrestin2 and casein kinase 2 in mouse immature cardiomyocytes.
    Toshihide Kashihara, Tsutomu Nakada, Katsuhiko Kojima, Toshikazu Takeshita, Mitsuhiko Yamada.
    The Journal of Physiology. April 20, 2017
    Key points Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca2+ transients of mouse immature but not mature cardiomyocytes by robustly activating CaV1.2 L‐type Ca2+ channels through a novel signalling pathway involving angiotensin type 1 (AT1) receptors, β‐arrestin2 and casein kinase 2. A β‐arrestin‐biased AT1 receptor agonist, TRV027, was as effective as AngII in activating L‐type Ca2+ channels. Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that β‐arrestin‐biased AT1 receptor agonists may be valuable therapeutics for paediatric heart failure. Abstract Angiotensin II (AngII), the main effector peptide of the renin–angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well‐known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca2+ transients by robustly activating L‐type CaV1.2 Ca2+ (CaV1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT1 receptors and β‐arrestin2. A β‐arrestin‐biased AT1 receptor agonist was as effective as AngII in activating CaV1.2 channels. Src‐family tyrosine kinases (SFKs) and casein kinase 2α’β (CK2α’β) were sequentially activated when AngII activated CaV1.2 channels. A cyclin‐dependent kinase inhibitor, p27Kip1 (p27), inhibited CK2α’β, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α’β but not c‐Src directly activated recombinant CaV1.2 channels composed of C‐terminally truncated α1C, the distal C‐terminus of α1C, β2C and α2δ1 subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C‐terminus of CaV1.2α1C subunits. Co‐immunoprecipitation revealed that CaV1.2 channels, CK2α’β and p27 formed a macromolecular complex. Therefore, stimulation of AT1 receptors by AngII activates CaV1.2 channels through β‐arrestin2 and CK2α’β, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that β‐arrestin‐biased AT1 receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.
    April 20, 2017   doi: 10.1113/JP273883   open full text
  • Chronic alcohol feeding potentiates hormone‐induced calcium signalling in hepatocytes.
    Paula J. Bartlett, Anil Noronha Antony, Amit Agarwal, Mauricette Hilly, Victoria L. Prince, Laurent Combettes, Jan B. Hoek, Lawrence D. Gaspers.
    The Journal of Physiology. April 18, 2017
    Key points Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+‐mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol‐dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone‐induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone‐evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol‐induced hepatocyte injury. Abstract ‘Adaptive’ responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide‐dependent cytosolic calcium ([Ca2+]i) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose–response for Ca2+‐mobilizing hormones resulting in more sustained and prolonged [Ca2+]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone‐induced calcium increases in control livers, but not after chronic alcohol‐feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone‐induced inositol 1,4,5 trisphosphate (IP3) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol‐fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone‐stimulated PLC activity, indicating calcium‐dependent PLCs are not upregulated by alcohol. We propose that the liver ‘adapts’ to chronic alcohol exposure by increasing hormone‐dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.
    April 18, 2017   doi: 10.1113/JP273891   open full text
  • Geniculohypothalamic GABAergic projections gate suprachiasmatic nucleus responses to retinal input.
    Lydia Hanna, Lauren Walmsley, Abigail Pienaar, Michael Howarth, Timothy M. Brown.
    The Journal of Physiology. April 11, 2017
    Key points Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt their physiology and behaviour in line with the solar day. In addition to direct retinal projections, the clock receives input from the visual thalamus, although the role of this geniculohypothalamic pathway in circadian photoreception is poorly understood. In the present study, we develop a novel brain slice preparation that preserves the geniculohypothalamic pathway to show that GABAergic thalamic neurons inhibit retinally‐driven activity in the central clock in a circadian time‐dependent manner. We also show that in vivo manipulation of thalamic signalling adjusts specific features of the hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily activated by crossed retinal inputs. Our data provide a mechanism by which geniculohypothalamic signals can adjust the magnitude of circadian and more acute hypothalamic light responses according to time‐of‐day and establish an important new model for future investigations of the circadian visual system. Abstract Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in allowing animals to optimize physiology and behaviour alongside daily changes in the environment. Retinal inputs encoding changes in external illumination provide the principle source of such information. The SCN also receives input from other retinorecipient brain regions, primarily via the geniculohypothalamic tract (GHT), although the contribution of these indirect projections to circadian photoreception is currently poorly understood. To address this deficit, in the present study, we established an in vitro mouse brain slice preparation that retains connectivity across the extended circadian system. Using multi‐electrode recordings, we first confirm that this preparation retains intact optic projections to the SCN, thalamus and pretectum and a functional GHT. We next show that optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, and also that this effect exhibits a pronounced day/night variation and involves a GABAergic mechanism. This inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside pharmacological inactivation or optogenetic excitation to show that GHT signalling actively modulates specific features of the SCN light response, indicating that GHT cells are primarily activated by crossed retinal projections. Taken together, our data establish a new model for studying network communication in the extended circadian system and provide novel insight into the roles of GHT‐signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the SCN according to time of day.
    April 11, 2017   doi: 10.1113/JP273850   open full text
  • Baroreflex control of renal sympathetic nerve activity in early heart failure assessed by the sequence method.
    Renata Maria Lataro, Luiz Eduardo Virgilio Silva, Carlos Alberto Aguiar Silva, Helio Cesar Salgado, Rubens Fazan.
    The Journal of Physiology. April 07, 2017
    Key points The integrity of the baroreflex control of sympathetic activity in heart failure (HF) remains under debate. We proposed the use of the sequence method to assess the baroreflex control of renal sympathetic nerve activity (RSNA). The sequence method assesses the spontaneous arterial pressure (AP) fluctuations and their related changes in heart rate (or other efferent responses), providing the sensitivity and the effectiveness of the baroreflex. Effectiveness refers to the fraction of spontaneous AP changes that elicits baroreflex‐mediated variations in the efferent response. Using three different approaches, we showed that the baroreflex sensitivity between AP and RSNA is not altered in early HF rats. However, the sequence method provided evidence that the effectiveness of baroreflex in changing RSNA in response to AP changes is markedly decreased in HF. The results help us better understand the baroreflex control of the sympathetic nerve activity. Abstract In heart failure (HF), the reflex control of the heart rate is known to be markedly impaired; however, the baroreceptor control of the sympathetic drive remains under debate. Applying the sequence method to a series of arterial pressure (AP) and renal sympathetic nerve activity (RSNA), we demonstrated a clear dysfunction in the baroreflex control of sympathetic activity in rats with early HF. We analysed the baroreflex control of the sympathetic drive using three different approaches: AP vs. RSNA curve, cross‐spectral analysis and sequence method between AP and RSNA. The sequence method also provides the baroreflex effectiveness index (BEI), which represents the percentage of AP ramps that actually produce a reflex response. The methods were applied to control rats and rats with HF induced by myocardial infarction. None of the methods employed to assess the sympathetic baroreflex gain were able to detect any differences between the control and the HF group. However, rats with HF exhibited a lower BEI compared to the controls. Moreover, an optimum delay of 1 beat was observed, i.e. 1 beat is required for the RSNA to respond after AP changing, which corroborates with the findings related to the timing between these two variables. For delay 1, the BEI of the controls was 0.45 ± 0.03, whereas the BEI of rats with HF was 0.29 ± 0.09 (P < 0.05). These data demonstrate that while the gain of the baroreflex is not affected in early HF, its effectiveness is markedly decreased. The analysis of the spontaneous changes in AP and RSNA using the sequence method provides novel insights into arterial baroreceptor reflex function.
    April 07, 2017   doi: 10.1113/JP274065   open full text
  • A Western‐style obesogenic diet alters maternal metabolic physiology with consequences for fetal nutrient acquisition in mice.
    Barbara Musial, Owen R. Vaughan, Denise S. Fernandez‐Twinn, Peter Voshol, Susan E. Ozanne, Abigail L. Fowden, Amanda N. Sferruzzi‐Perri.
    The Journal of Physiology. April 05, 2017
    Key points In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy, although their effects on the metabolism of the mother, in relation to feto‐placental glucose utilization and growth, are unknown. In the present study, the consumption of an obesogenic HFHS diet compromised maternal glucose tolerance and insulin sensitivity in late pregnancy in association with dysregulated lipid and glucose handling by the dam. These maternal metabolic changes induced by HFHS feeding were related to altered feto‐placental glucose metabolism and growth. A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth. These findings have implications for the health of women and their infants, who consume obesogenic diets during pregnancy. Abstract In the Western world, obesogenic diets containing high fat and high sugar (HFHS) are commonly consumed during pregnancy. However, the impacts of a HFHS diet during pregnancy on maternal insulin sensitivity and signalling in relation to feto‐placental growth and glucose utilization are unknown. The present study examined the effects of a HFHS diet during mouse pregnancy on maternal glucose tolerance and insulin resistance, as well as, on feto‐placental glucose metabolism. Female mice were fed a control or HFHS diet from day (D) 1 of pregnancy (term = D20.5). At D16 or D19, dams were assessed for body composition, metabolite and hormone concentrations, tissue abundance of growth and metabolic signalling pathways, glucose tolerance and utilization and insulin sensitivity. HFHS feeding perturbed maternal insulin sensitivity in late pregnancy; hepatic insulin sensitivity was higher, whereas sensitivity of the skeletal muscle and white adipose tissue was lower in HFHS than control dams. These changes were accompanied by increased adiposity and reduced glucose production and glucose tolerance of HFHS dams. The HFHS diet also disturbed the hormone and metabolite milieu and altered expression of growth and metabolic signalling pathways in maternal tissues. Furthermore, HFHS feeding was associated with impaired feto‐placental glucose metabolism and growth. A HFHS diet during pregnancy therefore causes maternal metabolic dysfunction with consequences for maternal nutrient allocation for fetal growth. These findings have implications for the health of women and their infants, who consume HFHS diets during pregnancy.
    April 05, 2017   doi: 10.1113/JP273684   open full text
  • mTOR folate sensing links folate availability to trophoblast cell function.
    Fredrick J. Rosario, Theresa L. Powell, Thomas Jansson.
    The Journal of Physiology. April 04, 2017
    Folate is a water‐soluble B vitamin that is essential for cellular methylation reactions and DNA synthesis and repair. Low maternal folate levels in pregnancy are associated with fetal growth restriction, however the underlying mechanisms are poorly understood. Mechanistic target of rapamycin (mTOR) links nutrient availability to cell growth and function by regulating gene expression and protein translation. Here we show that mTOR functions as a folate sensor in primary human trophoblast (PHT) cells. Folate deficiency in PHT cells caused inhibition of mTOR signalling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells involves both mTOR Complex 1 and 2 and requires the proton‐coupled folate transporter (PCFT, SLC46A1). The involvement of PCFT in mTOR folate sensing is not dependent on its function as a plasma membrane folate transporter. Increasing levels of homocysteine had no effect on PHT mTOR signalling, suggesting that mTOR senses low folate rather than high homocysteine. In addition, we demonstrate that maternal serum folate is positively correlated to placental mTORC1 and mTORC2 signalling activity in human pregnancy. We have identified a previously unknown molecular link between folate availability and cell function involving PCFT and mTOR signalling. We propose that mTOR folate sensing in trophoblast cells matches placental nutrient transport, and therefore fetal growth, to folate availability. These findings may have implications for our understanding of how altered folate availability causes human diseases such as fetal growth restriction, fetal malformations and cancer. This article is protected by copyright. All rights reserved
    April 04, 2017   doi: 10.1113/JP272424   open full text
  • Differential serotonergic modulation across the main and accessory olfactory bulbs.
    Zhenbo Huang, Nicolas Thiebaud, Debra Ann Fadool.
    The Journal of Physiology. March 31, 2017
    Key points There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current‐clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5‐HT2A receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5‐HT2 receptors on GABAergic interneurons and direct inhibition via the 5‐HT1 receptors on MCs. Differential 5‐HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression. Abstract Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5‐HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15–30 day mice. In the MOB, 5‐HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)– first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5‐HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5‐HT2A receptors through a direct activation. In comparison, 5‐HT‐evoked inhibitory responses in the AOB arose due to a polysynaptic, slow‐onset inhibition attributed to 5‐HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5‐HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.
    March 31, 2017   doi: 10.1113/JP273945   open full text
  • Computational analysis of the human sinus node action potential: model development and effects of mutations.
    Alan Fabbri, Matteo Fantini, Ronald Wilders, Stefano Severi.
    The Journal of Physiology. March 30, 2017
    Key points We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the ‘funny current’ (If) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart‐rate modulating drugs. Abstract The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (If) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss‐of‐function and gain‐of‐function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.
    March 30, 2017   doi: 10.1113/JP273259   open full text
  • Uteroplacental insufficiency reduces rat plasma leptin concentrations and alters placental leptin transporters: ameliorated with enhanced milk intake and nutrition.
    Jessica F. Briffa, Rachael O'Dowd, Karen M. Moritz, Tania Romano, Lisa R. Jedwab, Andrew J. McAinch, Deanne H. Hryciw, Mary E. Wlodek.
    The Journal of Physiology. March 29, 2017
    Key points Uteroplacental insufficiency compromises maternal mammary development, milk production and pup organ development; this is ameliorated by cross‐fostering, which improves pup growth and organ development and prevents adult diseases in growth‐restricted (Restricted) offspring by enhancing postnatal nutrition. Leptin is transported to the fetus from the mother by the placenta; we report reduced plasma leptin concentrations in Restricted fetuses associated with sex‐specific alterations in placental leptin transporter expression. Pup plasma leptin concentrations were also reduced during suckling, which may suggest reduced milk leptin transport or leptin reabsorption. Mothers suckled by Restricted pups had impaired mammary development and changes in milk fatty acid composition with no alterations in milk leptin; cross‐fostering restored pup plasma leptin concentrations, which may be correlated to improved milk composition and intake. Increased plasma leptin and altered milk fatty acid composition in Restricted pups suckling mothers with normal lactation may improve postnatal growth and prevent adult diseases. Abstract Uteroplacental insufficiency reduces birth weight and adversely affects fetal organ development, increasing adult disease risk. Cross‐fostering improves postnatal nutrition and restores these deficits. Mothers with growth‐restricted pups have compromised milk production and composition; however, the impact cross‐fostering has on milk production and composition is unknown. Plasma leptin concentrations peak during the completion of organogenesis, which occurs postnatally in rats. Leptin is transferred to the fetus via the placenta and to the pup via the lactating mammary gland. This study investigated the effect of uteroplacental insufficiency on pup plasma leptin concentrations and placental leptin transporters. We additionally examined whether cross‐fostering improves mammary development, milk composition and pup plasma leptin concentrations. Fetal growth restriction was induced by bilateral uterine vessel ligation surgery on gestation day 18 in Wistar Kyoto rats (termed uteroplacental insufficiency surgery mothers). Growth‐restricted (Restricted) fetuses had reduced plasma leptin concentrations, persisting throughout lactation, and sex‐specific alterations in placental leptin transporters. Mothers suckled by Restricted pups had impaired mammary development, altered milk fatty acid composition and increased plasma leptin concentrations, despite no changes in milk leptin. Milk intake was reduced in Restricted pups suckling uteroplacental insufficiency surgery mothers compared to Restricted pups suckling sham‐operated mothers. Cross‐fostering Restricted pups onto a sham‐operated mother improved postnatal growth and restored plasma leptin concentrations compared to Restricted pups suckling uteroplacental insufficiency surgery mothers. Uteroplacental insufficiency alters leptin homeostasis. This is ameliorated with cross‐fostering and enhanced milk fatty acid composition and consumption, which may protect the pups from developing adverse health conditions in adulthood.
    March 29, 2017   doi: 10.1113/JP273825   open full text
  • A calcium‐dependent pathway underlies activity‐dependent plasticity of electrical synapses in the thalamic reticular nucleus.
    Jessica Sevetson, Sarah Fittro, Emily Heckman, Julie S. Haas.
    The Journal of Physiology. March 29, 2017
    Recent results have demonstrated modification of electrical synapse strength by varied forms of neuronal activity. However, the mechanisms underlying plasticity induction in central mammalian neurons are unclear. Here we show that the two established inductors of plasticity at electrical synapses in the thalamic reticular nucleus – paired burst spiking in coupled neurons, and mGluR‐dependent tetanization of synaptic input – are separate pathways that converge at a common downstream endpoint. Using occlusion experiments and pharmacology in patched pairs of coupled neurons in vitro, we show that burst‐induced depression depends on calcium entry via voltage‐gated channels, is blocked by BAPTA chelation, and recruits intracellular calcium release on its way to activation of phosphatase activity. In contrast, mGluR‐dependent plasticity is independent of calcium entry or calcium dynamics. Together, these results show that the spiking‐initiated mechanisms underlying electrical synapse plasticity are similar to those that induce plasticity at chemical synapses, and offer the possibility that calcium‐regulated mechanisms may also lead to alternate outcomes, such as potentiation. Because these mechanistic elements are widely found in mature neurons, we expect them to apply broadly to electrical synapses across the brain, acting as the crucial link between neuronal activity and electrical synapse strength. This article is protected by copyright. All rights reserved
    March 29, 2017   doi: 10.1113/JP274049   open full text
  • Leg vascular and skeletal muscle mitochondrial adaptations to aerobic high‐intensity exercise training are enhanced in the early postmenopausal phase.
    Michael Nyberg, Jon Egelund, Camilla M. Mandrup, Caroline B. Andersen, Karen M. B. E. Hansen, Ida‐Marie F. Hergel, Nicholai Valbak‐Andersen, Ruth Frikke‐Schmidt, Bente Stallknecht, Jens Bangsbo, Ylva Hellsten.
    The Journal of Physiology. March 29, 2017
    Key points Exercise training effectively improves vascular and skeletal muscle function; however, these effects of training may be blunted in postmenopausal women as a result of the loss of oestrogens. Accordingly, the capacity to deliver oxygen to the active muscles may also be impaired in postmenopausal women. In both premenopausal and recent postmenopausal women, exercise training was shown to improve leg vascular and skeletal muscle mitochondrial function. Interestingly, these effects were more pronounced in postmenopausal women. Skeletal muscle oxygen supply and utilization were similar in the two groups of women. These findings suggest that the early postmenopausal phase is associated with an enhanced capacity of the leg vasculature and skeletal muscle mitochondria to adapt to exercise training and that the ability to deliver oxygen to match the demand of the active muscles is preserved in the early phase following the menopausal transition. Abstract Exercise training leads to favourable adaptations within skeletal muscle; however, this effect of exercise training may be blunted in postmenopausal women as a result of the loss of oestrogens. Furthermore, postmenopausal women may have an impaired vascular response to acute exercise. We examined the haemodynamic response to acute exercise in matched pre‐ and postmenopausal women before and after 12 weeks of aerobic high intensity exercise training. Twenty premenopausal and 16 early postmenopausal (mean ± SEM: 3.1 ± 0.5 years after final menstrual period) women only separated by 4 years of age (mean ± SEM: 50 ± 0 years vs. 54 ± 1 years) were included. Before training, leg blood flow, O2 delivery, O2 uptake and lactate release during knee‐extensor exercise were similar in pre‐ and postmenopausal women. Exercise training reduced (P < 0.05) leg blood flow, O2 delivery, O2 uptake, lactate release, blood pressure and heart rate during the same absolute workloads in postmenopausal women. These effects were not detected in premenopausal women. Quadriceps muscle protein contents of mitochondrial complex II, III and IV; endothelial nitric oxide synthase (eNOS); cyclooxygenase (COX)‐1; COX‐2; and oestrogen‐related receptor α (ERRα) were increased (P < 0.05) with training in postmenopausal women, whereas only the levels of mitochondrial complex V, eNOS and COX‐2 were increased (P < 0.05) in premenopausal women. These findings demonstrate that vascular and skeletal muscle mitochondrial adaptations to aerobic high intensity exercise training are more pronounced in recent post‐ compared to premenopausal women, possibly as an effect of enhanced ERRα signalling. Also, the hyperaemic response to acute exercise appears to be preserved in the early postmenopausal phase.
    March 29, 2017   doi: 10.1113/JP273871   open full text
  • The role played by oxidative stress in evoking the exercise pressor reflex in health and simulated peripheral artery disease.
    Jonathan E. Harms, J. Matthew Kuczmarski, Joyce Kim, Gail D. Thomas, Marc P. Kaufman.
    The Journal of Physiology. March 28, 2017
    Contraction of muscle evokes the exercise pressor reflex (EPR), which is expressed partly by increases in heart rate and arterial pressure. Patients with peripheral artery disease (PAD) show an exaggerated EPR, sometimes report pain when walking and are at risk for cardiac arrthymias. Previous research suggested that reactive oxygen species (ROS) mediate the exaggerated EPR associated with PAD. To examine the effects of ROS on the EPR, we infused a superoxide scavenger, tiron, into the superficial epigastric artery of decerebrated rats. In some, we simulated PAD by ligating a femoral artery for 72 h before the experiment. The peak EPR in “ligated” rats during saline infusion averaged 31 ± 4 mmHg, whereas the peak EPR in these rats during tiron infusion averaged 13 ± 2 mmHg (n = 12; P < 0.001); the attenuating effect of tiron on the EPR was partly reversed when saline was reinfused into the superficial epigastric artery (21 ± 2; P < 0.01 vs tiron). The peak EPR in “ligated” rats was also attenuated (n = 7; P < 0.01) by infusion of gp91ds‐tat, a peptide which blocks the activity of NAD(P)H oxidase. Tiron infusion had no effect on the EPR in rats with patent femoral arteries (n = 9). Western blots showed that the triceps surae muscles of “ligated” rats expressed more Nox2 and p67phox, which are components of NADPH oxidase, than did triceps surae muscles of “freely perfused” rats. Tiron added to muscle homogenates reduced ROS production in vitro. Our results provide further evidence that ROS mediates the exaggeration of EPR in rats with simulated PAD. This article is protected by copyright. All rights reserved
    March 28, 2017   doi: 10.1113/JP273816   open full text
  • Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.
    Daniel C. Bartos, Stefano Morotti, Kenneth S. Ginsburg, Eleonora Grandi, Donald M. Bers.
    The Journal of Physiology. March 28, 2017
    Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i. When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Abstract The slowly activating delayed rectifier K+ current (IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+]i dependence of IKs in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in IKs as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail IKs, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent IKs activation during normal Ca2+ transients. [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i. When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Membrane topology of a Kv7.1 α‐subunit and regulatory proteins.
    March 28, 2017   doi: 10.1113/JP273676   open full text
  • Nutritional status‐dependent endocannabinoid signalling regulates the integration of rat visceral information.
    Abdessattar Khlaifia, Isabelle Matias, Daniela Cota, Fabien Tell.
    The Journal of Physiology. March 27, 2017
    Key points Vagal sensory inputs transmit information from the viscera to brainstem neurones located in the nucleus tractus solitarii to set physiological parameters. These excitatory synapses exhibit a CB1 endocannabinoid‐induced long‐term depression (LTD) triggered by vagal fibre stimulation. We investigated the impact of nutritional status on long‐term changes in this long‐term synaptic plasticity. Food deprivation prevents LTD induction by disrupting CB1 receptor signalling. Short‐term refeeding restores the capacity of vagal synapses to express LTD. Ghrelin and cholecystokinin, respectively released during fasting and refeeding, play a key role in the control of LTD via the activation of energy sensing pathways such as AMPK and the mTOR and ERK pathways. Abstract Communication form the viscera to the brain is essential to set physiological homoeostatic parameters but also to drive more complex behaviours such as mood, memory and emotional states. Here we investigated the impact of the nutritional status on long‐term changes in excitatory synaptic transmission in the nucleus tractus solitarii, a neural hub integrating visceral signals. These excitatory synapses exhibit a CB1 endocannabinoid (eCB)‐induced long‐term depression (LTD) triggered by vagal fibre stimulation. Since eCB signalling is known to be an important component of homoeostatic regulation of the body and is regulated during various stressful conditions, we tested the hypothesis that food deprivation alters eCB signalling in central visceral afferent fibres. Food deprivation prevents eCB‐LTD induction due to the absence of eCB signalling. This loss was reversed by blockade of ghrelin receptors. Activation of the cellular fuel sensor AMP‐activated protein kinase or inhibition of the mechanistic target of rapamycin pathway abolished eCB‐LTD in free‐fed rats. Signals associated with energy surfeit, such as short‐term refeeding, restore eCB‐LTD induction, which in turn requires activation of cholecystokinin receptors and the extracellular signal‐regulated kinase pathway. These data suggest a tight link between eCB‐LTD in the NTS and nutritional status and shed light on the key role of eCB in the integration of visceral information.
    March 27, 2017   doi: 10.1113/JP273484   open full text
  • Article update.

    The Journal of Physiology. March 26, 2017
    There is no abstract available for this paper.
    March 26, 2017   doi: 10.1113/JP273893   open full text
  • Endothelin‐1 mediates natriuresis but not polyuria during vitamin D‐induced acute hypercalcaemia.
    Natsuko Tokonami, Lydie Cheval, Isabelle Monnay, Guillaume Meurice, Johannes Loffing, Eric Feraille, Pascal Houillier.
    The Journal of Physiology. March 23, 2017
    Key points Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism. A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)‐1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia‐induced effects. In the present study, we demonstrate that, during vitamin D‐induced hypercalcaemia, the activation of ET system by increased ET‐1 is responsible for natriuresis but not for polyuria. Vitamin D‐treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited. We have identified an original pathway that specifically mediates the effects of vitamin D‐induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. Abstract Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D‐induced hypercalcaemia increases the renal expression of endothelin (ET)‐1, we hypothesized that ET‐1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8‐week‐old, parathyroid hormone‐supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT‐treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET‐1 and the transcription factors CCAAT‐enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT‐treated mice. To examine the role of the ET system in hypercalcaemia‐induced natriuresis and polyuria, mice were treated with the ET‐1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT‐treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT‐treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D‐induced hypercalcaemia increases the renal production of ET‐1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria.
    March 23, 2017   doi: 10.1113/JP273610   open full text
  • Prolactin regulation of oxytocin neurone activity in pregnancy and lactation.
    Rachael A. Augustine, Sharon R. Ladyman, Gregory T. Bouwer, Yousif Alyousif, Tony J. Sapsford, Victoria Scott, Ilona C. Kokay, David R. Grattan, Colin H. Brown.
    The Journal of Physiology. March 23, 2017
    Key points During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin‐secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation. Abstract Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane‐anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s−1, respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s−1 in lactating rats but excited none in virgin or pregnant rats (χ22 = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 μg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s−1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect phosphorylation of extracellular regulated kinase 1 or 2, or of Akt in the supraoptic or paraventricular nuclei of virgin or lactating rats. Hence, prolactin inhibition of oxytocin neurones is lost in lactation, which might allow concurrent elevation of prolactin secretion from the pituitary gland and activation of oxytocin neurones for synthesis and delivery of milk to the newborn.
    March 23, 2017   doi: 10.1113/JP273712   open full text
  • Frequency and function in the basal ganglia: the origins of beta and gamma band activity.
    Alexander Blenkinsop, Sean Anderson, Kevin Gurney.
    The Journal of Physiology. March 23, 2017
    Neural oscillations in the basal ganglia are well studied yet remain poorly understood. Behavioural correlates of spectral activity are well described, yet a quantitative hypothesis linking time domain dynamics and spectral properties to basal ganglia function has been lacking. We show, for the first time, that a unified description is possible by interpreting previously ignored structure in data describing GPi responses to cortical stimulation. These data were used to expose a pair of distinctive neuronal responses to the stimulation. This observation formed the basis for a new mathematical model of the BG, quantitatively fitted to the data, which describes the dynamics in the data, and is validated against other stimulus protocol experiments. A key new result is that when the model is run using inputs hypothesised to occur during the performance of a motor task, beta and gamma frequency oscillations emerge naturally during static‐force and movement respectively, consistent with experimental local field potentials. This new model predicts that the pallido‐striatum connection has a key role in the generation of beta band activity, and that the gamma band activity associated with motor task performance has its origins in the pallido‐subthalamic feedback loop. The network's functionality as a selection‐mechanism also occurs as an emergent property, and closer fits to the data gave better selection properties. The model provides a coherent framework for the study of spectral, temporal and functional analyses of the BG and therefore lays the foundation for an integrated approach to study BG pathologies such as Parkinson's disease in silico. This article is protected by copyright. All rights reserved
    March 23, 2017   doi: 10.1113/JP273760   open full text
  • Protein kinase A regulates C‐terminally truncated CaV1.2 in Xenopus oocytes: roles of N‐ and C‐termini of the α1C subunit.
    Shimrit Oz, Ines Pankonien, Anouar Belkacemi, Veit Flockerzi, Enno Klussmann, Hannelore Haase, Nathan Dascal.
    The Journal of Physiology. March 23, 2017
    Key points β‐Adrenergic stimulation enhances Ca2+ entry via L‐type CaV1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV1.2 remain controversial despite extensive research. We show that PKA regulation of CaV1.2 can be reconstituted in Xenopus oocytes when the distal C‐terminus (dCT) of the main subunit, α1C, is truncated. The PKA upregulation of CaV1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase‐anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV1.2. The gating element within the initial segment of the N‐terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV1.2 in the heart. Abstract β‐Adrenergic stimulation enhances Ca2+ currents via L‐type, voltage‐gated CaV1.2 channels, strengthening cardiac contraction. The signalling via β‐adrenergic receptors (β‐ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post‐translational proteolytic truncation of the distal C‐terminus (dCT) of the main (α1C) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT‐truncated CaV1.2 in Xenopus oocytes, which previously failed with the non‐truncated α1C. cAMP and the purified catalytic subunit of PKA, PKA‐CS, injected into intact oocytes, enhanced CaV1.2 currents by ∼40% (rabbit α1C) to ∼130% (mouse α1C). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase‐anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaVβ2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long‐N‐terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV1.2 in the heart. We hypothesize that PKA is acting on CaV1.2, in part, by affecting a structural ‘scaffold’ comprising the interacting cytosolic N‐ and C‐termini of α1C.
    March 23, 2017   doi: 10.1113/JP274015   open full text
  • T‐type calcium channels contribute to NMDA receptor independent synaptic plasticity in hippocampal regular‐spiking oriens‐alveus interneurons.
    Elizabeth Nicholson, Dimitri M. Kullmann.
    The Journal of Physiology. March 22, 2017
    Key points Regular‐spiking interneurons in the hippocampal stratum oriens exhibit a form of long‐term potentiation of excitatory transmission that is independent of NMDA receptors but requires co‐activation of Ca2+‐permeable AMPA receptors and group I metabotropic glutamate receptors. We show that T‐type Ca2+ channels are present in such interneurons. Blockade of T‐type currents prevents the induction of long‐term potentiation, and also interferes with long‐lasting potentiation induced either by postsynaptic trains of action potentials or by pairing postsynaptic hyperpolarization with activation of group I metabotropic receptors. Several Ca2+ sources thus converge on the induction of NMDA receptor independent synaptic plasticity. Abstract NMDA receptor independent long‐term potentiation (LTP) in hippocampal stratum oriens‐alveus (O/A) interneurons requires co‐activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and Ca2+‐permeable AMPA receptors. The rectification properties of such AMPA receptors contribute to the preferential induction of LTP at hyperpolarized potentials. A persistent increase in excitatory transmission can also be triggered by exogenous activation of group I mGluRs at the same time as the interneuron is hyperpolarized, or by postsynaptic trains of action potentials in the absence of presynaptic stimulation. In the present study, we identify low‐threshold transient (T‐type) channels as a further source of Ca2+ that contributes to synaptic plasticity. T‐type Ca2+ currents were detected in mouse regular‐spiking O/A interneurons. Blocking T‐type currents pharmacologically prevented LTP induced by high‐frequency stimulation of glutamatergic axons, or by application of the group I mGluR agonist dihydroxyphenylglycine, paired with postsynaptic hyperpolarization. T‐type current blockade also prevented synaptic potentiation induced by postsynaptic action potential trains. Several sources of Ca2+ thus converge on NMDA receptor independent LTP induction in O/A interneurons.
    March 22, 2017   doi: 10.1113/JP273695   open full text
  • Voltage‐sensitive conductances increase the sensitivity of rod photoresponses following pigment bleaching.
    Johan Pahlberg, Rikard Frederiksen, Gabriel E. Pollock, Kiyoharu J. Miyagishima, Alapakkam P. Sampath, M. Carter Cornwall.
    The Journal of Physiology. March 22, 2017
    Key points Following substantial bleaching of the visual pigment, the desensitization of the rod photovoltage is not as substantial as the desensitization of the rod outer segment photocurrent. The block of cation conductances during the internal dialysis of Cs+ further desensitizes the photovoltage thereby eliminating its difference in desensitization with the rod outer segment photocurrent. Bleached visual pigment produced an acceleration of the rod photovoltage with respect to the outer segment photocurrent, which is eliminated upon internal dialysis of Cs+. Abstract A majority of our visual experience occurs during the day when a substantial fraction of the visual pigment in our photoreceptor cells is bleached. Under these conditions it is widely believed that rods are saturated and do not contribute substantially to downstream signalling. However, behavioural experiments on subjects with only rod function reveals that these individuals unexpectedly retain substantial vision in daylight. We sought to understand this discrepancy by characterizing the sensitivity of rod photoresponses following exposure to bright bleaching light. Measurements of the rod outer segment photocurrent in transgenic mice, which have only rod function, revealed the well‐studied reduction in the sensitivity of rod photoresponses following pigment bleaching. However, membrane voltage measurements showed that the desensitization of the photovoltage was considerably less than that of the outer segment photocurrent following equivalent pigment bleaching. This discrepancy was largely eliminated during the blockade of cation channels due to the internal dialysis of Cs+, which increased the bleach‐induced desensitization of the photovoltage and slowed its temporal characteristics. Thus, sensitization of the photovoltage by rod inner segment conductances appears to extend the operating range of rod phototransduction following pigment bleaching.
    March 22, 2017   doi: 10.1113/JP273398   open full text
  • Diuretic‐sensitive electroneutral Na+ movement and temperature effects on central axons.
    Meneka Kanagaratnam, Christopher Pendleton, Danilo Almeida Souza, Joseph Pettit, James Howells, Mark D. Baker.
    The Journal of Physiology. March 22, 2017
    Key points Optic nerve axons get less excitable with warming. F‐fibre latency does not shorten at temperatures above 30°C. Action potential amplitude falls when the Na+‐pump is blocked, an effect speeded by warming. Diuretics reduce the rate of action potential fall in the presence of ouabain. Our data are consistent with electroneutral entry of Na+ occurring in axons and contributing to setting the resting potential. Abstract Raising the temperature of optic nerve from room temperature to near physiological has effects on the threshold, refractoriness and superexcitability of the shortest latency (fast, F) nerve fibres, consistent with hyperpolarization. The temperature dependence of peak impulse latency was weakened at temperatures above 30°C suggesting a temperature‐sensitive process that slows impulse propagation. The amplitude of the supramaximal compound action potential gets larger on warming, whereas in the presence of bumetanide and amiloride (blockers of electroneutral Na+ movement), the action potential amplitude consistently falls. This suggests a warming‐induced hyperpolarization that is reduced by blocking electroneutral Na+ movement. In the presence of ouabain, the action potential collapses. This collapse is speeded by warming, and exposure to bumetanide and amiloride slows the temperature‐dependent amplitude decline, consistent with a warming‐induced increase in electroneutral Na+ entry. Blocking electroneutral Na+ movement is predicted to be useful in the treatment of temperature‐dependent symptoms under conditions with reduced safety factor (Uhthoff's phenomenon) and provide a route to neuroprotection.
    March 22, 2017   doi: 10.1113/JP273963   open full text
  • Lysophosphatidic acid‐induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.
    Hiroki Kittaka, Kunitoshi Uchida, Naomi Fukuta, Makoto Tominaga.
    The Journal of Physiology. March 22, 2017
    Key points Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA‐induced itch‐related behaviours are decreased in TRPA1‐knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+‐independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672–673 and KR977–978 (K: lysine, R: arginine). Abstract Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch‐related behaviours but not pain‐related behaviours. The LPA‐induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA‐induced itch is mediated by LPA5, PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.
    March 22, 2017   doi: 10.1113/JP273961   open full text
  • Requirement of extracellular Ca2+ binding to specific amino acids for heat‐evoked activation of TRPA1.
    Erkin Kurganov, Shigeru Saito, Claire Tanaka Saito, Makoto Tominaga.
    The Journal of Physiology. March 22, 2017
    Key points We found that extracellular Ca2+, but not other divalent cations (Mg2+ and Ba2+) or intracellular Ca2+, is involved in heat‐evoked activation of green anole (ga) TRPA1. Heat‐evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+. Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat‐evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. Abstract Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non‐selective cation‐permeable channel that has six transmembrane domains and cytoplasmic N‐ and C‐termini. The N‐terminus is characterized by an unusually large number of ankyrin repeats. Although the 3‐dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature‐dependent TRPA1 activation is unclear. We previously reported that extracellular Ca2+, but not intracellular Ca2+, plays an important role in heat‐evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+‐dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat‐activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca2+, rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat‐evoked TRPA1 activation in the presence of extracellular Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat‐evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat‐evoked channel activation.
    March 22, 2017   doi: 10.1113/JP274083   open full text
  • Cardiac diastolic and autonomic dysfunction are aggravated by central chemoreflex activation in heart failure with preserved ejection fraction rats.
    Camilo Toledo, David C. Andrade, Claudia Lucero, Alexis Arce‐Alvarez, Hugo S. Díaz, Valentín Aliaga, Harold D. Schultz, Noah J. Marcus, Mónica Manríquez, Marcelo Faúndez, Rodrigo Del Rio.
    The Journal of Physiology. March 19, 2017
    Key points Heart failure with preserved ejection fraction (HFpEF) is associated with disordered breathing patterns, and sympatho‐vagal imbalance. Although it is well accepted that altered peripheral chemoreflex control plays a role in the progression of heart failure with reduced ejection fraction (HFrEF), the pathophysiological mechanisms underlying deterioration of cardiac function in HFpEF are poorly understood. We found that central chemoreflex is enhanced in HFpEF and neuronal activation is increased in pre‐sympathetic regions of the brainstem. Our data showed that activation of the central chemoreflex pathway in HFpEF exacerbates diastolic dysfunction, worsens sympatho‐vagal imbalance and markedly increases the incidence of cardiac arrhythmias in rats with HFpEF. Abstract Heart failure (HF) patients with preserved ejection fraction (HFpEF) display irregular breathing, sympatho‐vagal imbalance, arrhythmias and diastolic dysfunction. It has been shown that tonic activation of the central and peripheral chemoreflex pathway plays a pivotal role in the pathophysiology of HF with reduced ejection fraction. In contrast, no studies to date have addressed chemoreflex function or its effect on cardiac function in HFpEF. Therefore, we tested whether peripheral and central chemoreflexes are hyperactive in HFpEF and if chemoreflex activation exacerbates cardiac dysfunction and autonomic imbalance. Sprague‐Dawley rats (n = 32) were subjected to sham or volume overload to induce HFpEF. Resting breathing variability, chemoreflex gain, cardiac function and sympatho‐vagal balance, and arrhythmia incidence were studied. HFpEF rats displayed [mean ± SD; chronic heart failure (CHF) vs. Sham, respectively] a marked increase in the incidence of apnoeas/hypopnoeas (20.2 ± 4.0 vs. 9.7 ± 2.6 events h−1), autonomic imbalance [0.6 ± 0.2 vs. 0.2 ± 0.1 low/high frequency heart rate variability (LF/HFHRV)] and cardiac arrhythmias (196.0 ± 239.9 vs. 19.8 ± 21.7 events h−1). Furthermore, HFpEF rats showed increase central chemoreflex sensitivity but not peripheral chemosensitivity. Accordingly, hypercapnic stimulation in HFpEF rats exacerbated increases in sympathetic outflow to the heart (229.6 ± 43.2% vs. 296.0 ± 43.9% LF/HFHRV, normoxia vs. hypercapnia, respectively), incidence of cardiac arrhythmias (196.0 ± 239.9 vs. 576.7 ± 472.9 events h−1) and diastolic dysfunction (0.008 ± 0.004 vs. 0.027 ± 0.027 mmHg μl−1). Importantly, the cardiovascular consequences of central chemoreflex activation were related to sympathoexcitation since these effects were abolished by propranolol. The present results show that the central chemoreflex is enhanced in HFpEF and that acute activation of central chemoreceptors leads to increases of cardiac sympathetic outflow, cardiac arrhythmogenesis and impairment in cardiac function in rats with HFpEF.
    March 19, 2017   doi: 10.1113/JP273558   open full text
  • The influence of adrenergic stimulation on sex differences in left ventricular twist mechanics.
    Alexandra M. Williams, Rob E. Shave, William S. Cheyne, Neil D. Eves.
    The Journal of Physiology. March 19, 2017
    Key points Sex differences in left ventricular (LV) mechanics occur during acute physiological challenges; however, it is unknown whether sex differences in LV mechanics are fundamentally regulated by differences in adrenergic control. Using two‐dimensional echocardiography and speckle tracking analysis, this study compared LV mechanics in males and females matched for LV length during post‐exercise ischaemia (PEI) and β1‐adrenergic receptor blockade. Our data demonstrate that while basal rotation was increased in males, LV twist was not significantly different between the sexes during PEI. In contrast, during β1‐adrenergic receptor blockade, LV apical rotation, twist and untwisting velocity were reduced in males compared to females. Significant relationships were observed between LV twist and LV internal diameter and sphericity index in females, but not males. These findings suggest that LV twist mechanics may be more sensitive to alterations in adrenergic stimulation in males, but more highly influenced by ventricular structure and geometry in females. Abstract Sex differences in left ventricular (LV) mechanics exist at rest and during acute physiological stress. Differences in cardiac autonomic and adrenergic control may contribute to sex differences in LV mechanics and LV haemodynamics. Accordingly, this study aimed to investigate sex differences in LV mechanics with altered adrenergic stimulation achieved through post‐handgrip‐exercise ischaemia (PEI) and β1‐adrenergic receptor (AR) blockade. Twenty males (23 ± 5 years) and 20 females (22 ± 3 years) were specifically matched for LV length (males: 8.5 ± 0.5 cm, females: 8.2 ± 0.6 cm, P = 0.163), and two‐dimensional speckle‐tracking echocardiography was used to assess LV structure and function at baseline, during PEI and following administration of 5 mg bisoprolol (β1‐AR antagonist). During PEI, LV end‐diastolic volume and stroke volume were increased in both groups (P < 0.001), as was end‐systolic wall stress (P < 0.001). LV twist and apical rotation were not altered from baseline or different between the sexes; however, basal rotation increased in males (P = 0.035). During β1‐AR blockade, LV volumes were unchanged but blood pressure and heart rate were reduced in both groups (P < 0.001). LV apical rotation (P = 0.036) and twist (P = 0.029) were reduced in males with β1‐AR blockade but not females, resulting in lower apical rotation (males: 6.8 ± 2.1 deg, females: 8.8 ± 2.3 deg, P = 0.007) and twist (males: 8.6 ± 1.9 deg, females: 10.7 ± 2.8 deg, P = 0.008), and slower untwisting velocity (males: 68.2 ± 22.1 deg s−1, females: 82.0 ± 18.7 deg s−1, P = 0.046) compared to females. LV twist mechanics are reduced in males compared to females during reductions to adrenergic stimulation, providing preliminary evidence that LV twist mechanics may be more sensitive to adrenergic control in males than in females.
    March 19, 2017   doi: 10.1113/JP273368   open full text
  • Mechanisms of pruritogen‐induced activation of itch nerves in isolated mouse skin.
    F. Ru, H. Sun, D. Jurcakova, R. A. Herbstsomer, J. Meixong, X. Dong, B. J. Undem.
    The Journal of Physiology. March 19, 2017
    Key points Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G‐protein coupled receptor (GPCR) MrgprA3; it is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that transient receptor potential A1 (TRPA1) gene deletion blocks CQ‐induced scratching. In the present study we used a novel dorsal skin–nerve preparation to evaluate mechanisms underlying CQ‐ and histamine‐induced action potential discharge in itch nerve terminals. We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, but TRPA1 or other TRP channel are not required. Evidence is provided for a role for calcium‐activated chloride channels such as TMEM16a in GPCR‐activation of itch nerve terminals. The mechanism by which TRP channels participate in pruritogen‐induced scratching may involve sites of action other than the primary afferent terminals. Abstract Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin–nerve preparation was used to evaluate chloroquine (CQ)‐ and histamine‐induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C‐fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr‐cluster Δ−/− mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ‐induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild‐type and TRPA1‐deficient mice. A selective inhibitor of the calcium‐activated chloride channel TMEM16A, N‐((4‐methoxy)‐2‐naphthyl)‐5‐nitroanthranilic acid (MONNA), inhibited CQ‐induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role.
    March 19, 2017   doi: 10.1113/JP273795   open full text
  • Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short‐term facilitation at mossy fibre to CA3 pyramidal cell synapses.
    Sam A. Booker, Graham R. Campbell, Karolina S. Mysiak, Peter J. Brophy, Peter C. Kind, Don J. Mahad, David J. A. Wyllie.
    The Journal of Physiology. March 15, 2017
    Key points Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission. High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice. Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function. Abstract Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole‐cell patch‐clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV‐deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy‐fibre synapse because the amplitude, input–output relationship and 50 ms paired‐pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short‐term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired‐pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy‐fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short‐term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction.
    March 15, 2017   doi: 10.1113/JP273581   open full text
  • The role of dentate nuclei in human oculomotor control: insights from cerebrotendinous xanthomatosis.
    Francesca Rosini, Elena Pretegiani, Andrea Mignarri, Lance M. Optican, Valeria Serchi, Nicola Stefano, Marco Battaglini, Lucia Monti, Maria T. Dotti, Antonio Federico, Alessandra Rufa.
    The Journal of Physiology. March 14, 2017
    Key points A cerebellar dentate nuclei (DN) contribution to volitional oculomotor control has recently been hypothesized but not fully understood. Cerebrotendinous xanthomatosis (CTX) is a rare neurometabolic disease typically characterized by DN damage. In this study, we compared the ocular movement characteristics of two sets of CTX patients, with and without brain MRI evidence of DN involvement, with a set of healthy subjects. Our results suggest that DN participate in voluntary behaviour, such as the execution of antisaccades, and moreover are involved in controlling the precision of the ocular movement. The saccadic abnormalities related to DN involvement were independent of global and regional brain atrophy. Our study confirms the relevant role of DN in voluntary aspects of oculomotion and delineates specific saccadic abnormalities that could be used to detect the involvement of DN in other cerebellar disorders. Abstract It is well known that the medial cerebellum controls saccadic speed and accuracy. In contrast, the role of the lateral cerebellum (cerebellar hemispheres and dentate nuclei, DN) is less well understood. Cerebrotendinous xanthomatosis (CTX) is a lipid storage disorder due to mutations in CYP27A1, typically characterized by DN damage. CTX thus provides a unique opportunity to study DN in human oculomotor control. We analysed horizontal and vertical visually guided saccades and horizontal antisaccades of 19 CTX patients. Results were related to the presence/absence of DN involvement and compared with those of healthy subjects. To evaluate the contribution of other areas, abnormal saccadic parameters were compared with global and regional brain volumes. CTX patients executed normally accurate saccades with normal main sequence relationships, indicating that the brainstem and medial cerebellar structures were functionally spared. Patients with CTX executed more frequent multistep saccades and directional errors during the antisaccade task than controls. CTX patients with DN damage showed less precise saccades with longer latencies, and more frequent directional errors, usually not followed by corrections, than either controls or patients without DN involvement. These saccadic abnormalities related to DN involvement but were independent of global and regional brain atrophy. We hypothesize that two different cerebellar networks contribute to the metrics of a movement: the medial cerebellar structures determine accuracy, whereas the lateral cerebellar structures control precision. The lateral cerebellum (hemispheres and DN) also participates in modulating goal directed gaze behaviour, by prioritizing volitional over reflexive movements.
    March 14, 2017   doi: 10.1113/JP273670   open full text
  • Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel.
    Etsuko Minobe, Masayuki X. Mori, Masaki Kameyama.
    The Journal of Physiology. March 13, 2017
    Key points Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent. Abstract Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed.
    March 13, 2017   doi: 10.1113/JP273736   open full text
  • Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation.
    Shelley E. Harris, Miles J. Blasio, Melissa A. Davis, Amy C. Kelly, Hailey M. Davenport, F. B. Peter Wooding, Dominique Blache, David Meredith, Miranda Anderson, Abigail L. Fowden, Sean W. Limesand, Alison J. Forhead.
    The Journal of Physiology. March 13, 2017
    Key points Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose‐dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas. Abstract Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long‐term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T3), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic islets isolated from intact fetal sheep, beta cell proliferation in vitro was reduced by T3 in a dose‐dependent manner and increased by insulin at high concentrations only. Leptin induced a bimodal response whereby beta cell proliferation was suppressed at the lowest, and increased at the highest, concentrations. Therefore, proliferation of beta cells isolated from the ovine fetal pancreas is sensitive to physiological concentrations of T3, insulin and leptin. Alterations in these hormones may be responsible for the increased beta cell proliferation and mass observed in the hypothyroid sheep fetus and may have consequences for pancreatic function in later life.
    March 13, 2017   doi: 10.1113/JP273555   open full text
  • KATP channel inhibition blunts electromechanical decline during hypoxia in left ventricular working rabbit hearts.
    Kara Garrott, Sarah Kuzmiak‐Glancy, Anastasia Wengrowski, Hanyu Zhang, Jack Rogers, Matthew W. Kay.
    The Journal of Physiology. March 13, 2017
    Key points Heart function is critically dependent upon the balance of energy production and utilization. Sarcolemmal ATP‐sensitive potassium channels (KATP channels) in cardiac myocytes adjust contractile function to compensate for the level of available energy. Understanding the activation of KATP channels in working myocardium during high‐stress situations is crucial to the treatment of cardiovascular disease, especially ischaemic heart disease. Using a new optical mapping approach, we measured action potentials from the surface of excised contracting rabbit hearts to assess when sarcolemmal KATP channels were activated during physiologically relevant workloads and during gradual reductions in myocardial oxygenation. We demonstrate that left ventricular pressure is closely linked to KATP channel activation and that KATP channel inhibition with a low concentration of tolbutamide prevents electromechanical decline when oxygen availability is reduced. As a result, KATP channel inhibition probably exacerbates a mismatch between energy demand and energy production when myocardial oxygenation is low. Abstract Sarcolemmal ATP‐sensitive potassium channel (KATP channel) activation in isolated cells is generally understood, although the relationship between myocardial oxygenation and KATP activation in excised working rabbit hearts remains unknown. We optically mapped action potentials (APs) in excised rabbit hearts to test the hypothesis that hypoxic changes would be more severe in left ventricular (LV) working hearts (LWHs) than Langendorff (LANG) perfused hearts. We further hypothesized that KATP inhibition would prevent those changes. Optical APs were mapped when measuring LV developed pressure (LVDP), coronary flow rate and oxygen consumption in LANG and LWHs. Hearts were paced to increase workload and perfusate was deoxygenated to study the effects of myocardial hypoxia. A subset of hearts was perfused with 1 μm tolbutamide (TOLB) to identify the level of AP duration (APD) shortening attributed to KATP channel activation. During sinus rhythm, APD was shorter in LWHs compared to LANG hearts. APD in both LWHs and LANG hearts dropped steadily during deoxygenation. With TOLB, APDs in LWHs were longer at all workloads and APD reductions during deoxygenation were blunted in both LWHs and LANG hearts. At 50% perfusate oxygenation, APD and LVDP were significantly higher in LWHs perfused with TOLB (199 ± 16 ms; 92 ± 5.3 mmHg) than in LWHs without TOLB (109 ± 14 ms, P = 0.005; 65 ± 6.5 mmHg, P = 0.01). Our results indicate that KATP channels are activated to a greater extent in perfused hearts when the LV performs pressure–volume work. The results of the present study demonstrate the critical role of KATP channels in modulating myocardial function over a wide range of physiological conditions.
    March 13, 2017   doi: 10.1113/JP273873   open full text
  • Relationship between cortical state and spiking activity in the lateral geniculate nucleus of marmosets.
    Alexander N.J. Pietersen, Soon Keen Cheong, Brandon Munn, Pulin Gong, Paul R. Martin, Samuel G. Solomon.
    The Journal of Physiology. March 10, 2017
    Key points How parallel are the primate visual pathways? In the present study, we demonstrate that parallel visual pathways in the dorsal lateral geniculate nucleus (LGN) show distinct patterns of interaction with rhythmic activity in the primary visual cortex (V1). In the V1 of anaesthetized marmosets, the EEG frequency spectrum undergoes transient changes that are characterized by fluctuations in delta‐band EEG power. We show that, on multisecond timescales, spiking activity in an evolutionary primitive (koniocellular) LGN pathway is specifically linked to these slow EEG spectrum changes. By contrast, on subsecond (delta frequency) timescales, cortical oscillations can entrain spiking activity throughout the entire LGN. Our results are consistent with the hypothesis that, in waking animals, the koniocellular pathway selectively participates in brain circuits controlling vigilance and attention. Abstract The major afferent cortical pathway in the visual system passes through the dorsal lateral geniculate nucleus (LGN), where nerve signals originating in the eye can first interact with brain circuits regulating visual processing, vigilance and attention. In the present study, we investigated how ongoing and visually driven activity in magnocellular (M), parvocellular (P) and koniocellular (K) layers of the LGN are related to cortical state. We recorded extracellular spiking activity in the LGN simultaneously with local field potentials (LFP) in primary visual cortex, in sufentanil‐anaesthetized marmoset monkeys. We found that asynchronous cortical states (marked by low power in delta‐band LFPs) are linked to high spike rates in K cells (but not P cells or M cells), on multisecond timescales. Cortical asynchrony precedes the increases in K cell spike rates by 1–3 s, implying causality. At subsecond timescales, the spiking activity in many cells of all (M, P and K) classes is phase‐locked to delta waves in the cortical LFP, and more cells are phase‐locked during synchronous cortical states than during asynchronous cortical states. The switch from low‐to‐high spike rates in K cells does not degrade their visual signalling capacity. By contrast, during asynchronous cortical states, the fidelity of visual signals transmitted by K cells is improved, probably because K cell responses become less rectified. Overall, the data show that slow fluctuations in cortical state are selectively linked to K pathway spiking activity, whereas delta‐frequency cortical oscillations entrain spiking activity throughout the entire LGN, in anaesthetized marmosets.
    March 10, 2017   doi: 10.1113/JP273569   open full text
  • The TRPM7 channel kinase regulates store‐operated calcium entry.
    Malika Faouzi, Tatiana Kilch, F. David Horgen, Andrea Fleig, Reinhold Penner.
    The Journal of Physiology. March 10, 2017
    Key points Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store‐operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7−/− cells restores SOCE. TRPM7 is not a store‐operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. Abstract The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store‐operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7‐deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild‐type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell‐cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild‐type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store‐operated channel itself. Using kinase‐deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.
    March 10, 2017   doi: 10.1113/JP274006   open full text
  • Perinatal nicotine exposure impairs the maturation of glutamatergic inputs in the auditory brainstem.
    Veronika J. Baumann, Ursula Koch.
    The Journal of Physiology. March 10, 2017
    Key points Chronic perinatal nicotine exposure causes abnormal auditory brainstem responses and auditory processing deficits in children and animal models. The effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem was investigated in granule cells in the ventral nucleus of the lateral lemniscus, which receive a single calyx‐like input from the cochlear nucleus. Perinatal nicotine exposure caused a massive reduction in the amplitude of the excitatory input current. This caused a profound decrease in the number and temporal precision of spikes in these neurons. Perinatal nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons. Abstract Maternal smoking causes chronic nicotine exposure during early development and results in auditory processing deficits including delayed speech development and learning difficulties. Using a mouse model of chronic, perinatal nicotine exposure we explored to what extent synaptic inputs to granule cells in the ventral nucleus of the lateral lemniscus are affected by developmental nicotine treatment. These neurons receive one large calyx‐like input from octopus cells in the cochlear nucleus and play a role in sound pattern analysis, including speech sounds. In addition, they exhibit high levels of α7 nicotinic acetylcholine receptors, especially during early development. Our whole‐cell patch‐clamp experiments show that perinatal nicotine exposure causes a profound reduction in synaptic input amplitude. In contrast, the number of inputs innervating each neuron and synaptic release properties of this calyx‐like synapse remained unaltered. Spike number and spiking precision in response to synaptic stimulation were greatly diminished, especially for later stimuli during a stimulus train. Moreover, chronic nicotine exposure delayed the developmental downregulation of functional nicotinic acetylcholine receptors on these neurons, indicating a direct action of nicotine in this brain area. This presumably direct effect of perinatal nicotine exposure on synaptic maturation in the auditory brainstem might be one of the underlying causes for auditory processing difficulties in children of heavy smoking mothers.
    March 10, 2017   doi: 10.1113/JP274059   open full text
  • Exploratory assessment of left ventricular strain–volume loops in severe aortic valve diseases.
    Hugo G. Hulshof, Arie P. Dijk, Keith P. George, Maria T. E. Hopman, Dick H. J. Thijssen, David L. Oxborough.
    The Journal of Physiology. March 09, 2017
    Key points Severe aortic valve diseases are common cardiac abnormalities that are associated with poor long‐term survival. Before any reduction in left ventricular (LV) function, the left ventricle undergoes structural remodelling under the influence of changing haemodynamic conditions. In this study, we combined temporal changes in LV structure (volume) with alterations in LV functional characteristics (strain, ԑ) into a ԑ–volume loop, in order to provide novel insight into the haemodynamic cardiac consequences of aortic valve diseases in those with preserved LV ejection fraction. We showed that our novel ԑ–volume loop and the specific loop characteristics provide additional insight into the functional and mechanical haemodynamic consequences of severe aortic valve diseases (with preserved LV ejection fraction). Finally, we showed that the ԑ–volume loop characteristics provide discriminative capacity compared with conventional measures of LV function. Abstract The purpose of this study was to examine left ventricular (LV) strain (ԑ)–volume loops to provide novel insight into the haemodynamic cardiac consequences of aortic valve stenosis (AS) and aortic valve regurgitation (AR). Twenty‐seven participants were retrospectively recruited: AR (n = 7), AS (n = 10) and control subjects (n = 10). Standard transthoracic echocardiography was used to obtain apical four‐chamber images to construct ԑ–volume relationships, which were assessed using the following parameters: early systolic ԑ (ԑ_ES); slope of ԑ–volume relationship during systole (Sslope); end‐systolic peak ԑ (peak ԑ); and diastolic uncoupling (systolic ԑ–diastolic ԑ at same volume) during early diastole (UNCOUP_ED) and late diastole (UNCOUP_LD). Receiver operating characteristic curves were used to determine the ability to detect impaired LV function. Although LV ejection fraction was comparable between groups, longitudinal peak ԑ was reduced compared with control subjects. In contrast, ԑ_ES and Sslope were lower in both pathologies compared with control subejcts (P < 0.01), but also different between AS and AR (P < 0.05). UNCOUP_ED and UNCOUP_LD were significantly higher in both patient groups compared with control subjects (P < 0.05). Receiver operating characteristic curves revealed that loop characteristics (AUC = 0.99, 1.00 and 1.00; all P < 0.01) were better able then peak ԑ (AUC = 0.75, 0.89 and 0.76; P = 0.06, <0.01 and 0.08, respectively) and LV ejection fraction (AUC = 0.56, 0.69 and 0.69; all P > 0.05) to distinguish AS vs control, AR vs control and AS vs AR groups, respectively. Temporal changes in ԑ–volume characteristics provide novel insight into the haemodynamic cardiac impact of AS and AR. Contrary to traditional measures (i.e. ejection fraction, peak ԑ), these novel measures successfully distinguish between the haemodynamic cardiac impact of AS and AR.
    March 09, 2017   doi: 10.1113/JP273526   open full text
  • Low pHo boosts burst firing and catecholamine release by blocking TASK‐1 and BK channels while preserving Cav1 channels in mouse chromaffin cells.
    Laura Guarina, David H. F. Vandael, Valentina Carabelli, Emilio Carbone.
    The Journal of Physiology. March 02, 2017
    Key points Mouse chromaffin cells (MCCs) generate spontaneous burst‐firing that causes large increases of Ca2+‐dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs. With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo) from 7.4 to 6.6 induces cell depolarizations of 10–15 mV that generate bursts of ∼330 ms at 1–2 Hz and a 7.4‐fold increase of cumulative catecholamine‐release. Burst‐firing originates from the inhibition of the pH‐sensitive TASK‐1‐channels and a 60% reduction of BK‐channel conductance at pHo 6.6. Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine. MCCs act as pH‐sensors. At low pHo, they depolarize, undergo burst‐firing and increase catecholamine‐secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue. Abstract Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+‐dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common ‘tonic‐irregular’ to the less frequent ‘burst’ firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1β2‐subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+‐entry and potentiates CA release by ∼3.5‐fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst‐firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10–15 mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330 ms, occurred at 1–2 Hz and caused an ∼7‐fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH‐sensitive TASK‐1/TASK‐3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK‐1 blocker A1899 (300 nm) and BK blocker paxilline (300 nm) and could be prevented by blocking L‐type channels by adding 3 μm nifedipine. Mixtures of the two blockers raised cumulative CA‐secretion even more than low pHo (∼12‐fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+‐entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA‐secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.
    March 02, 2017   doi: 10.1113/JP273735   open full text
  • Calcium‐calmodulin‐dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.
    Marilen Federico, Enrique L. Portiansky, Leandro Sommese, Francisco J. Alvarado, Paula G. Blanco, Carolina N. Zanuzzi, John Dedman, Marcia Kaetzel, Xander H. T. Wehrens, Alicia Mattiazzi, Julieta Palomeque.
    The Journal of Physiology. March 02, 2017
    Key points Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose‐rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD‐WT mice, which was prevented by co‐treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD‐SR‐autocamide inhibitory peptide (AIP) mice, expressing the SR‐targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD‐SR‐AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD‐WT mice but not in FRD‐S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. Abstract The impact of cardiac apoptosis in pre‐diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose‐rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin‐protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre‐diabetes. We generated a pre‐diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control‐diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia‐induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD‐WT mice. Co‐treatment with the reactive oxygen species scavenger Tempol prevented FRD‐induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD‐SR‐AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD‐WT hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre‐diabetic heart. The FRD‐induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.
    March 02, 2017   doi: 10.1113/JP273714   open full text
  • Visceral and somatic pain modalities reveal NaV1.7‐independent visceral nociceptive pathways.
    James R. F. Hockley, Rafael González‐Cano, Sheridan McMurray, Miguel A. Tejada‐Giraldez, Cian McGuire, Antonio Torres, Anna L. Wilbrey, Vincent Cibert‐Goton, Francisco R. Nieto, Thomas Pitcher, Charles H. Knowles, José Manuel Baeyens, John N. Wood, Wendy J. Winchester, David C. Bulmer, Cruz Miguel Cendán, Gordon McMurray.
    The Journal of Physiology. March 01, 2017
    Key points Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics. Abstract Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.
    March 01, 2017   doi: 10.1113/JP272837   open full text
  • Pre‐ischaemic mitochondrial substrate constraint by inhibition of malate‐aspartate shuttle preserves mitochondrial function after ischaemia–reperfusion.
    Nichlas Riise Jespersen, Takashi Yokota, Nicolaj Brejnholt Støttrup, Andreas Bergdahl, Kim Bolther Pælestik, Jonas Agerlund Povlsen, Flemming Dela, Hans Erik Bøtker.
    The Journal of Physiology. February 27, 2017
    Key points Pre‐ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate‐aspartate shuttle (MAS), provides cardi