Release of ATP in the ventral medulla during hypoxia in rats: role in hypoxic ventilatory response

P2X2 receptor subunits of the ATP-gated ion channels are expressed by physiologically identified respiratory neurons in the ventral respiratory column, implicating ATP in the control of respiratory activity. We now show that, during hypoxia, release of ATP in the ventrolateral medulla (VLM) plays an important role in the hypoxic ventilatory response in rats. By measuring ATP release in real time at the ventral surface of the medulla with novel amperometric biosensors, we found that hypoxia (10% O2; 5 min) induced a marked increase in the concentration of ATP (~3 µM). This ATP release occurred after the initiation of enhanced respiratory activity but coincided with the later hypoxia-induced slowing of the respiratory rhythm. ATP was also released at the ventral surface of the medulla during hypoxia in peripherally chemodenervated animals (vagi, aortic, and carotid sinus nerve sectioned). By using horizontal slices of the rat medulla, we found that, during hypoxia, ATP is produced throughout the VLM in the locations corresponding to the ventral respiratory column. Blockade of ATP receptors in the VLM (microinjection of P2 receptor antagonist pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid; 100 µM) augmented the hypoxia-induced secondary slowing of the respiratory rhythm. Our findings suggest that ATP released within the ventral respiratory column is involved in maintenance of the respiratory activity in conditions when hypoxia-induced slowing of respiration occurs. These data illustrate a new functional role for ATP-mediated purinergic signaling in the medullary mechanisms controlling respiratory activity

Glucagon-like peptide-1 (GLP-1) mediates cardioprotection by remote ischaemic conditioning

This work was supported by the British Heart Foundation (Ref: RG/14/4/30736), Medical Research Council (MR/N02589X/1) and The Wellcome Trust (Ref: 200893/Z/16/Z). A.V.G. is a Wellcome Trust Senior Research Fellow. S.M. is a Marie Skłodowska-Curie Research Fellow (Ref: 654691)

The role of parafacial neurons in the control of breathing during exercise

Neuronal cell groups residing within the retrotrapezoid nucleus (RTN) and C1 area of the rostral ventrolateral medulla oblongata contribute to the maintenance of resting respiratory activity and arterial blood pressure, and play an important role in the development of cardiorespiratory responses to metabolic challenges (such as hypercapnia and hypoxia). In rats, acute silencing of neurons within the parafacial region which includes the RTN and the rostral aspect of the C1 circuit (pFRTN/C1), transduced to express HM4D (Gi-coupled) receptors, was found to dramatically reduce exercise capacity (by 60%), determined by an intensity controlled treadmill running test. In a model of simulated exercise (electrical stimulation of the sciatic or femoral nerve in urethane anaesthetised spontaneously breathing rats) silencing of the pFRTN/C1 neurons had no effect on cardiovascular changes, but significantly reduced the respiratory response during steady state exercise. These results identify a neuronal cell group in the lower brainstem which is critically important for the development of the respiratory response to exercise and, determines exercise capacity

Impaired CO₂ sensitivity of astrocytes in a mouse model of Rett syndrome

Rett syndrome, a prototypical neurological disorder caused by loss of function of the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2) gene, is associated with a severely disordered breathing pattern and reduced ventilatory CO(2) sensitivity. In a mouse model of Rett syndrome (MeCP2 knockout), re-introduction of the MeCP2 gene selectively in astrocytes rescues normal respiratory phenotype. In the present study we determined whether the metabolic and/or signalling functions of astrocytes are affected by testing the hypotheses that in conditions of MeCP2 deficiency, medullary astrocytes are unable to produce/release appropriate amounts of lactate or detect changes in [Image: see text]/[H(+)], or both. No differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex in brain slices of MeCP2-knockout and wild-type mice were found. In brainstem slices of wild-type mice, respiratory acidosis triggered robust elevations in [Ca(2+)](i) in astrocytes residing near the ventral surface of the medulla oblongata. The magnitude of CO(2)-induced [Ca(2+)](i) responses in medullary astrocytes was markedly reduced in conditions of MeCP2 deficiency, whereas [Ca(2+)](i) responses to ATP were unaffected. These data suggest that (i) metabolic function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in [Image: see text]/[H(+)]. Taken together with the evidence of severely blunted ventilatory sensitivity to CO(2) in mice with conditional MeCP2 deletion in astroglia, these data support the hypothesis of an important role played by astrocytes in central respiratory CO(2)/pH chemosensitivity. KEY POINTS: Rett syndrome is a prototypical neurological disorder characterised by abnormal breathing pattern and reduced ventilatory CO(2) sensitivity. Medullary astrocytes are a crucial component of central CO(2)/pH chemosensitivity. . This study tested the hypotheses that methyl-CpG-binding protein 2 (MeCP2) deficient medullary astrocytes are (i) unable to produce/release appropriate amounts of lactate, and/or (ii) unable to sense changes in [Image: see text]/[H(+)]. . We found no differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex between MeCP2-knockout and wild-type mice. . Respiratory acidosis triggered robust [Ca(2+)](i) responses in wild-type astrocytes residing near the ventral surface of the medulla oblongata. CO(2)-induced [Ca(2+)](i) responses in astrocytes were dramatically reduced in conditions of MeCP2 deficiency. . These data suggest that (i) ‘metabolic’ function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in [Image: see text]/[H(+)].

Contributions of carotid bodies, retrotrapezoid nucleus neurons and preBötzinger complex astrocytes to the CO2-sensitive drive for breathing

Current models of respiratory CO2 chemosensitivity are centred around the function of a specific population of neurons residing in the medullary retrotrapezoid nucleus (RTN). However, there is significant evidence suggesting that chemosensitive neurons exist in other brainstem areas, including the rhythm-generating region of the medulla oblongata – the preBötzinger complex (preBötC). There is also evidence that astrocytes, non-neuronal brain cells, contribute to central CO2 chemosensitivity. In this study, we reevaluated the relative contributions of the RTN neurons, the preBötC astrocytes, and the carotid body chemoreceptors in mediating the respiratory responses to CO2 in experimental animals (adult laboratory rats). To block astroglial signalling via exocytotic release of transmitters, preBötC astrocytes were targeted to express the tetanus toxin light chain (TeLC). Bilateral expression of TeLC in preBötC astrocytes was associated with ∼20% and ∼30% reduction of the respiratory response to CO2 in conscious and anaesthetized animals, respectively. Carotid body denervation reduced the CO2 respiratory response by ∼25%. Bilateral inhibition of RTN neurons transduced to express Gi-coupled designer receptors exclusively activated by designer drug (DREADDGi) by application of clozapine-N-oxide reduced the CO2 response by ∼20% and ∼40% in conscious and anaesthetized rats, respectively. Combined blockade of astroglial signalling in the preBötC, inhibition of RTN neurons and carotid body denervation reduced the CO2-induced respiratory response by ∼70%. These data further support the hypothesis that the CO2-sensitive drive to breathe requires inputs from the peripheral chemoreceptors and several central chemoreceptor sites. At the preBötC level, astrocytes modulate the activity of the respiratory network in response to CO2, either by relaying chemosensory information (i.e. they act as CO2 sensors) or by enhancing the preBötC network excitability to chemosensory inputs

Immediate and sustained increases in the activity of vagal preganglionic neurons during exercise and after exercise training

BACKGROUND: The brain controls the heart by dynamic recruitment and withdrawal of cardiac parasympathetic (vagal) and sympathetic activity. Autonomic control is essential for the development of cardiovascular responses during exercise, however, the patterns of changes in the activity of the two autonomic limbs, and their functional interactions in orchestrating physiological responses during exercise, are not fully understood. METHODS AND RESULTS: The aim of this study was to characterise changes in vagal parasympathetic drive in response to exercise and exercise training by directly recording the electrical activity of vagal preganglionic neurons in experimental animals (rats). Single unit recordings were made using carbon-fibre microelectrodes from the populations of vagal preganglionic neurons of the nucleus ambiguus and the dorsal vagal motor nucleus of the brainstem.It was found that (i) vagal preganglionic neurons of the nucleus ambiguus and the dorsal vagal motor nucleus are strongly activated during bouts of acute exercise, and (ii) exercise training markedly increases the resting activity of both populations of vagal preganglionic neurons and augments the excitatory responses of nucleus ambiguus neurons during exercise. CONCLUSIONS: These data show that central vagal drive increases during exercise and provide the first direct neurophysiological evidence that exercise training increases vagal tone. The data argue against the notion of exercise-induced central vagal withdrawal during exercise. We propose that robust increases in the activity of vagal preganglionic neurons during bouts of exercise underlie activity-dependent plasticity, leading to higher resting vagal tone that confers multiple health benefits associated with regular exercise

Volumetric Spatial Correlations of Neurovascular Coupling Studied using Single Pulse Opto-fMRI

Neurovascular coupling describes the link between neuronal activity and cerebral blood flow. This relationship has been the subject of intense scrutiny, with most previous work seeking to understand temporal correlations that describe neurovascular coupling. However, to date, the study of spatial correlations has been limited to two-dimensional mapping of neuronal or vascular derived signals emanating from the brain's surface, using optical imaging techniques. Here, we investigate spatial correlations of neurovascular coupling in three dimensions, by applying a single 10 ms pulse of light to trigger optogenetic activation of cortical neurons transduced to express channelrhodopsin2, with concurrent fMRI. We estimated the spatial extent of increased neuronal activity using a model that takes into the account the scattering and absorption of blue light in brain tissue together with the relative density of channelrhodopsin2 expression across cortical layers. This method allows precise modulation of the volume of activated tissue in the cerebral cortex with concurrent three-dimensional mapping of functional hyperemia. Single pulse opto-fMRI minimizes adaptation, avoids heating artefacts and enables confined recruitment of the neuronal activity. Using this novel method, we present evidence for direct proportionality of volumetric spatial neurovascular coupling in the cerebral cortex

Respiratory responses to hypercapnia and hypoxia in mice with genetic ablation of Kir5.1 (Kcnj16)

Inward rectifier (Kir) potassium channels contribute to the control of electrical activity in excitable tissues and their activity is modulated by many biochemical factors, including protons. Heteromeric Kir4.1–Kir5.1 channels are highly pH sensitive within the physiological range of pH changes and are strongly expressed by the peripheral chemosensors as well as in the brainstem pH-sensitive areas which mediate respiratory responses to changes in blood and brain levels of /[H+]. In the present study, Kir5.1 knockout mice (Kir5.1−/−) were used to determine the role of these channels in the chemosensory control of breathing. We found that Kir5.1−/− mice presented with persistent metabolic acidosis and a clear respiratory phenotype. Despite metabolic acidosis, ventilation at rest and in hyperoxic hypercapnia were similar in wild-type and Kir5.1−/− mice. Ventilatory responses to hypoxia and normoxic hypercapnia were significantly reduced in Kir5.1−/− mice; however, carotid body chemoafferent responses to hypoxia and CO2 were not affected. In the in situ brainstem–spinal cord preparations with denervated peripheral chemoreceptors, resting phrenic nerve activity and phrenic nerve responses to respiratory acidosis or isohydric hypercapnia were also similar in Kir5.1−/− and wild-type mice. In in situ preparations of Kir5.1−/− mice with intact peripheral chemoreceptors, application of CN− resulted in a significantly reduced phrenic nerve response, suggesting that the relay of peripheral chemosensory information to the CNS is compromised. We suggest that this compensatory modulation of the peripheral chemosensory inputs develops in Kir5.1−/− mice in order to counteract the effect of continuing metabolic acidosis on the activity of the peripheral chemoreceptors. These results therefore suggest that despite their intrinsic pH sensitivity, Kir4.1–Kir5.1 channels are dispensable for functional central and peripheral respiratory chemosensitivity

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release

Summary Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior