Alexander V. Gourine

Astrocytes Control Breathing Through pH-dependent Release of ATP

Astrocytes, ATP, Brainstem, and Breathing Astrocytes (or glial cells), previously assumed to be passive players in brain physiology, may play a functional role in a number of complex behaviors. The central chemosensory control of breathing involves highly specialized neuronal populations in the brainstem, but what about astrocytes? Gourine et al. (p. 571, published online 15 July) now present evidence that glial cells may help to control breathing. A number of techniques were used to reveal glial calcium rises in vitro that elicit a depolarization of neurons in the primary locus for central respiratory chemosensitivity. The depolarization in these neurons is evoked by vesicular release of ATP in neighboring astrocytes in response to the fall in extracellular pH. Thus, brainstem astrocytes have the ability to sense changes in blood and brain CO2, and pH directly, and may control the activity of the respiratory neuronal networks to regulate breathing. Central nervous system glial cells are key players in the chemo-reflex essential for breathing. Astrocytes provide structural and metabolic support for neuronal networks, but direct evidence demonstrating their active role in complex behaviors is limited. Central respiratory chemosensitivity is an essential mechanism that, via regulation of breathing, maintains constant levels of blood and brain pH and partial pressure of CO2. We found that astrocytes of the brainstem chemoreceptor areas are highly chemosensitive. They responded to physiological decreases in pH with vigorous elevations in intracellular Ca2+ and release of adenosine triphosphate (ATP). ATP propagated astrocytic Ca2+ excitation, activated chemoreceptor neurons, and induced adaptive increases in breathing. Mimicking pH-evoked Ca2+ responses by means of optogenetic stimulation of astrocytes expressing channelrhodopsin-2 activated chemoreceptor neurons via an ATP-dependent mechanism and triggered robust respiratory responses in vivo. This demonstrates a potentially crucial role for brain glial cells in mediating a fundamental physiological reflex.

The Carotid Body as a Therapeutic Target for the Treatment of Sympathetically Mediated Diseases

Hypertension, heart failure (HF), type II diabetes mellitus, and chronic kidney disease represent significant and growing global health issues.1 The rates of control of blood pressure and the therapeutic efforts to prevent progression of HF, chronic kidney disease, diabetes mellitus, and their sequelae remain unsatisfactory.2–5 Although patient nonadherence and nonpersistence with medications participate in this failure, especially in asymptomatic disorders, the inherent complexity of drug titration, drug interactions, and both the real and perceived adverse events collectively contribute to the failure of lifelong polypharmacy. Furthermore, therapy targeting the potentially unique contribution of autonomic imbalance is limited by the poorly tolerated systemic adverse effects of adrenergic blocking agents. Recent introduction of medical procedures, such as renal denervation,6,7 and devices such as deep brain stimulation,8 baroreceptor stimulation,9 and direct vagus nerve stimulation10 begin to address these gaps in selective patients. The contribution of excessive sympathetic nerve activity to the development and progression of hypertension, insulin resistance, and HF has been demonstrated in both preclinical and human experiments. Preclinical experiments in models of these diseases have successfully used sympathetic or parasympathetic modifications to alter the time course of their progression.11,12 Reduction of blood pressure after dorsal rhizotomy in rats with renal hypertension and reduced total body noradrenaline and muscle sympathetic nerve activity in humans after renal denervation confirm that the afferent signals from the kidney underlie some of the excessive sympathetic drive seen in these states.13,14 However, additional afferent signals may arise from sites elsewhere in the body and in particular the carotid body (CB). We propose targeting the CB in patients with increased chemosensitivity to address the underlying autonomic imbalance seen in hypertension, HF, insulin resistance, and chronic kidney disorders. ### The CB: A Peripheral Chemosensor The CB (Figure 1), the dominant …

Connexin hemichannel-mediated CO2-dependent release of ATP in the medulla oblongata contributes to central respiratory chemosensitivity

Arterial , a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of in the blood and brain, yet the mechanisms by which the brain senses CO2 remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO2‐dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO2‐dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of . ATP release is independent of extracellular Ca2+ and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin‐1 have no effect, we conclude that a connexin hemichannel is involved in CO2‐dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO2‐dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO2‐dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)‐positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia‐evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26‐mediated release of ATP in response to changes in is an important mechanism contributing to central respiratory chemosensitivity.

Hypertension is critically dependent on the carotid body input in the spontaneously hypertensive rat

Peripheral chemoreflex sensitivity is enhanced in hypertension yet the role of these receptors in the development and maintenance of high blood pressure remains unknown. Carotid chemoreceptors were denervated in both young and adult spontaneously hypertensive rats (SHRs) by sectioning the carotid sinus nerves bilaterally while recording arterial blood pressure chronically using radio telemetry. Carotid sinus denervation (CSD) in the young animals prevented arterial pressure from reaching the hypertensive levels observed in sham‐operated animals whereas in adult SHRs arterial pressure fell by ∼20 mmHg. After CSD there was a decrease in sympathetic activity, measured indirectly using power spectral analysis and hexamethonium, and an improvement in baroreceptor reflex gain. Carotid bodies are active in the SHR and contribute to both the development and maintenance of hypertension; whether carotid body ablation is a useful anti‐hypertensive intervention in drug‐resistant hypertensive patients remains to be resolved.

Lactate-mediated glia-neuronal signalling in the mammalian brain

Astrocytes produce and release L-lactate as a potential source of energy for neurons. Here we present evidence that L-lactate, independently of its caloric value, serves as an astrocytic signalling molecule in the locus coeruleus (LC). The LC is the principal source of norepinephrine to the frontal brain and thus one of the most influential modulatory centers of the brain. Optogenetically activated astrocytes release L-lactate, which excites LC neurons and triggers release of norepinephrine. Exogenous L-lactate within the physiologically relevant concentration range mimics these effects. L-lactate effects are concentration-dependent, stereo-selective, independent of L-lactate uptake into neurons and involve a cAMP-mediated step. In vivo injections of L-lactate in the LC evokes arousal similar to the excitatory transmitter, L-glutamate. Our results imply the existence of an unknown receptor for this ‘glio-transmitter’.

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.

Cardioprotection evoked by remote ischaemic preconditioning is critically dependent on the activity of vagal pre-ganglionic neurones

Aims Innate mechanisms of inter-organ protection underlie the phenomenon of remote ischaemic preconditioning (RPc) in which episode(s) of ischaemia and reperfusion in tissues remote from the heart reduce myocardial ischaemia/reperfusion injury. The uncertainty surrounding the mechanism(s) underlying RPc centres on whether humoral factor(s) produced during ischaemia/reperfusion of remote tissue and released into the systemic circulation mediate RPc, or whether a neural signal is required. While these two hypotheses may not be incompatible, one approach to clarify the potential role of a neural pathway requires targeted disruption or activation of discrete central nervous substrate(s). Methods and results Using a rat model of myocardial ischaemia/reperfusion injury in combination with viral gene transfer, pharmaco-, and optogenetics, we tested the hypothesis that RPc cardioprotection depends on the activity of vagal pre-ganglionic neurones and consequently an intact parasympathetic drive. For cell-specific silencing or activation, neurones of the brainstem dorsal motor nucleus of the vagus nerve (DVMN) were targeted using viral vectors to express a Drosophila allatostatin receptor (AlstR) or light-sensitive fast channelrhodopsin variant (ChIEF), respectively. RPc cardioprotection, elicited by ischaemia/reperfusion of the limbs, was abolished when DVMN neurones transduced to express AlstR were silenced by selective ligand allatostatin or in conditions of systemic muscarinic receptor blockade with atropine. In the absence of remote ischaemia/reperfusion, optogenetic activation of DVMN neurones transduced to express ChIEF reduced infarct size, mimicking the effect of RPc. Conclusion These data indicate a crucial dependence of RPc cardioprotection against ischaemia/reperfusion injury upon the activity of a distinct population of vagal pre-ganglionic neurones.

A Role for TASK-1 (KCNK3) Channels in the Chemosensory Control of Breathing

Acid-sensitive K+ channels of the tandem P-domain K+-channel family (TASK-1 and TASK-3) have been implicated in peripheral and central respiratory chemosensitivity; however, because of the lack of decisive pharmacological agents, the final proof of the role of the TASK channel in the chemosensory control of breathing has been missing. In the mouse, TASK-1 and TASK-3 channels are dispensable for central respiratory chemosensitivity (Mulkey et al., 2007). Here, we have used knock-out animals to determine whether TASK-1 and TASK-3 channels play a role in the carotid body function and chemosensory control of breathing exerted by the carotid body chemoreceptors. Ventilatory responses to hypoxia (10% O2 in inspired air) and moderate normoxic hypercapnia (3–6% CO2 in inspired air) were significantly reduced in TASK-1 knock-out mice. In contrast, TASK-3-deficient mice showed responses to both stimuli that were similar to those developed by their wild-type counterparts. TASK-1 channel deficiency resulted in a marked reduction of the hypoxia (by 49%)- and CO2 (by 68%)-evoked increases in the carotid sinus nerve chemoafferent discharge recorded in the in vitro superfused carotid body/carotid sinus nerve preparations. Deficiency in both TASK-1 and TASK-3 channels increased baseline chemoafferent activity but did not cause a further reduction of the carotid body chemosensory responses. These observations provide direct evidence that TASK-1 channels contribute significantly to the increases in the carotid body chemoafferent discharge in response to a decrease in arterial PO2 or an increase in PCO2/[H+]. TASK-1 channels therefore play a key role in the control of ventilation by peripheral chemoreceptors.

Essential role of Phox2b-expressing ventrolateral brainstem neurons in the chemosensory control of inspiration and expiration.

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO2/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem–spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO2-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO2-evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO2 in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO2-evoked increases of inspiratory activity.

Cardioprotection, attenuated systemic inflammation, and survival benefit of β1-adrenoceptor blockade in severe sepsis in rats*

Objective: To explore the hypothesis that beta-1 adrenoreceptor blockade may be protective through the attenuation of sympathetic hyperactivity and catecholaminergic inflammatory effects on cardiac and hepatic function. Design: Prospective, randomized, controlled study. Setting: Animal laboratory in a university medical center. Subjects: Male adult Wistar rats. Interventions: Peripheral &bgr;1-adrenoceptor blockade through daily intraperitoneal injection (metoprolol, 100 mg·kg−1; atenolol, 6 mg·kg−1) or central nervous system &bgr;1-adrenoceptor blockade (intracerebroventricular metoprolol, 25 &mgr;g) to achieve ∼20% heart rate reduction in rats for 2 days before or after the induction of lethal endotoxemia, cecal ligation and puncture, or fecal peritonitis. Measurements and Main Results: Peripheral &bgr;1-adrenoceptor blockade established for 2 days before lethal endotoxemia markedly improved survival in both metoprolol-treated (n = 16; log rank test, p = .002) and atenolol-treated (n = 15; p = .03) rats. Overall mortality in cecal ligation and puncture was similar between metoprolol (40%; n = 10) and saline (50%; n = 10) pretreatment (p = .56), but the median time to death was increased by 33 hrs in metoprolol-treated rats (p = .03). Metoprolol pretreatment reduced hepatic expression of proinflammatory cytokines and lowered plasma interleukin-6 (both p < .05). Myocardial protein expression of interleukin-18 and monocyte chemoattractant protein-1, key mediators of cardiac dysfunction in sepsis, were also reduced (p < .05). Peripheral &bgr;1-adrenoceptor blockade commenced 6 hrs after lethal endotoxemia or fecal peritonitis did not improve survival. However, arterial blood pressure was preserved and left ventricular contractility restored similar to that found in nonseptic controls. Central nervous system &bgr;1-adrenoceptor blockade (metoprolol) did not reduce plasma cytokines or mortality, despite enhancing parasympathetic tone. Conclusions: Peripheral &bgr;1-adrenoceptor blockade offers anti-inflammatory and cardioprotective effects, with mortality reduction if commenced before a septic insult. Its role in sepsis should be explored further.