Geoff Burnstock, purinergic signalling, and chemosensory control of breathing.

This article is the authors' contribution to the tribute issue in honour of Geoffrey Burnstock, the founder of this journal and the field of purinergic signalling. We give a brief account of the results of experimental studies which at the beginning received valuable input from Geoff, who both directly and indirectly influenced our research undertaken over the last two decades. Research into the mechanisms controlling breathing identified ATP as the common mediator of the central and peripheral chemosensory transduction. Studies of the sources and mechanisms of chemosensory ATP release in the CNS suggested that this signalling pathway is universally engaged in conditions of increased metabolic demand by brain glial cells - astrocytes. Astrocytes appear to function as versatile CNS metabolic sensors that detect changes in brain tissue pH, CO2, oxygen, and cerebral perfusion pressure. Experimental studies on various aspects of astrocyte biology generated data indicating that the function of these omnipresent glial cells and communication between astrocytes and neurons are governed by purinergic signalling, - first discovered by Geoff Burnstock in the 70's and researched through his entire scientific career

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

Rett syndrome is a prototypical neurological disorder characterised by abnormal breathing pattern and reduced ventilatory CO2 sensitivity. Medullary astrocytes are a crucial component of central CO2 /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 PCO2/[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. CO2 -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 PCO2/[H(+) ]

What is the key mediator of the neurovascular coupling response?

The mechanisms of neurovascular coupling contribute to ensuring brain energy supply is sufficient to meet demand. Despite significant research interest, the mechanisms underlying increases in regional blood flow that follow heightened neuronal activity are not completely understood. This article presents a systematic review and analysis of published data reporting the effects of pharmacological or genetic blockade of all hypothesised signalling pathways of neurovascular coupling. Our primary outcome measure was the percent reduction of the neurovascular response assessed using in vivo animal models. Selection criteria were met by 50 primary sources reporting the effects of 79 treatments. Experimental conditions were grouped into categories targeting mechanisms mediated by nitric oxide (NO), prostanoids, purines, potassium, amongst others. Blockade of neuronal NO synthase was found to have the largest effect of inhibiting any individual target, reducing the neurovascular response by 64% (average of 11 studies). Inhibition of multiple targets in combination with nNOS blockade had no further effect. This analysis points to the existence of an unknown signalling mechanism accounting for approximately one third of the neurovascular response

On the existence of a central respiratory oxygen sensor

A commonly held view that dominates both the scientific and educational literature is that in terrestrial mammals the central nervous system lacks a physiological hypoxia sensor capable of triggering increases in lung ventilation in response to decreases in PO2 of the brain parenchyma. Indeed, a normocapnic hypoxic ventilatory response has never been observed in humans following bilateral resection of the carotid bodies. In contrast, almost complete or partial recovery of the hypoxic ventilatory response after denervation/removal of the peripheral respiratory oxygen chemoreceptors has been demonstrated in many experimental animals when assessed in an awake state. In this essay we review the experimental evidence obtained using in vitro and in vivo animal models, results of human studies, and discuss potential mechanisms underlying the effects of CNS hypoxia on breathing. We consider experimental limitations and discuss potential reasons why the recovery of the hypoxic ventilatory response has not been observed in humans. We review recent experimental evidence suggesting that the lower brainstem contains functional respiratory oxygen sensitive elements capable of stimulating respiratory activity independently of peripheral chemoreceptor input

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

Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles

Active neurons increase their energy supply by dilating nearby arterioles and capillaries. This neurovascular coupling underlies blood oxygen level-dependent functional imaging signals, but its mechanism is controversial. Canonically, neurons release glutamate to activate metabotropic glutamate receptor 5 (mGluR5) on astrocytes, evoking Ca(2+) release from internal stores, activating phospholipase A2 and generating vasodilatory arachidonic acid derivatives. However, adult astrocytes lack mGluR5, and knockout of the inositol 1,4,5-trisphosphate receptors that release Ca(2+) from stores does not affect neurovascular coupling. We now show that buffering astrocyte Ca(2+) inhibits neuronally evoked capillary dilation, that astrocyte [Ca(2+)]i is raised not by release from stores but by entry through ATP-gated channels, and that Ca(2+) generates arachidonic acid via phospholipase D2 and diacylglycerol kinase rather than phospholipase A2. In contrast, dilation of arterioles depends on NMDA receptor activation and Ca(2+)-dependent NO generation by interneurons. These results reveal that different signaling cascades regulate cerebral blood flow at the capillary and arteriole levels

Origins of the vagal drive controlling left ventricular contractility

The strength, functional significance and origins of direct parasympathetic innervation of the left ventricle (LV) remain controversial. In the present study we used an anaesthetized rat model to first confirm the presence of tonic inhibitory vagal influence on LV inotropy. Using genetic neuronal targeting and functional neuroanatomical mapping we tested the hypothesis that parasympathetic control of LV contractility is provided by vagal preganglionic neurones located in the dorsal motor nucleus (DVMN). It was found that under systemic β-adrenoceptor blockade (atenolol) combined with spinal cord (C1) transection (to remove sympathetic influences), intravenous administration of atropine increases LV contractility in rats anaesthetized with urethane, but not in animals anaesthetized with pentobarbital. Increased LV contractility in rats anaesthetized with urethane was also observed when DVMN neurones targeted bilaterally to express an inhibitory Drosophila allatostatin receptor were silenced by application of an insect peptide allatostatin. Microinjections of glutamate and muscimol to activate or inhibit neuronal cell bodies in distinct locations along the rostro-caudal extent of the left and right DVMN revealed that vagal preganglionic neurones which have an impact on LV contractility are located in the caudal region of the left DVMN. Changes in LV contractility were only observed when this subpopulation of DVMN neurones was activated or inhibited. These data confirm the existence of a tonic inhibitory muscarinic influence on LV contractility. Activity of a subpopulation of DVMN neurones provides functionally significant parasympathetic control of LV contractile function. This article is protected by copyright. All rights reserved

Respiratory rhythm irregularity after carotid body denervation in rats

Respiratory activity is controlled by inputs from the peripheral and central chemoreceptors. Since overactivity of the carotid bodies, the main peripheral chemoreceptors, is linked to the pathophysiology of disparate metabolic and cardiovascular diseases, carotid body denervation (CBD) has been proposed as a potential treatment. However, long-term effects of CBD on the respiratory rhythm and regularity of breathing remain unknown. Here, we show that five weeks after bilateral CBD in rats, the respiratory rhythm was slower and less regular. Ten weeks after bilateral CBD, the respiratory frequency was not different from the sham-operated group, but the regularity of the respiratory rhythm was still reduced. Increased frequency of randomly occurring apneas is likely to be responsible for the irregular breathing pattern after CBD. These results should be taken into consideration since any treatment that reduces the stability of the respiratory rhythm might exacerbate the cardio-respiratory instability and worsen the cardiovascular outcomes

Glucagon-Like Peptide-1 (GLP-1) Mediates Cardioprotection by Remote Ischaemic Conditioning

AIMS: Although the nature of the humoral factor which mediates cardioprotection established by remote ischaemic conditioning (RIc) remains unknown, parasympathetic (vagal) mechanisms appear to play a critical role. As the production and release of many gut hormones is modulated by the vagus nerve, here we tested the hypothesis that RIc cardioprotection is mediated by the actions of glucagon-like peptide-1 (GLP-1). METHODS AND RESULTS: A rat model of myocardial infarction (coronary artery occlusion followed by reperfusion) was used. Remote ischaemic pre- (RIPre) and perconditioning (RIPer) was induced by 15 min occlusion of femoral arteries applied prior to or during the myocardial ischaemia. The degree of RIPre and RIPer cardioprotection was determined in conditions of cervical or subdiaphragmatic vagotomy, or following blockade of GLP-1 receptors (GLP-1R) using specific antagonist Exendin(9-39). Phosphorylation of PI3K/AKT and STAT3 was assessed. RIPre and RIPer reduced infarct size by ~50%. In conditions of bilateral cervical or subdiaphragmatic vagotomy RIPer failed to establish cardioprotection. GLP-1R blockade abolished cardioprotection induced by either RIPre or RIPer. Exendin(9-39) also prevented RIPre-induced AKT phosphorylation. Cardioprotection induced by GLP-1R agonist Exendin-4 was preserved following cervical vagotomy, but was abolished in conditions of M3 muscarinic receptor blockade. CONCLUSIONS: These data strongly suggest that GLP-1 functions as a humoral factor of remote ischaemic conditioning cardioprotection. This phenomenon requires intact vagal innervation of the visceral organs and recruitment of GLP-1R-mediated signalling. Cardioprotection induced by GLP-1R agonism is mediated by a mechanism involving M3 muscarinic receptors