Sergey Kasparov

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.

Adenoviral vector demonstrates that angiotensin II‐induced depression of the cardiac baroreflex is mediated by endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat

1 Angiotensin II (ANGII) acting on ANGII type 1 (AT1) receptors in the solitary tract nucleus (NTS) depresses the baroreflex. Since ANGII stimulates the release of nitric oxide (NO), we tested whether the ANGII‐mediated depression of the baroreflex in the NTS depended on NO release. 2 In a working heart‐brainstem preparation (WHBP) of rat NTS microinjection of either ANGII (500 fmol) or a NO donor (diethylamine nonoate, 500 pmol) both depressed baroreflex gain by ‐56 and ‐67 %, respectively (P < 0.01). In contrast, whilst ANGII potentiated the peripheral chemoreflex, the NO donor was without effect. 3 NTS microinjection of non‐selective NO synthase (NOS) inhibitors (l‐NAME; 50 pmol) or (l‐NMMA; 200 pmol) prevented the ANGII‐induced baroreflex attenuation (P > 0.1). In contrast, a neurone‐specific NOS inhibitor, TRIM (50 pmol), was without effect. 4 Using an adenoviral vector, a dominant negative mutant of endothelial NOS (TeNOS) was expressed bilaterally in the NTS. Expression of TeNOS affected neither baseline cardiovascular parameters nor baroreflex sensitivity. However, ANGII microinjected into the transfected region failed to affect the baroreflex. 5 Immunostaining revealed that eNOS‐positive neurones were more numerous than those labelled for AT1 receptors. Neurones double labelled for both AT1 receptors and eNOS comprised 23 ± 5.4 % of the eNOS‐positive cells and 57 ± 9.2 % of the AT1 receptor‐positive cells. Endothelial cells were also double labelled for eNOS and AT1 receptors. 6 We suggest that ANGII activates eNOS located in either neurones and/or endothelial cells to release NO, which acts selectively to depress the baroreflex.

Nitric oxide and autonomic control of heart rate: a question of specificity.

Despite its highly diffusible nature, the gaseous signalling molecule nitric oxide (NO) can exert specific effects within the CNS and PNS. To date, the specificity of the actions of NO remains an unsolved puzzle. There are several plausible mechanisms that might account for this specificity in the context of autonomic regulation of heart rate. NO acts at distinct levels within the autonomic nervous system to control cardiac rate, with opposing effects at different sites. We discuss factors that might contribute to this diversity of action, and conclude that the isoform of enzyme involved in producing NO, the spatial proximity of the NO source to the target, and differences in the intracellular coupling within the target cell are all crucial for encoding the functional action of NO.

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.

Differential effects of angiotensin II on cardiorespiratory reflexes mediated by nucleus tractus solitarii – a microinjection study in the rat

1 The effect of microinjecting angiotensin II (ANGII) into the nucleus of the solitary tract (NTS) on both baroreceptor and peripheral chemoreceptor reflexes was compared. 2 Experiments were performed in a working heart‐brainstem preparation of rat. Baroreceptors were stimulated by raising perfusion pressure and chemoreceptors were activated with aortic injections of sodium cyanide (0·025%, 25–75 μl). Reflex changes in phrenic nerve activity and heart rate were measured after bilateral NTS microinjection (50 nl) of ANGII (0·5–5000 fmol). 3 NTS microinjection of 5 fmol ANGII elicited a transient (28·2 ± 6 s; mean ± s.e.m.) bradycardia (‐18 ± 3 beats min−1), and decreased phrenic nerve activity cycle length and amplitude (P < 0·05). At higher doses of ANGII a similar respiratory response was seen but heart rate changes were inconsistent. 4 The baroreceptor reflex bradycardia was depressed significantly by NTS microinjections of ANGII (5–5000 fmol) in a dose‐dependent manner with the reflex gain decreasing from 1·7 ± 0·16 to 0·66 ± 0·1 beats min−1 mmHg−1 (P < 0·01) at 5000 fmol. Although the chemoreceptor reflex bradycardia was depressed at a low dose of ANGII (5 fmol), all higher doses (50–5000 fmol) produced a dose‐dependent potentiation of the reflex bradycardia (maximally +64 ± 8%). The respiratory component was unaffected. The effects of ANGII on both reflexes were blocked by an ANGII type 1 (AT1) receptor antagonist, losartan (20 μM). 5 The potentiating action of ANGII on the chemoreceptor reflex cardiac response was abolished by a neurokinin type 1 (NK1) receptor blocker (CP‐99,994, 5 μM) but this had no effect on the baroreceptor reflex. 6 AT1 receptors in the NTS can depress the baroreceptor reflex bradycardia which is independent of NK1 receptors. The ANGII effect on the cardiac component of the chemoreceptor reflex is bi‐directional being inhibited at low concentrations and potentiated at higher concentrations; the latter involves NK1 receptors and presumably results from release of substance P.

Chronic inhibition of endothelial nitric oxide synthase activity in nucleus tractus solitarii enhances baroreceptor reflex in conscious rats

In acute experiments, we demonstrated previously that nitric oxide (NO) donors exogenously applied to the nucleus tractus solitarii (NTS) depressed the baroreceptor cardiac reflex. In this study, we determined a role for endogenous endothelial nitric oxide synthase (eNOS) activity in the NTS for chronically regulating baroreceptor reflex function in conscious rats. A recombinant adenoviral vector directing expression of a truncated form of eNOS was microinjected bilaterally into the NTS to inhibit endogenous eNOS activity. Arterial pressure was monitored continuously using radio‐telemetry in freely moving animals and spontaneous baroreceptor reflex gain (sBRG) determined by a time‐series method. sBRG showed a gradual increase from day 7 to 21 after gene transfer and the value at day 21 (1.68 ± 0.20 ms mmHg−1, n= 6) was significantly higher than that before gene transfer (1.13 ± 0.09 ms mmHg−1, P < 0.001). This value was also significantly higher than that in rats in which enhanced green fluorescent protein (eGFP) was expressed in the NTS (1.04 ± 0.21 ms mmHg−1; n= 6, P < 0.01) and saline‐treated groups (1.12 ± 0.15 ms mmHg−1; n= 4, P < 0.05), which did not change from control levels. In addition, heart rate decreased from 336 ± 6 to 318 ± 8 b.p.m. (P < 0.05) 21 days after gene transfer. This value was also significantly lower than that in control groups (eGFP: 348 ± 9 b.p.m., n= 6, P < 0.01; saline: 347 ± 5 b.p.m., n= 4, P < 0.05). Gene transfer did not affect arterial pressure. These findings suggest that in the conscious rat eNOS is constitutively active within the NTS and is a factor regulating baroreceptor reflex gain and heart rate.

Junctional adhesion molecule-1 is upregulated in spontaneously hypertensive rats: evidence for a prohypertensive role within the brain stem.

Junctional adhesion molecule-1 (JAM-1) forms part of the tight junction between adjacent endothelial cells. Using microarray technology, we showed previously that JAM-1 was differentially expressed in the brain stem of spontaneously hypertensive rats compared with normotensive Wistar–Kyoto (WKY) rats. In this study, we quantified the expression of JAM-1 in the brain stem of spontaneously hypertensive rats and WKY rats and established whether any differential expression was confined to this region of the brain or was ubiquitous throughout the central nervous system and, indeed, the whole body. Because the nucleus tractus solitarii plays a pivotal role in arterial pressure regulation, we assessed whether JAM-1 in this region affects the chronic regulation of arterial pressure. Real time RT-PCR revealed that JAM-1 mRNA was upregulated in multiple regions of the brain and all of the peripheral vascular beds studied. In the nucleus tractus solitarii, the level of JAM-1 mRNA was significantly higher in both young (3-week–old, prehypertensive) and adult male spontaneously hypertensive rats (15 to 18 weeks old) than that of age-matched WKY rats (fold differences; prehypertensives: 1.01±0.06 versus 1.59±0.13; n=10; P<0.01; adult: 1.08±0.14 versus 2.86±0.57; n=10; P<0.01). After adenoviral-mediated expression of JAM-1 in the nucleus tractus solitarii of adult WKY rats (15 weeks old; n=6), systolic pressure was increased from 120±4 to 132±4 mm Hg (P<0.01). Our data suggest that JAM-1 expression in the spontaneously hypertensive rat is upregulated throughout the body compared with the WKY rat and that this is not secondary to the hypertension. When JAM-1 is expressed in the nucleus tractus solitarii, it raises arterial pressure, suggesting a novel prohypertensive role for this protein within the brain stem.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

BackgroundGenetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).ResultsHere we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons.ConclusionWe believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

Functional Oxygen Sensitivity of Astrocytes

In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2. Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca2+]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca2+ from the intracellular stores. Hypoxia-induced [Ca2+]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. SIGNIFICANCE STATEMENT Most, if not all, animal cells possess mechanisms that allow them to detect decreases in oxygen availability leading to slow-timescale, adaptive changes in gene expression and cell physiology. To date, only two types of mammalian cells have been demonstrated to be specialized for rapid functional oxygen sensing: glomus cells of the carotid body (peripheral respiratory chemoreceptors) that stimulate breathing when oxygenation of the arterial blood decreases; and pulmonary arterial smooth muscle cells responsible for hypoxic pulmonary vasoconstriction to limit perfusion of poorly ventilated regions of the lungs. Results of the present study suggest that there is another specialized oxygen-sensitive cell type in the body, the astrocyte, that is tuned for rapid detection of physiological changes in brain oxygenation.

Differential effects of angiotensin II in the nucleus tractus solitarii of the rat – plausible neuronal mechanisms

1 Cellular mechanisms of the actions of angiotensin II (ANGII) within the nucleus of the solitary tract (NTS) were studied using rat brain slices in 78 neurones recorded in the whole‐cell configuration. Twenty‐nine per cent of cells had an on‐going activity and with only one exception these cells responded to tractus solitarii (TS) stimulation with a monophasic excitatory postsynaptic potential (EPSP). In approximately half of the silent cells, TS stimulation evoked an EPSP‐inhibitory postsynaptic potential (IPSP) complex. 2 The ANGII (200 or 1000 nM) effect on TS‐evoked EPSPs depended on the cell subpopulation. In cells with on‐going activity, ANGII (1000 nM) increased evoked EPSP amplitude by +70 ± 13% (means ± s.e.m., n= 5) but reduced it (200 and 1000 nM) in silent cells where both evoked EPSPs and IPSPs were present. ANGII either increased TS‐evoked IPSP conductances in cells where they were detectable or revealed an evoked IPSP (200 nM ANGII: IPSP conductance increased from 70 ± 29 to 241 ± 34 pS; n= 11). All ANGII effects were prevented by the ANGII type 1 (AT1) receptor blocker losartan. Since 200 nM ANGII did not increase responses to iontophoretically applied GABA, the effect of ANGII on TS‐evoked IPSPs may occur presynaptically. 3 The neurokinin type 1 (NK1) receptor antagonist CP‐99,994 (5 μM) blocked the ANGII‐induced increase in EPSPs but had no effect on TS‐evoked IPSP potentiation by ANGII. 4 Thus, ANGII can potentiate both inhibitory and excitatory synaptic transmission within different subpopulations of NTS neurones. Potentiation of evoked EPSPs, but not of IPSPs, involves activation of NK1 receptors. The balance of these actions of ANGII could be reflex specific: for the baroreflex circuitry the inhibitory action might predominate while the peripheral chemoreceptor reflex may be facilitated due to enhanced excitatory transmission.