Salt-loading promotes extracellular ATP release mediated by glial cells in the hypothalamic paraventricular nucleus of rats.

Previously, we have shown that purinergic signalling is involved in the control of hyperosmotic-induced sympathoexcitation at the level of the PVN, via activation of P2X receptors. However, the source(s) of ATP that drives osmotically-induced increases in sympathetic outflow remained undetermined. Here, we tested the two competing hypotheses that either (1) higher extracellular ATP in PVN during salt loading (SL) is a result of a failure of ectonucleotidases to metabolize ATP; and/or (2) SL can stimulate PVN astrocytes to release ATP. Rats were salt loaded with a 2 % NaCl solution replacing drinking water up to 4 days, an experimental model known to cause a gradual increase in blood pressure and plasma osmolarity. Immunohistochemical assessment of glial-fibrillary acidic protein (GFAP) revealed increased glial cell reactivity in the PVN of rats after 4 days of high salt exposure. ATP and adenosine release measurements via biosensors in hypothalamic slices showed that baseline ATP release was increased 17-fold in the PVN while adenosine remained unchanged. Disruption of Ca2+-dependent vesicular release mechanisms in PVN astrocytes by virally-driven expression of a dominant-negative SNARE protein decreased the release of ATP. The activity of ectonucleotidases quantified in vitro by production of adenosine from ATP was increased in SL group. Our results showed that SL stimulates the release of ATP in the PVN, at least in part, from glial cells by a vesicle-mediated route and likely contributes to the neural control of circulation during osmotic challenges.

Glucagon-like peptide-1 (GLP-1) receptor activation dilates cerebral arterioles, increases cerebral blood flow, and mediates remote (pre)conditioning neuroprotection against ischaemic stroke.

Stroke remains one of the most common causes of death and disability worldwide. Several preclinical studies demonstrated that the brain can be effectively protected against ischaemic stroke by two seemingly distinct treatments: remote ischaemic conditioning (RIC), involving cycles of ischaemia/reperfusion applied to a peripheral organ or tissue, or by systemic administration of glucagon-like-peptide-1 (GLP-1) receptor (GLP-1R) agonists. The mechanisms underlying RIC- and GLP-1-induced neuroprotection are not completely understood. In this study, we tested the hypothesis that GLP-1 mediates neuroprotection induced by RIC and investigated the effect of GLP-1R activation on cerebral blood vessels, as a potential mechanism of GLP-1-induced protection against ischaemic stroke. A rat model of ischaemic stroke (90 min of middle cerebral artery occlusion followed by 24-h reperfusion) was used. RIC was induced by 4 cycles of 5 min left hind limb ischaemia interleaved with 5-min reperfusion periods. RIC markedly (by ~ 80%) reduced the cerebral infarct size and improved the neurological score. The neuroprotection established by RIC was abolished by systemic blockade of GLP-1R with a specific antagonist Exendin(9-39). In the cerebral cortex of GLP-1R reporter mice, ~ 70% of cortical arterioles displayed GLP-1R expression. In acute brain slices of the rat cerebral cortex, activation of GLP-1R with an agonist Exendin-4 had a strong dilatory effect on cortical arterioles and effectively reversed arteriolar constrictions induced by metabolite lactate or oxygen and glucose deprivation, as an ex vivo model of ischaemic stroke. In anaesthetised rats, Exendin-4 induced lasting increases in brain tissue PO2, indicative of increased cerebral blood flow. These results demonstrate that neuroprotection against ischaemic stroke established by remote ischaemic conditioning is mediated by a mechanism involving GLP-1R signalling. Potent dilatory effect of GLP-1R activation on cortical arterioles suggests that the neuroprotection in this model is mediated via modulation of cerebral blood flow and improved brain perfusion.

Brain metabolic sensing and metabolic signaling at the level of an astrocyte.

Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals-hormones like ghrelin, glucagon-like peptide-1 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever-changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension.

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 ([Ca(2+)]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 Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]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.

A critical role for purinergic signalling in the mechanisms underlying generation of BOLD fMRI responses.

The mechanisms of neurovascular coupling underlying generation of BOLD fMRI signals remain incompletely understood. It has been proposed that release of vasoactive substances by astrocytes couples neuronal activity to changes in cerebrovascular blood flow. However, the role of astrocytes in fMRI responses remains controversial. Astrocytes communicate via release of ATP, and here we tested the hypothesis that purinergic signaling plays a role in the mechanisms underlying fMRI. An established fMRI paradigm was used to trigger BOLD responses in the forepaw region of the somatosensory cortex (SSFP) of an anesthetized rat. Forepaw stimulation induced release of ATP in the SSFP region. To interfere with purinergic signaling by promoting rapid breakdown of the vesicular and/or released ATP, a lentiviral vector was used to express a potent ectonucleotidase, transmembrane prostatic acid phosphatase (TMPAP), in the SSFP region. TMPAP expression had no effect on resting cerebral blood flow, cerebrovascular reactivity, and neuronal responses to sensory stimulation. However, TMPAP catalytic activity markedly reduced the magnitude of BOLD fMRI responses triggered in the SSFP region by forepaw stimulation. Facilitated ATP breakdown could result in accumulation of adenosine. However, blockade of A1 receptors had no effect on BOLD responses and did not reverse the effect of TMPAP. These results suggest that purinergic signaling plays a significant role in generation of BOLD fMRI signals. We hypothesize that astrocytes activated during periods of enhanced neuronal activity release ATP, which propagates astrocytic activation, stimulates release of vasoactive substances and dilation of cerebral vasculature.

fMRI mapping of the visual system in the mouse brain with interleaved snapshot GE-EPI.

The use of functional magnetic resonance imaging (fMRI) in mice is increasingly prevalent, providing a means to non-invasively characterise functional abnormalities associated with genetic models of human diseases. The predominant stimulus used in task-based fMRI in the mouse is electrical stimulation of the paw. Task-based fMRI in mice using visual stimuli remains underexplored, despite visual stimuli being common in human fMRI studies. In this study, we map the mouse brain visual system with BOLD measurements at 9.4T using flashing light stimuli with medetomidine anaesthesia. BOLD responses were observed in the lateral geniculate nucleus, the superior colliculus and the primary visual area of the cortex, and were modulated by the flashing frequency, diffuse vs focussed light and stimulus context. Negative BOLD responses were measured in the visual cortex at 10Hz flashing frequency; but turned positive below 5Hz. In addition, the use of interleaved snapshot GE-EPI improved fMRI image quality without diminishing the temporal contrast-noise-ratio. Taken together, this work demonstrates a novel methodological protocol in which the mouse brain visual system can be non-invasively investigated using BOLD fMRI.

fMRI response to blue light delivery in the naïve brain: implications for combined optogenetic fMRI studies.

The combination of optogenetics and functional magnetic resonance imaging (fMRI) is referred to as opto-fMRI. Optogenetics utilises genetic engineering to introduce light sensitive actuator proteins into cells. Functional MRI (fMRI) is a specialist form of magnetic resonance imaging concerned with imaging changes in blood flow and oxygenation, linked to regional variation in metabolic activity, in the brain. This study describes a methodological concern regarding the effects of light delivery into the brain for the purposes of opto-fMRI. We show that blue light delivery to the naïve rat brain causes profound fMRI responses, despite the absence of optogenetic activation. We demonstrate that these fMRI responses are dependent upon laser power and show that the laser causes significant heating. We identify how heating impacts upon the MR signal causing NMR frequency shifts, and T1 and T2* changes. This study brings attention to a possible confounder which must be taken into account when opto-fMRI experiments are designed.

Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via α2 adrenergic receptors.

Noradrenaline (NA) release from locus coeruleus axons generates vascular contractile tone in arteriolar smooth muscle and contractile capillary pericytes. This tone allows neuronal activity to evoke vasodilation that increases local cerebral blood flow (CBF). Much of the vascular resistance within the brain is located in capillaries and locus coeruleus axons have NA release sites closer to pericytes than to arterioles. In acute brain slices, NA contracted pericytes but did not raise the pericyte cytoplasmic Ca2+ concentration, while the α1 agonist phenylephrine did not evoke contraction. Blocking α2 adrenergic receptors (α2Rs, which induce contraction by inhibiting cAMP production), greatly reduced the NA-evoked pericyte contraction, whereas stimulating α2Rs using xylazine (a sedative) or clonidine (an anti-hypertensive drug) evoked pericyte contraction. Noradrenaline-evoked pericyte contraction and capillary constriction are thus mediated via α2Rs. Consequently, α2Rs may not only modulate CBF in health and pathological conditions, but also contribute to CBF changes evoked by α2R ligands administered in research, veterinary and clinical settings.

Astrocytes produce nitric oxide via nitrite reduction in mitochondria to regulate cerebral blood flow during brain hypoxia.

During hypoxia, increases in cerebral blood flow maintain brain oxygen delivery. Here, we describe a mechanism of brain oxygen sensing that mediates the dilation of intraparenchymal cerebral blood vessels in response to reductions in oxygen supply. In vitro and in vivo experiments conducted in rodent models show that during hypoxia, cortical astrocytes produce the potent vasodilator nitric oxide (NO) via nitrite reduction in mitochondria. Inhibition of mitochondrial respiration mimics, but also occludes, the effect of hypoxia on NO production in astrocytes. Astrocytes display high expression of the molybdenum-cofactor-containing mitochondrial enzyme sulfite oxidase, which can catalyze nitrite reduction in hypoxia. Replacement of molybdenum with tungsten or knockdown of sulfite oxidase expression in astrocytes blocks hypoxia-induced NO production by these glial cells and reduces the cerebrovascular response to hypoxia. These data identify astrocyte mitochondria as brain oxygen sensors that regulate cerebral blood flow during hypoxia via release of nitric oxide.

Cerebral perfusion in untreated, controlled, and uncontrolled hypertension.

This study evaluated the association between systemic arterial blood pressure and cerebral perfusion in 740 participants of the UK's largest tri-ethnic study with measurements of cerebral blood flow (CBF) performed using arterial spin labelling MRI. A significant negative correlation between blood pressure, age and CBF was observed across the patient cohort. The lowest CBF values were recorded in the group of patients with hypertension that were prescribed with anti-hypertensive drugs, but uncontrolled on medication. These findings confirm that hypertension is associated with reduced cerebral perfusion and highlight the importance of blood pressure control for the benefit of maintaining brain blood flow.

CO2 signaling mediates neurovascular coupling in the cerebral cortex.

Neurovascular coupling is a fundamental brain mechanism that regulates local cerebral blood flow (CBF) in response to changes in neuronal activity. Functional imaging techniques are commonly used to record these changes in CBF as a proxy of neuronal activity to study the human brain. However, the mechanisms of neurovascular coupling remain incompletely understood. Here we show in experimental animal models (laboratory rats and mice) that the neuronal activity-dependent increases in local CBF in the somatosensory cortex are prevented by saturation of the CO2-sensitive vasodilatory brain mechanism with surplus of exogenous CO2 or disruption of brain CO2/HCO3- transport by genetic knockdown of electrogenic sodium-bicarbonate cotransporter 1 (NBCe1) expression in astrocytes. A systematic review of the literature data shows that CO2 and increased neuronal activity recruit the same vasodilatory signaling pathways. These results and analysis suggest that CO2 mediates signaling between neurons and the cerebral vasculature to regulate brain blood flow in accord with changes in the neuronal activity.

Remote and Selective Control of Astrocytes by Magnetomechanical Stimulation.

Astrocytes play crucial and diverse roles in brain health and disease. The ability to selectively control astrocytes provides a valuable tool for understanding their function and has the therapeutic potential to correct dysfunction. Existing technologies such as optogenetics and chemogenetics require the introduction of foreign proteins, which adds a layer of complication and hinders their clinical translation. A novel technique, magnetomechanical stimulation (MMS), that enables remote and selective control of astrocytes without genetic modification is described here. MMS exploits the mechanosensitivity of astrocytes and triggers mechanogated Ca2+ and adenosine triphosphate (ATP) signaling by applying a magnetic field to antibody-functionalized magnetic particles that are targeted to astrocytes. Using purpose-built magnetic devices, the mechanosensory threshold of astrocytes is determined, a sub-micrometer particle for effective MMS is identified, the in vivo fate of the particles is established, and cardiovascular responses are induced in rats after particles are delivered to specific brainstem astrocytes. By eliminating the need for device implantation and genetic modification, MMS is a method for controlling astroglial activity with an improved prospect for clinical application than existing technologies.

Astrocytes monitor cerebral perfusion and control systemic circulation to maintain brain blood flow.

Astrocytes provide neurons with essential metabolic and structural support, modulate neuronal circuit activity and may also function as versatile surveyors of brain milieu, tuned to sense conditions of potential metabolic insufficiency. Here we show that astrocytes detect falling cerebral perfusion pressure and activate CNS autonomic sympathetic control circuits to increase systemic arterial blood pressure and heart rate with the purpose of maintaining brain blood flow and oxygen delivery. Studies conducted in experimental animals (laboratory rats) show that astrocytes respond to acute decreases in brain perfusion with elevations in intracellular [Ca2+]. Blockade of Ca2+-dependent signaling mechanisms in populations of astrocytes that reside alongside CNS sympathetic control circuits prevents compensatory increases in sympathetic nerve activity, heart rate and arterial blood pressure induced by reductions in cerebral perfusion. These data suggest that astrocytes function as intracranial baroreceptors and play an important role in homeostatic control of arterial blood pressure and brain blood flow.

Electrochemical carbon fiber-based technique for simultaneous recordings of brain tissue PO2, pH, and extracellular field potentials.

A method for simultaneous electrochemical detection of brain tissue PO2 (PtO2) and pH changes together with neuronal activity using a modified form of fast cyclic voltammetry with carbon fiber electrodes is described. This technique has been developed for in vivo applications and recordings from discrete brain nuclei in experimental animals. The small size of the carbon fiber electrode (⌀7 µm, length <100 µm) ensures minimal disruption of the brain tissue and allows recordings from small brain areas. Sample rate (up to 4 Hz) is sufficient to resolve rapid changes in PtO2 and pH that follow changes in neuronal activity and metabolism. Rapid switching between current and voltage recordings allows combined electrochemical detection and monitoring of extracellular action potentials. For simultaneous electrochemical detection of PtO2 and pH, two consecutive trapezoidal voltage ramps are applied with double differential-subtraction of the background current. This enables changes in current caused by protons and oxygen to be detected separately with minimal interference between the two. The profile of PtO2 changes evoked by increases in local neuronal activity recorded using the described technique was very similar to that of blood-oxygen-level-dependent responses recorded using fMRI. This voltammetric technique can be combined with fMRI and brain vessel imaging to study the metabolic mechanisms underlying neurovascular coupling response with much greater spatial and temporal resolution than is currently possible.

A critical role for the ATP-sensitive potassium channel subunit KIR6.1 in the control of cerebral blood flow.

KIR6.1 (KCNJ8) is a subunit of ATP sensitive potassium channel (KATP) that plays an important role in the control of peripheral vascular tone and is highly expressed in brain contractile cells (vascular smooth muscle cells and pericytes). This study determined the effect of global deletion of the KIR6.1 subunit on cerebral blood flow, neurovascular coupling and cerebral oxygenation in mice. In KIR6.1 deficient mice resting cerebral blood flow and brain parenchymal partial pressure of oxygen (PO2) were found to be markedly lower compared to that in their wildtype littermates. However, cortical blood oxygen level dependent responses triggered by visual stimuli were not affected in conditions of KIR6.1 deficiency. These data suggest that KATP channels containing KIR6.1 subunit are critically important for the maintenance of normal cerebral perfusion and parenchymal PO2 but play no significant role in the mechanisms underlying functional changes in brain blood flow.

Evidence for serotonin synthesis-dependent regulation of in vitro neuronal firing rates in the midbrain raphe complex.

Evidence suggests that 5-hydroxytryptamine 1A (5-HT(1A)) receptor-mediated autoregulation of serotonergic neuronal firing rates is impaired in stress-related neuropsychiatric disorders. In vitro models may provide insight into neural mechanisms underlying regulation of serotonergic systems. However, serotonin synthesis and tonic autoregulation of serotonergic neuronal firing rates are impaired in in vitro preparations lacking tryptophan. We describe the effects of perfusion of living rat brain slices with tryptophan on both 1) tissue concentrations of serotonin metabolites and 2) neuronal firing rates within the dorsal raphe nucleus. Brain slices were perfused with artificial cerebrospinal fluid lacking tryptophan for 4 h, followed by exposure to 1) 40 microM tryptophan (0-60 min) or 2) 0-400 microM tryptophan (23 min) and microdissected for analysis of indole concentrations. Parallel studies examined effects of tryptophan on neuronal firing rates and interactions with drugs expected to alter synaptic concentrations of serotonin. Tryptophan resulted in time-dependent and concentration-dependent increases in serotonin and serotonin metabolites, effects that were correlated with restoration of tonic autoinhibition of dorsal raphe nucleus neuronal firing rates. Inhibition of serotonin synthesis resulted in time-dependent and concentration-dependent increases in 5-hydroxtryptophan that correlated with reversal of the tryptophan-mediated autoinhibition of neuronal firing rates. Tryptophan modulated effects of several drugs on neuronal firing rates, including a selective 5-HT(1A) receptor antagonist (WAY-100635), a monoamine oxidase inhibitor (pargyline), a selective serotonin reuptake inhibitor (fluoxetine), and a serotonin-releasing agent (methylenedioxymethamphetamine). These studies support the hypothesis that tonic autoregulation of serotonergic neuronal firing rates is dependent on tryptophan availability and characterise conditions necessary to study this process in vitro.

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.

In vitro assessment of the agonist properties of the novel 5-HT1A receptor ligand, CUMI-101 (MMP), in rat brain tissue.

INTRODUCTION: Development of agonist positron emission tomography (PET) radioligands for the 5-HT neurotransmitter system is an important target to enable the understanding of human 5-HT function in vivo. [(11)C]CUMI-101, proposed as the first 5-HT(1A) receptor agonist PET ligand, has been reported to behave as a potent 5-HT(1A) agonist in a cellular system stably expressing human recombinant 5-HT(1A) receptors. In this study, we investigate the agonist properties of CUMI-101 in rat brain tissue. METHODS: [(35)S]-GTPγS binding studies were used to determine receptor function in HEK (human embryonic kidney) 293 cells transfected with human recombinant 5-HT(1A) receptors and in rat cortex and rat hippocampal tissue, following administration of CUMI-101 and standard 5-HT1A antagonists (5-HT, 5-CT and 8-OH-DPAT). RESULTS: CUMI-101 behaved as an agonist at human recombinant 5-HT(1A) receptors (pEC(50) 9.2). However, CUMI-101 did not show agonist activity in either rat cortex or hippocampus at concentrations up to 10 µM. In these tissues, CUMI-behaved as an antagonist with pK(B)s of 9.2 and 9.3, respectively. CONCLUSIONS: Our studies demonstrate that as opposed to its behavior in human recombinant system, in rat brain tissue CUMI-101 behaves as a potent 5-HT(1A) receptor antagonist.