Precapillary sphincters and pericytes at first-order capillaries as key regulators for brain capillary perfusion.

Rises in local neural activity trigger local increases of cerebral blood flow, which is essential to match local energy demands. However, the specific location of microvascular flow control is incompletely understood. Here, we used two-photon microscopy to observe brain microvasculature in vivo. Small spatial movement of a three-dimensional (3D) vasculature makes it challenging to precisely measure vessel diameter at a single x-y plane. To overcome this problem, we carried out four-dimensional (x-y-z-t) imaging of brain microvessels during exposure to vasoactive molecules in order to constrain the impact of brain movements on the recordings. We demonstrate that rises in synaptic activity, acetylcholine, nitric oxide, cyclic guanosine monophosphate, ATP-sensitive potassium channels, and endothelin-1 exert far greater effects on brain precapillary sphincters and first-order capillaries than on penetrating arterioles or downstream capillaries, but with similar kinetics. The high level of responsiveness at precapillary sphincters and first-order capillaries was matched by a higher level of α-smooth muscle actin in pericytes as compared to penetrating arterioles and downstream capillaries. Mathematical modeling based on 3D vasculature reconstruction showed that precapillary sphincters predominantly regulate capillary blood flow and pressure as compared to penetrating arterioles and downstream capillaries. Our results confirm a key role for precapillary sphincters and pericytes on first-order capillaries as sensors and effectors of endothelium- or brain-derived vascular signals.

PV interneurons evoke astrocytic Ca2+ responses in awake mice, which contributes to neurovascular coupling.

Neurovascular coupling (NVC) modulates cerebral blood flow to match increased metabolic demand during neuronal excitation. Activation of inhibitory interneurons also increase blood flow, but the basis for NVC caused by interneurons is unclear. While astrocyte Ca2+ levels rise with excitatory neural transmission, much less is known with regards to astrocytic sensitivity to inhibitory neurotransmission. We performed two-photon microscopy in awake mice to examine the correlation between astrocytic Ca2+ and NVC, evoked by activation of either all (VGATIN ) or only parvalbumin-positive GABAergic interneurons (PVIN ). Optogenetic stimulation of VGATIN and PVIN in the somatosensory cortex triggered astrocytic Ca2+ increases that were abolished by anesthesia. In awake mice, PVIN evoked astrocytic Ca2+ responses with a short latency that preceded NVC, whereas VGATIN evoked Ca2+ increases that were delayed relative to the NVC response. The early onset of PVIN evoked astrocytic Ca2+ increases depended on noradrenaline release from locus coeruleus as did the subsequent NVC response. Though the relationship between interneuron activity and astrocytic Ca2+ responses is complex, we suggest that the rapid astrocyte Ca2+ responses to increased PVIN activity shaped the NVC. Our results underline that interneuron and astrocyte-dependent mechanisms should be studied in awake mice.

Spreading Depression, Spreading Depolarizations, and the Cerebral Vasculature.

Spreading depression (SD) is a transient wave of near-complete neuronal and glial depolarization associated with massive transmembrane ionic and water shifts. It is evolutionarily conserved in the central nervous systems of a wide variety of species from locust to human. The depolarization spreads slowly at a rate of only millimeters per minute by way of grey matter contiguity, irrespective of functional or vascular divisions, and lasts up to a minute in otherwise normal tissue. As such, SD is a radically different breed of electrophysiological activity compared with everyday neural activity, such as action potentials and synaptic transmission. Seventy years after its discovery by Leão, the mechanisms of SD and its profound metabolic and hemodynamic effects are still debated. What we did learn of consequence, however, is that SD plays a central role in the pathophysiology of a number of diseases including migraine, ischemic stroke, intracranial hemorrhage, and traumatic brain injury. An intriguing overlap among them is that they are all neurovascular disorders. Therefore, the interplay between neurons and vascular elements is critical for our understanding of the impact of this homeostatic breakdown in patients. The challenges of translating experimental data into human pathophysiology notwithstanding, this review provides a detailed account of bidirectional interactions between brain parenchyma and the cerebral vasculature during SD and puts this in the context of neurovascular diseases.

Shedding Light on the Blood-Brain Barrier Transport with Two-Photon Microscopy In Vivo.

Treatment of brain disorders relies on efficient delivery of therapeutics to the brain, which is hindered by the blood-brain barrier (BBB). The work of Prof. Margareta Hammarlund-Udenaes was instrumental in understanding the principles of drug delivery to the brain and developing new tools to study it. Here, we show how some of the concepts developed in her research can be translated to in vivo 2-photon microscopy (2PM) studies of the BBB. We primarily focus on the methods developed in our laboratory to characterize the paracellular diffusion, adsorptive-mediated transcytosis, and receptor-mediated transcytosis of drug nanocarriers at the microscale, illustrating how 2PM can deepen our understanding of the mechanisms of drug delivery to the brain.

Brain barriers and their potential role in migraine pathophysiology.

Migraine is a ubiquitous neurologic disease that afflicts people of all ages. Its molecular pathogenesis involves peptides that promote intracranial vasodilation and modulate nociceptive transmission upon release from sensory afferents of cells in the trigeminal ganglion and parasympathetic efferents of cells in the sphenopalatine ganglion. Experimental data have confirmed that intravenous infusion of these vasoactive peptides induce migraine attacks in people with migraine, but it remains a point of scientific contention whether their site of action lies outside or within the central nervous system. In this context, it has been hypothesized that transient dysfunction of brain barriers before or during migraine attacks might facilitate the passage of migraine-inducing peptides into the central nervous system. Here, we review evidence suggestive of brain barrier dysfunction in migraine pathogenesis and conclude with lessons learned in order to provide directions for future research efforts.

ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors.

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood-brain barrier.

The endothelial cells that form the blood-brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules-sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran-from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

Stimulation-induced increases in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses.

Functional neuroimaging, such as fMRI, is based on coupling neuronal activity and accompanying changes in cerebral blood flow (CBF) and metabolism. However, the relationship between CBF and events at the level of the penetrating arterioles and capillaries is not well established. Recent findings suggest an active role of capillaries in CBF control, and pericytes on capillaries may be major regulators of CBF and initiators of functional imaging signals. Here, using two-photon microscopy of brains in living mice, we demonstrate that stimulation-evoked increases in synaptic activity in the mouse somatosensory cortex evokes capillary dilation starting mostly at the first- or second-order capillary, propagating upstream and downstream at 5-20 µm/s. Therefore, our data support an active role of pericytes in cerebrovascular control. The gliotransmitter ATP applied to first- and second-order capillaries by micropipette puffing induced dilation, followed by constriction, which also propagated at 5-20 µm/s. ATP-induced capillary constriction was blocked by purinergic P2 receptors. Thus, conducted vascular responses in capillaries may be a previously unidentified modulator of cerebrovascular function and functional neuroimaging signals.

Brain capillary pericytes and neurovascular coupling.

The neurovascular coupling ensures that cerebral activity is matched by the relevant blood flow. The control of the blood flow is mediated by capillaries and by the precapillary aterioles. It is the tone of the mural cells, which include pericytes, smooth muscle cells and cells with intermediate phenotypes between pericytes and smooth muscle cells, that determine the the diameter of the blood vessels and consequently the flow. Here we discuss the structure of these blood vessels and the excitationcontraction coupling of the mural cells.

Capillary pericytes regulate cerebral blood flow in health and disease.

Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.

Continuous EEG Monitoring in a Consecutive Patient Cohort with Sepsis and Delirium.

BACKGROUND: Delirium is common during sepsis, although under-recognized. We aimed to assess the value of continuous electroencephalography (cEEG) to aid in the diagnosis of delirium in septic patients. METHODS: We prospectively evaluated 102 consecutive patients in a medical intensive care unit (ICU), who had sepsis or septic shock, without evidence of acute primary central nervous system disease. We initiated cEEG recording immediately after identification. The median cEEG time per patient was 44 h (interquartile range 21-99 h). A total of 6723 h of cEEG recordings were examined. The Confusion Assessment Method for the ICU (CAM-ICU) was administered six times daily to identify delirium. We analyzed the correlation between cEEG and delirium using 1252 two-minute EEG sequences recorded simultaneously with the CAM-ICU scorings. RESULTS: Of the 102 included patients, 66 (65%) had at least one delirium episode during their ICU stay, 30 (29%) remained delirium-free, and 6 (6%) were not assessable due to deep sedation or coma. The absence of delirium was independently associated with preserved high-frequency beta activity (> 13 Hz) (P < 10-7) and cEEG reactivity (P < 0.001). Delirium was associated with preponderance of low-frequency cEEG activity and absence of high-frequency cEEG activity. Sporadic periodic cEEG discharges occurred in 15 patients, 13 of whom were delirious. No patient showed clinical or electrographic evidence of non-convulsive status epilepticus. CONCLUSIONS: Our findings indicate that cEEG can help distinguish septic patients with delirium from non-delirious patients.

Spontaneous astrocytic Ca2+ activity abounds in electrically suppressed ischemic penumbra of aged mice.

Experimental focal cortical ischemic lesions consist of an ischemic core and a potentially salvageable peri-ischemic region, the ischemic penumbra. The activity of neurons and astrocytes is assumed to be suppressed in the penumbra because the electrical function is interrupted, but this is incompletely elucidated. Most experimental stroke studies used young adult animals, whereas stroke is prevalent in the elderly population. Using two-photon imaging in vivo, we here demonstrate extensive but electrically silent, spontaneous Ca2+ activity in neurons and astrocytes in the ischemic penumbra of 18- to 24-month-old mice 2-4 hr after middle cerebral artery occlusion. In comparison, stroke reduced spontaneous Ca2+ activity in neurons and astrocytes in adult mice (3-4 months of age). In aged mice, stroke increased astrocytic spontaneous Ca2+ activity considerably while neuronal spontaneous Ca2+ activity was unchanged. Blockade of action potentials and of purinergic receptors strongly reduced spontaneous Ca2+ activity in both neurons and astrocytes in the penumbra of old stroke mice. This indicates that stroke had a direct influence on mechanisms in presynaptic terminals and on purinergic signaling. Thus, highly dynamic variations in spontaneous Ca2+ activity characterize the electrically compromised penumbra, with remarkable differences between adult and old mice. The data are consistent with the notion that aged neurons and astrocytes take on a different phenotype than young mice. The increased activity of the aged astrocyte phenotype may be harmful to neurons. We suggest that the abundant spontaneous Ca2+ activity in astrocytes in the ischemic penumbra of old mice may be a novel target for neuroprotection strategies. A video abstract of this article can be found at https://youtu.be/AKlwKFsz1qE.

Sleep efficiency and neurophysiological patterns in middle-aged men are associated with cognitive change over their adult life course.

Disrupted sleep is a contributing factor to cognitive ageing, while also being associated with neurodegenerative disorders. Little is known, however, about the relation of sleep and the gradual cognitive changes over the adult life course. Sleep electroencephalogram (EEG) patterns are potential markers of the cognitive progress. To test this hypothesis, we assessed sleep architecture and EEG of 167 men born in the Copenhagen Metropolitan Area in 1953, who, based on individual cognitive testing from early (~18 years) to late adulthood (~58 years), were divided into 85 subjects with negative and 82 with positive cognitive change over their adult life. Participants underwent standard polysomnography, including manual sleep scoring at age ~58 years. Features of sleep macrostructure were combined with a number of EEG features to distinguish between the two groups. EEG rhythmicity was assessed by spectral power analysis in frontal, central and occipital sites. Functional connectivity was measured by inter-hemispheric EEG coherence. Group differences were assessed by analysis of covariance (p < 0.05), including education and severity of depression as potential covariates. Subjects with cognitive decline exhibited lower sleep efficiency, reduced inter-hemispheric connectivity during rapid eye movement (REM) sleep, and slower EEG rhythms during stage 2 non-REM sleep. Individually, none of these tendencies remained significant after multiple test correction; however, by combining them in a machine learning approach, the groups were separated with 72% accuracy (75% sensitivity, 67% specificity). Ongoing medical screenings are required to confirm the potential of sleep efficiency and sleep EEG patterns as signs of individual cognitive progress.

CaMKII-dependent endoplasmic reticulum fission by whisker stimulation and during cortical spreading depolarization.

Cortical spreading depolarization waves, the cause underlying migraine aura, are also the markers and mechanism of pathology in the acutely injured human brain. Propagation of spreading depolarization wave uniquely depends on the interaction between presynaptic and postsynaptic glutamate N-methyl-d-aspartate receptors (NMDARs). In the normally perfused brain, even a single wave causes a massive depolarization of neurons and glia, which results in transient loss of neuronal function and depression of the ongoing electrocorticographic activity. Endoplasmic reticulum is the cellular organelle of particular importance for modulation of neurotransmission. Neuronal endoplasmic reticulum structure is assumed to be persistently continuous in neurons, but is rapidly lost within 1 to 2 min of global cerebral ischaemia, i.e. the organelle disintegrates by fission. This phenomenon appears to be timed with the cardiac arrest-induced cortical spreading depolarizations, rather than ensuing cell death. To what extent NMDAR-dependent processes may trigger neuronal endoplasmic reticulum fission and whether fission is reversible in the normally perfused brain is unknown. We used two-photon microscopy to examine neuronal endoplasmic reticulum structural dynamics during whisker stimulation and cortical spreading depolarizations in vivo. Somatosensory stimulation triggered loss of endoplasmic reticulum continuity, a likely outcome of constriction and fission, in dendritic spines within less than 10 s of stimulation, which was spontaneously reversible and recovery to normal took 5 min. The endoplasmic reticulum fission was inhibited by blockade of NMDAR and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activated downstream of the NMDARs, whereas inhibition of guanosine triphosphate hydrolases hindered regain of endoplasmic reticulum continuity, i.e. fusion. In contrast to somatosensory stimulation, endoplasmic reticulum fission during spreading depolarization was widespread and present in dendrites and spines, and was preceded by dramatic rise in intracellular Ca2+. The endoplasmic reticulum fission during spreading depolarization was more persistent, as 1 h after the depolarization cortical neurons still exhibited loss of endoplasmic reticulum continuity. Notably, endoplasmic reticulum fission was accompanied with loss of electrocorticographic activity, whereas subsequent regain of synaptic function paralleled the organelle fusion. Furthermore, blocking CaMKII activity partly rescued endoplasmic reticulum fission and markedly shortened the recovery time of brain spontaneous activity. Thus, prevention of endoplasmic reticulum fission with CaMKII inhibitors may be a novel strategy to rescue brain function in patients with migraine and a promising therapeutic avenue in the acutely injured brain.

Active role of capillary pericytes during stimulation-induced activity and spreading depolarization.

Spreading depolarization is assumed to be the mechanism of migraine with aura, which is accompanied by an initial predominant hyperaemic response followed by persistent vasoconstriction. Cerebral blood flow responses are impaired in patients and in experimental animals after spreading depolarization. Understanding the regulation of cortical blood vessels during and after spreading depolarization could help patients with migraine attacks, but our knowledge of these vascular mechanisms is still incomplete. Recent findings show that control of cerebral blood flow does not only occur at the arteriole level but also at capillaries. Pericytes are vascular mural cells that can constrict or relax around capillaries, mediating local cerebral blood flow control. They participate in the constriction observed during brain ischaemia and might be involved the disruption of the microcirculation during spreading depolarization. To further understand the regulation of cerebral blood flow in spreading depolarization, we examined penetrating arterioles and capillaries with respect to vascular branching order, pericyte location and pericyte calcium responses during somatosensory stimulation and spreading depolarization. Mice expressing a red fluorescent indicator and intravenous injections of FITC-dextran were used to visualize pericytes and vessels, respectively, under two-photon microscopy. By engineering a genetically encoded calcium indicator we could record calcium changes in both pericytes around capillaries and vascular smooth muscle cells around arterioles. We show that somatosensory stimulation evoked a decrease in cytosolic calcium in pericytes located on dilating capillaries, up to the second order capillaries. Furthermore, we show that prolonged vasoconstriction following spreading depolarization is strongest in first order capillaries, with a persistent increase in pericyte calcium. We suggest that the persistence of the 'spreading cortical oligaemia' in migraine could be caused by this constriction of cortical capillaries. After spreading depolarization, somatosensory stimulation no longer evoked changes in capillary diameter and pericyte calcium. Thus, calcium changes in pericytes located on first order capillaries may be a key determinant in local blood flow control and a novel vascular mechanism in migraine. We suggest that prevention or treatment of capillary constriction in migraine with aura, which is an independent risk factor for stroke, may be clinically useful.

Neurostereologic Lesion Volumes and Spreading Depolarizations in Severe Traumatic Brain Injury Patients: A Pilot Study.

BACKGROUND: Spreading depolarizations (SDs) occur in 50-60% of patients after surgical treatment of severe traumatic brain injury (TBI) and are independently associated with unfavorable outcomes. Here we performed a pilot study to examine the relationship between SDs and various types of intracranial lesions, progression of parenchymal damage, and outcomes. METHODS: In a multicenter study, fifty patients (76% male; median age 40) were monitored for SD by continuous electrocorticography (ECoG; median duration 79 h) following surgical treatment of severe TBI. Volumes of hemorrhage and parenchymal damage were estimated using unbiased stereologic assessment of preoperative, postoperative, and post-ECoG serial computed tomography (CT) studies. Neurologic outcomes were assessed at 6 months by the Glasgow Outcome Scale-Extended. RESULTS: Preoperative volumes of subdural and subarachnoid hemorrhage, but not parenchymal damage, were significantly associated with the occurrence of SDs (P's < 0.05). Parenchymal damage increased significantly (median 34 ml [Interquartile range (IQR) - 2, 74]) over 7 (5, 8) days from preoperative to post-ECoG CT studies. Patients with and without SDs did not differ in extent of parenchymal damage increase [47 ml (3, 101) vs. 30 ml (- 2, 50), P = 0.27], but those exhibiting the isoelectric subtype of SDs had greater initial parenchymal damage and greater increases than other patients (P's < 0.05). Patients with temporal clusters of SDs (≥ 3 in 2 h; n = 10 patients), which included those with isoelectric SDs, had worse outcomes than those without clusters (P = 0.03), and parenchymal damage expansion also correlated with worse outcomes (P = 0.01). In multivariate regression with imputation, both clusters and lesion expansion were significant outcome predictors. CONCLUSIONS: These results suggest that subarachnoid and subdural blood are important primary injury factors in provoking SDs and that clustered SDs and parenchymal lesion expansion contribute independently to worse patient outcomes. These results warrant future prospective studies using detailed quantification of TBI lesion types to better understand the relationship between anatomic and physiologic measures of secondary injury.

A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain.

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO2, arterial O2, and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E2 (PgE2) plays a key role for cerebrovascular CO2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2) evokes an increase in astrocyte [Ca2+]i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2 is decreased and vasodilation triggered by increased astrocyte [Ca2+]iin vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO2 Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO2, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE2 We demonstrate that hypercapnia (increased CO2) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO2-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.

Spinal dorsal horn astrocytes release GABA in response to synaptic activation.

KEY POINTS: GABA is an essential molecule for sensory information processing. It is usually assumed to be released by neurons. Here we show that in the dorsal horn of the spinal cord, astrocytes respond to glutamate by releasing GABA. Our findings suggest a novel role for astrocytes in somatosensory information processing. ABSTRACT: Astrocytes participate in neuronal signalling by releasing gliotransmitters in response to neurotransmitters. We investigated if astrocytes from the dorsal horn of the spinal cord of adult red-eared turtles (Trachemys scripta elegans) release GABA in response to glutamatergic receptor activation. For this, we developed a GABA sensor consisting of HEK cells expressing GABAA receptors. By positioning the sensor recorded in the whole-cell patch-clamp configuration within the dorsal horn of a spinal cord slice, we could detect GABA in the extracellular space. Puff application of glutamate induced GABA release events with time courses that exceeded the duration of inhibitory postsynaptic currents by one order of magnitude. Because the events were neither affected by extracellular addition of nickel, cadmium and tetrodotoxin nor by removal of Ca2+ , we concluded that they originated from non-neuronal cells. Immunohistochemical staining allowed the detection of GABA in a fraction of dorsal horn astrocytes. The selective stimulation of A∂ and C fibres in a dorsal root filament induced a Ca2+ increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca2+ in a single astrocyte was sufficient to prevent the GABA release evoked by glutamate. Our results indicate that glutamate triggers the release of GABA from dorsal horn astrocytes with a time course compatible with the integration of sensory inputs.

Fast Ca2+ responses in astrocyte end-feet and neurovascular coupling in mice.

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity-dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca2+ changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca2+ changes in neuropil, astrocyte processes, and astrocyte end-feet in response to whisker pad stimulation in mice. We also developed a pixel-based analysis to improve the detection of rapid Ca2+ signals in the subcellular compartments of astrocytes. Fast Ca2+ responses were observed using both chemical and genetically encoded Ca2+ indicators in astrocyte end-feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK801) attenuated fast Ca2+ responses in the neuropil and astrocyte processes, but not in astrocyte end-feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), an agonist for the extrasynaptic GABAA receptor (GABAA R), increased CBF responses and the fast Ca2+ response in astrocyte end-feet but did not affect Ca2+ responses in astrocyte processes and neuropil. These results suggest that fast Ca2+ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca2+ responses in astrocyte end-feet are unaffected by MK801 but increase via GABAA R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca2+ increases in end-feet adjust CBF during synaptic activity.

Interneuron Deficit Associates Attenuated Network Synchronization to Mismatch of Energy Supply and Demand in Aging Mouse Brains.

Higher cognitive functions depend critically on synchronized network activity in the gamma range (30-100 Hz), which results from activity of fast-spiking parvalbumin-positive (PV) interneurons. Here, we examined synaptic activity in the gamma band in relation to PV interneuron activity, stimulation-induced calcium activity in neurons and astrocytes, and cerebral blood flow and oxygen responses in the somatosensory cortex of young adult and old adult mice in vivo using electrical whisker pad stimulation. Gamma activity was reduced in old adult mice, and associated with reduced calcium activity of PV interneurons, whereas the overall responses of neurons and astrocytes were unchanged. Hemodynamic responses were highly correlated to the power of synaptic activity in both young adult and old adult mice, but the hemodynamic response amplitude attained was lower in old adult mice. In comparison, the work-dependent rise in O2 use, that is, the rise in the cerebral metabolic rate of oxygen (CMRO2) evoked by excitatory postsynaptic currents almost doubled in old adult mice. We conclude that PV interneuron function and gamma activity are particularly affected in old adult mice. Alterations in neurovascular coupling and CMRO2 responses may contribute to increased frailty and risk of cognitive decline in aged brains.