The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.

BACKGROUND: Neurovascular coupling (NVC) is a key process in cerebral blood flow regulation. NVC ensures adequate brain perfusion to changes in local metabolic demands. Neuronal nitric oxide synthase (nNOS) is suspected to be involved in NVC; however, this has not been tested in humans. Our objective was to investigate the effects of nNOS inhibition on NVC in humans. METHODS: We performed a 3-visit partially randomized, double-blinded, placebo-controlled, crossover study in 12 healthy subjects. On each visit, subjects received an intravenous infusion of either S-methyl-L-thiocitrulline (a selective nNOS-inhibitor), 0.9% saline (placebo control), or phenylephrine (pressor control). The NVC assessment involved eliciting posterior circulation hyperemia through visual stimulation while measuring posterior and middle cerebral arteries blood velocity. RESULTS: nNOS inhibition blunted the rapidity of the NVC response versus pressor control, evidenced by a reduced initial rise in mean posterior cerebral artery velocity (-3.3% [-6.5, -0.01], P=0.049), and a reduced rate of increase (ie, acceleration) in posterior cerebral artery velocity (slope reduced -4.3% [-8.5, -0.1], P=0.045). The overall magnitude of posterior cerebral artery response relative to placebo control or pressor control was not affected. Changes in BP parameters were well-matched between the S-methyl-L-thiocitrulline and pressor control arms. CONCLUSIONS: Neuronal NOS plays a role in dynamic cerebral blood flow control in healthy adults, particularly the rapidity of the NVC response to visual stimulation. This work opens the way to further investigation of the role of nNOS in conditions of impaired NVC, potentially revealing a therapeutic target.

Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

OBJECTIVE: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. CONCLUSIONS: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Cholecystokinin Switches the Plasticity of GABA Synapses in the Dorsomedial Hypothalamus via Astrocytic ATP Release.

Whether synapses in appetite-regulatory brain regions undergo long-term changes in strength in response to satiety peptides is poorly understood. Here we show that following bursts of afferent activity, the neuromodulator and satiety peptide cholecystokinin (CCK) shifts the plasticity of GABA synapses in the dorsomedial nucleus of the hypothalamus of male Sprague Dawley rats from long-term depression to long-term potentiation (LTP). This LTP requires the activation of both type 2 CCK receptors and group 5 metabotropic glutamate receptors, resulting in a rise in astrocytic intracellular calcium and subsequent ATP release. ATP then acts on presynaptic P2X receptors to trigger a prolonged increase in GABA release. Our observations demonstrate a novel form of CCK-mediated plasticity that requires astrocytic ATP release, and could serve as a mechanism for appetite regulation.SIGNIFICANCE STATEMENT Satiety peptides, like cholecystokinin, play an important role in the central regulation of appetite, but their effect on synaptic plasticity is not well understood. The current data provide novel evidence that cholecystokinin shifts the plasticity from long-term depression to long-term potentiation at GABA synapses in the rat dorsomedial nucleus of the hypothalamus. We also demonstrate that this plasticity requires the concerted action of cholecystokinin and glutamate on astrocytes, triggering the release of the gliotransmitter ATP, which subsequently increases GABA release from neighboring inhibitory terminals. This research reveals a novel neuropeptide-induced switch in the direction of synaptic plasticity that requires astrocytes, and could represent a new mechanism by which cholecystokinin regulates appetite.

Stress-induced structural and functional modifications of astrocytes-Further implicating glia in the central response to stress.

An organism's response to stress requires activation of multiple brain regions. This can have long-lasting effects on synaptic transmission and plasticity that likely provide adaptive benefits. Recent evidence implicates not only neurones, but also glial cells in the regulation of the central response to stress. Intense, repeated or uncontrolled stress has been implicated in the emergence of multiple neuropsychiatric conditions. Human studies have consistently observed glial dysfunction in mood and stress disorders such as major depression. Interestingly animal models of stress have recapitulated glial abnormalities that are comparable to the human condition, validating the use of rodent models for the study of stress disorders. In this review we will focus upon one family of glia, the astrocytes, and describe the evidence to date that links astrocytes to possible stress-related disorders.

Cellular and Ionic Mechanisms of Arterial Vasomotion.

Rhythmical contractility of blood vessels was first observed in bat wing veins by Jones (Philos Trans R Soc Lond 1852:142, 131-136), and subsequently described in arteries and arterioles of multiple vascular beds in several species. Despite an abundance of descriptive literature regarding the presence of vasomotion, to date we do not have an accurate picture of the cellular and ionic basis of these oscillations in tone, or the physiological relevance of the changes in pulsatile blood flow arising from vasomotion. This chapter reviews our current understanding of the cellular and ionic mechanisms underlying vasomotion in resistance arteries and arterioles. Focus is directed to the ion channels, changes in cytosolic Ca2+ concentration, and involvement of intercellular gap junctions in the development and synchronization of rhythmic changes in membrane potential and cytosolic Ca2+ concentration within the vessel wall that contribute to vasomotion. The physiological consequences of vasomotion are discussed with a focus on the cerebral vasculature, as recent advances show that rhythmic oscillations in cerebral arteriolar diameter appear to be entrained by cortical neural activity to increase the local supply of blood flow to active regions of the brain.

Steady-State Free Ca2+ in Astrocytes Is Decreased by Experience and Impacts Arteriole Tone.

Astrocytes can control basal synaptic strength and arteriole tone via their resting Ca2+ activity. However, whether resting astrocyte Ca2+ can adjust to a new steady-state level, with an impact on surrounding brain cells, remains unknown. Using two-photon Ca2+ imaging in male rat acute brain slices of the somatosensory neocortex, we found that theta burst neural activity produced an unexpected long-lasting reduction in astrocyte free Ca2+ in the soma and endfeet. The drop in intracellular Ca2+ was attenuated by antagonists targeting multiple ionotropic and metabotropic glutamate receptors, and intracellular cascades involved Ca2+ stores and nitric oxide. The reduction in astrocyte endfoot Ca2+ was coincident with an increase in arteriole tone, and both the Ca2+ drop and the tone change were prevented by an NMDA receptor antagonist. Astrocyte patch-clamp experiments verified that the glutamate receptors in question were located on astrocytes and that Ca2+ changes within astrocytes were responsible for the long-lasting change in arteriole diameter caused by theta burst neural activity. In astrocytes from animals that lived in an enriched environment, we measured a relatively lower resting Ca2+ level that occluded any further drop in Ca2+ in response to theta burst activity. These data suggest that electrically evoked patterns of neural activity or natural experience can adjust steady-state resting astrocyte Ca2+ and that the effect has an impact on basal arteriole diameter.SIGNIFICANCE STATEMENT The field of astrocyte-neuron and astrocyte-arteriole interactions is currently in a state of refinement. Experimental evidence ex vivo suggests that direct manipulation of astrocyte-free Ca2+ regulates synaptic signaling and local blood flow control; however, in vivo experiments fail to link synaptically evoked astrocyte Ca2+ transients and immediate changes to various astrocyte-mediated processes. To clarify this discrepancy, we examined a different aspect of astrocyte Ca2+: the resting, steady-state free Ca2+ of astrocytes, its modulation, and its potential role in the tonic regulation of surrounding brain cells. We found that ex vivo or in vivo neural activity induced a long-lasting reduction in resting free astrocyte Ca2+ and that this phenomenon changed arteriole tone.

Tonic Local Brain Blood Flow Control by Astrocytes Independent of Phasic Neurovascular Coupling.

According to the current model of neurovascular coupling, blood flow is controlled regionally through phasic changes in the activity of neurons and astrocytes that signal to alter arteriole diameter. Absent in this model, however, is how brain blood flow is tonically regulated independent of regional changes in activity. This is important because a large fraction of brain blood flow is required to maintain basal metabolic needs. Using two-photon fluorescence imaging combined with patch-clamp in acute rat brain slices of sensory-motor cortex, we demonstrate that reducing resting Ca(2+) in astrocytes with intracellular BAPTA causes vasoconstriction in adjacent arterioles. BAPTA-induced vasoconstriction was eliminated by a general COX blocker and the effect is mimicked by a COX-1, but not COX-2, antagonist, suggesting that astrocytes provide tonic, steady-state vasodilation by releasing prostaglandin messengers. Tonic vasodilation was insensitive to TTX, as well as a variety of synaptic and extrasynaptic receptor antagonists, indicating that the phenomenon operates largely independent of neural activity. Using in vivo two-photon fluorescence imaging of the barrel cortex in fully awake mice, we reveal that acute COX-1 inhibition reduces resting arteriole diameter but fails to affect vasodilation in response to vibrissae stimulation. Our findings demonstrate that astrocytes provide tonic regulation of arterioles using resting intracellular Ca(2+) in a manner that is independent of phasic, neuronal-evoked vasodilation. Significance statement: The brain requires both phasic and tonic regulation of its blood supply to service energy needs over various temporal windows. While many mechanisms have been described for phasic blood flow regulation, how the brain accomplishes tonic control is largely unknown. Here we describe a way in which astrocytes contribute to the management of basal brain blood flow by providing steady-state vasodilation to arterioles via resting astrocyte Ca(2+) and the continuous release of prostaglandin messengers. This phenomenon may be important for understanding the declines in basal brain blood flow that occur in aging and dementia, as well as for the interpretation of fMRI data.

Bidirectional Control of Blood Flow by Astrocytes: A Role for Tissue Oxygen and Other Metabolic Factors.

Altering cerebral blood flow through the control of cerebral vessel diameter is critical so that the delivery of molecules important for proper brain functioning is matched to the activity level of neurons. Although the close relationship of brain glia known as astrocytes with cerebral blood vessels has long been recognized, it is only recently that these cells have been demonstrated to translate information on the activity level and energy demands of neurons to the vasculature. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq-protein coupled receptors elevate intracellular calcium via IP3 signaling. A close examination of astrocyte endfeet calcium signals has been shown to cause either vasoconstriction or vasodilation. Common to both vasomotor responses is the generation of arachidonic acid in astrocytes by calcium sensitive phospholipase A2. Vasoconstriction ensues from the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid, while vasodilation ensues from the production of epoxyeicosatrienoic acids or prostaglandins. Factors that determine whether constrictor or dilatory pathways predominate include brain oxygen, lactate, adenosine as well as nitric oxide. Changing the oxygen level itself leads to many downstream changes that facilitate the switch from vasoconstriction at high oxygen to vasodilation at low oxygen. These findings highlight the importance of astrocytes as sensors of neural activity and metabolism to coordinate the delivery of essential nutrients via the blood to the working cells.

Targeting skeletal muscle mitochondria to prevent type 2 diabetes in youth.

The prevalence of type 2 diabetes (T2D) has increased dramatically over the past two decades, not only among adults but also among adolescents. T2D is a systemic disorder affecting every organ system and is especially damaging to the cardiovascular system, predisposing individuals to severe cardiac and vascular complications. The precise mechanisms that cause T2D are an area of active research. Most current theories suggest that the process begins with peripheral insulin resistance that precedes failure of the pancreatic ß-cells to secrete sufficient insulin to maintain normoglycemia. A growing body of literature has highlighted multiple aspects of mitochondrial function, including oxidative phosphorylation, lipid homeostasis, and mitochondrial quality control in the regulation of peripheral insulin sensitivity. Whether the cellular mechanisms of insulin resistance in adults are comparable to that in adolescents remains unclear. This review will summarize both clinical and basic studies that shed light on how alterations in skeletal muscle mitochondrial function contribute to whole body insulin resistance and will discuss the evidence supporting high-intensity exercise training as a therapy to circumvent skeletal muscle mitochondrial dysfunction to restore insulin sensitivity in both adults and adolescents.

Astrocyte and microvascular imaging in awake animals using two-photon microscopy.

Neurovascular coupling is an important control mechanism in CBF regulation. New insights into the integrated relationship between synaptic activity, astrocytes Ca(2+) , and cerebral blood vessels using two-photon fluorescence imaging are slowly emerging. Here, we provide a brief overview of the current understandings and controversies regarding astrocytes in activity-dependent vasodilation. We highlight the key advantages and disadvantages of the in vitro and in vivo methodologies used to study this topic. In particular, we emphasize some of the drawbacks of acute brain slices as well as the confounding effects of anesthesia in in vivo preparations. To overcome these limitations, we discuss an emerging and important trend in imaging cell Ca(2+) and blood flow control in awake and behaving animals. This new approach may help resolve existing controversies on astrocyte control of arteriole diameter by providing a more physiologically relevant preparation to study CBF regulation.

Brain metabolism dictates the polarity of astrocyte control over arterioles.

Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E(2), leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.

Astrocyte Regulation of Cerebral Blood Flow in Health and Disease.

Astrocytes play an important role in controlling microvascular diameter and regulating local cerebral blood flow (CBF) in several physiological and pathological scenarios. Neurotransmitters released from active neurons evoke Ca2+ increases in astrocytes, leading to the release of vasoactive metabolites of arachidonic acid (AA) from astrocyte endfeet. Synthesis of prostaglandin E2 (PGE2) and epoxyeicosatrienoic acids (EETs) dilate blood vessels while 20-hydroxyeicosatetraenoic acid (20-HETE) constricts vessels. The release of K+ from astrocyte endfeet also contributes to vasodilation or constriction in a concentration-dependent manner. Whether astrocytes exert a vasodilation or vasoconstriction depends on the local microenvironment, including the metabolic status, the concentration of Ca2+ reached in the endfoot, and the resting vascular tone. Astrocytes also contribute to the generation of steady-state vascular tone. Tonic release of both 20-HETE and ATP from astrocytes constricts vascular smooth muscle cells, generating vessel tone, whereas tone-dependent elevations in endfoot Ca2+ produce tonic prostaglandin dilators to limit the degree of constriction. Under pathological conditions, including Alzheimer's disease, epilepsy, stroke, and diabetes, disruption of normal astrocyte physiology can compromise the regulation of blood flow, with negative consequences for neurological function.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

Photonics tools begin to clarify astrocyte calcium transients.

Astrocytes integrate information from neurons and the microvasculature to coordinate brain activity and metabolism. Using a variety of calcium-dependent cellular mechanisms, these cells impact numerous aspects of neurophysiology in health and disease. Astrocyte calcium signaling is highly diverse, with complex spatiotemporal features. Here, we review astrocyte calcium dynamics and the optical imaging tools used to measure and analyze these events. We briefly cover historical calcium measurements, followed by our current understanding of how calcium transients relate to the structure of astrocytes. We then explore newer photonics tools including super-resolution techniques and genetically encoded calcium indicators targeted to specific cellular compartments and how these have been applied to astrocyte biology. Finally, we provide a brief overview of analysis software used to accurately quantify the data and ultimately aid in our interpretation of the various functions of astrocyte calcium transients.

Astrocytes amplify neurovascular coupling to sustained activation of neocortex in awake mice.

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca2+ signaling in vivo by expressing a plasma membrane Ca2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca2+ is to amplify functional hyperemia when neuronal activation is prolonged.

Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Bidirectional control of arteriole diameter by astrocytes.

Astrocytes are the most numerous cells in the CNS. It is a defining feature of brain anatomy that every astrocyte has at least one contact with the vasculature, termed an endfoot. Collectively, all endfeet completely circumscribe all vessels in the brain. This unique anatomical feature has profound functional significance, as astrocyte endfeet have been discovered to release diffusible messengers that communicate directly with underlying smooth muscle cells to change arterial diameter and thereby regulate cerebral blood flow. A growing body of data now demonstrates that astrocytes serve as a bridge, relaying information on the level of neural activity to blood vessels in order to co-ordinate oxygen and glucose delivery with the energy demands of the tissue. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq-coupled receptors elevate intracellular calcium via IP(3) signalling, which activates phospholipase A2 and generates arachidonic acid. Arachidonic acid acts as a signalling molecule or is converted to several lipid derivates, including prostaglandin E(2) and epoxyeicosatrienoic acids. Each of these lipids acts on vascular smooth muscle cells via different mechanisms to affect vessel diameter. Arachidonic acid initiates the production of 20-hydroxyeicosatetraenoic acid to cause vasoconstriction, whereas prostaglandin E(2) and epoxyeicosatrienoic acids cause vasodilatation. Factors that determine whether constrictor or dilatory pathways predominate involve nitric oxide and brain metabolic elements, such as oxygen, lactate and adenosine. Thus, astrocytes are thought to be capable of bidirectional control of arterial diameter, and the type of influence depends on the state of brain activity.

Transient swelling, acidification, and mitochondrial depolarization occurs in neurons but not astrocytes during spreading depression.

Cortical spreading depression (SD) is a propagating wave of neuronal and glial depolarization that manifests in several brain disorders. However, the relative contribution of neurons and astrocytes to SD genesis has remained controversial. This is in part due to a lack of utilizing sophisticated experimental methodologies simultaneously to quantify multiple cellular parameters. To address this, we used simultaneous two-photon imaging, intrinsic optical imaging, and electrophysiological recordings to ascertain the changes in cellular processes that are fundamental to both cell types including cell volume, pH, and metabolism during SD propagation. We found that SD was correlated in neurons with robust yet transient increased volume, intracellular acidification, and mitochondrial depolarization. Our data indicated that a propagating large conductance during SD generated neuronal depolarization, which led to both calcium influx triggering metabolic changes and H(+) entry. Notably, astrocytes did not exhibit changes in cell volume, pH, or mitochondrial membrane potentials associated with SD, but they did show alterations induced by changing external [K(+)]. This suggests that astrocytes are not the primary contributor to SD propagation but are instead activated passively by extracellular potassium accumulation. These data support the hypothesis that neurons are the crucial cell type contributing to the pathophysiological responses of SD.

Obesity-induced astrocyte dysfunction impairs heterosynaptic plasticity in the orbitofrontal cortex.

Overconsumption of highly palatable, energy-dense food is considered a key driver of the obesity pandemic. The orbitofrontal cortex (OFC) is critical for reward valuation of gustatory signals, yet how the OFC adapts to obesogenic diets is poorly understood. Here, we show that extended access to a cafeteria diet impairs astrocyte glutamate clearance, which leads to a heterosynaptic depression of GABA transmission onto pyramidal neurons of the OFC. This decrease in GABA tone is due to an increase in extrasynaptic glutamate, which acts via metabotropic glutamate receptors to liberate endocannabinoids. This impairs the induction of endocannabinoid-mediated long-term plasticity. The nutritional supplement, N-acetylcysteine rescues this cascade of synaptic impairments by restoring astrocytic glutamate transport. Together, our findings indicate that obesity targets astrocytes to disrupt the delicate balance between excitatory and inhibitory transmission in the OFC.

Whole brain irradiation in mice causes long-term impairment in astrocytic calcium signaling but preserves astrocyte-astrocyte coupling.

Whole brain irradiation (WBI) therapy is an important treatment for brain metastases and potential microscopic malignancies. WBI promotes progressive cognitive dysfunction in over half of surviving patients, yet, the underlying mechanisms remain obscure. Astrocytes play critical roles in the regulation of neuronal activity, brain metabolism, and cerebral blood flow, and while neurons are considered radioresistant, astrocytes are sensitive to γ-irradiation. Hallmarks of astrocyte function are the ability to generate stimulus-induced intercellular Ca2+ signals and to move metabolic substrates through the connected astrocyte network. We tested the hypothesis that WBI-induced cognitive impairment associates with persistent impairment of astrocytic Ca2+ signaling and/or gap junctional coupling. Mice were subjected to a clinically relevant protocol of fractionated WBI, and 12 to 15 months after irradiation, we confirmed persistent cognitive impairment compared to controls. To test the integrity of astrocyte-to-astrocyte gap junctional coupling postWBI, astrocytes were loaded with Alexa-488-hydrazide by patch-based dye infusion, and the increase of fluorescence signal in neighboring astrocyte cell bodies was assessed with 2-photon microscopy in acute slices of the sensory-motor cortex. We found that WBI did not affect astrocyte-to-astrocyte gap junctional coupling. Astrocytic Ca2+ responses induced by bath administration of phenylephrine (detected with Rhod-2/AM) were also unaltered by WBI. However, an electrical stimulation protocol used in long-term potentiation (theta burst), revealed attenuated astrocyte Ca2+ responses in the astrocyte arbor and soma in WBI. Our data show that WBI causes a long-lasting decrement in synaptic-evoked astrocyte Ca2+ signals 12-15 months postirradiation, which may be an important contributor to cognitive decline seen after WBI.