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.

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.

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.

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.

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.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

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.

Astrocytes regulate ultra-slow arteriole oscillations via stretch-mediated TRPV4-COX-1 feedback.

Very-low-frequency oscillations in microvascular diameter cause fluctuations in oxygen delivery that are important for fueling the brain and for functional imaging. However, little is known about how the brain regulates ongoing oscillations in cerebral blood flow. In mouse and rat cortical brain slice arterioles, we find that selectively enhancing tone is sufficient to recruit a TRPV4-mediated Ca2+ elevation in adjacent astrocyte endfeet. This endfoot Ca2+ signal triggers COX-1-mediated "feedback vasodilators" that limit the extent of evoked vasoconstriction, as well as constrain fictive vasomotion in slices. Astrocyte-Ptgs1 knockdown in vivo increases the power of arteriole oscillations across a broad range of very low frequencies (0.01-0.3 Hz), including ultra-slow vasomotion (∼0.1 Hz). Conversely, clamping astrocyte Ca2+in vivo reduces the power of vasomotion. These data demonstrate bidirectional communication between arterioles and astrocyte endfeet to regulate oscillatory microvasculature activity.

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.

Heterogeneity of Sensory-Induced Astrocytic Ca2+ Dynamics During Functional Hyperemia.

Astrocytic Ca2+ fluctuations associated with functional hyperemia have typically been measured from large cellular compartments such as the soma, the whole arbor and the endfoot. The most prominent Ca2+ event is a large magnitude, delayed signal that follows vasodilation. However, previous work has provided little information about the spatio-temporal properties of such Ca2+ transients or their heterogeneity. Here, using an awake, in vivo two-photon fluorescence-imaging model, we performed detailed profiling of delayed astrocytic Ca2+ signals across astrocytes or within individual astrocyte compartments using small regions of interest next to penetrating arterioles and capillaries along with vasomotor responses to vibrissae stimulation. We demonstrated that while a 5-s air puff that stimulates all whiskers predominantly generated reproducible functional hyperemia in the presence or absence of astrocytic Ca2+ changes, whisker stimulation inconsistently produced astrocytic Ca2+ responses. More importantly, these Ca2+ responses were heterogeneous among subcellular structures of the astrocyte and across different astrocytes that resided within the same field of view. Furthermore, we found that whisker stimulation induced discrete Ca2+ "hot spots" that spread regionally within the endfoot. These data reveal that astrocytic Ca2+ dynamics associated with the microvasculature are more complex than previously thought, and highlight the importance of considering the heterogeneity of astrocytic Ca2+ activity to fully understanding neurovascular coupling.

Seizures elevate gliovascular unit Ca2+ and cause sustained vasoconstriction.

Seizures can result in a severe hypoperfusion/hypoxic attack that causes postictal memory and behavioral impairments. However, neither postictal changes to microvasculature nor Ca2+ changes in key cell types controlling blood perfusion have been visualized in vivo, leaving essential components of the underlying cellular mechanisms unclear. Here, we use 2-photon microvascular and Ca2+ imaging in awake mice to show that seizures result in a robust vasoconstriction of cortical penetrating arterioles, which temporally mirrors the prolonged postictal hypoxia. The vascular effect was dependent on cyclooxygenase 2, as pretreatment with ibuprofen prevented postictal vasoconstriction. Moreover, seizures caused a rapid elevation in astrocyte endfoot Ca2+ that was confined to the seizure period, and vascular smooth muscle cells displayed a significant increase in Ca2+ both during and following seizures, lasting up to 75 minutes. Our data show enduring postictal vasoconstriction and temporal activities of 2 cell types within the neurovascular unit that are associated with seizure-induced hypoperfusion/hypoxia. These findings support prevention of this event may be a novel and tractable treatment strategy in patients with epilepsy who experience extended postseizure impairments.

Limiting RyR2 Open Time Prevents Alzheimer's Disease-Related Neuronal Hyperactivity and Memory Loss but Not ß-Amyloid Accumulation.

Neuronal hyperactivity is an early primary dysfunction in Alzheimer's disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued ß-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-ß-amyloid-targeted anti-AD strategy.

Author Correction: Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.

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.

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.

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.

Astrocytes Integrate Behavioral State and Vascular Signals during Functional Hyperemia.

Dynamic changes in astrocyte free Ca2+ regulate synaptic signaling and local blood flow. Although astrocytes are poised to integrate signals from synapses and the vasculature to perform their functional roles, it remains unclear what dictates astrocyte responses during neurovascular coupling under realistic conditions. We examined peri-arteriole and peri-capillary astrocytes in the barrel cortex of active mice in response to sensory stimulation or volitional behaviors. We observed an AMPA and NMDA receptor-dependent elevation in astrocyte endfoot Ca2+ that followed functional hyperemia onset. This delayed astrocyte Ca2+ signal was dependent on the animal's action at the time of measurement as well as a neurovascular pathway that linked to endothelial-derived nitric oxide. A similar elevation in endfoot Ca2+ was evoked using vascular chemogenetics or optogenetics, and opto-stimulated dilation recruited the same nitric oxide pathway as functional hyperemia. These data show that behavioral state and microvasculature influence astrocyte Ca2+ in active mice. VIDEO ABSTRACT.