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.