Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulationinduced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca 2+ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca 2+ signals is due to a signal-enhancing analysis of the Ca 2+ activity. The rapid stimulation-evoked Ca 2+ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca 2+ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca 2+ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity. and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1-5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca 2+ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7-10). Ca 2+ responses are of interest because intracellular Ca 2+ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca 2+ -dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca 2+ changes (11). Astrocytic Ca 2+ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8,10,12,13). Furthermore, uncaging of Ca 2+ in astrocytes triggers vascular responses in brain slices through specific Ca 2+ -dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca 2+ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16,17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca 2+ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca 2+ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca 2+ responses were brief en...

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 Ca changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca 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 Ca signals in the subcellular compartments of astrocytes. Fast Ca responses were observed using both chemical and genetically encoded Ca 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 Ca 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 GABA receptor (GABA R), increased CBF responses and the fast Ca response in astrocyte end-feet but did not affect Ca responses in astrocyte processes and neuropil. These results suggest that fast Ca increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca responses in astrocyte end-feet are unaffected by MK801 but increase via GABA R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca increases in end-feet adjust CBF during synaptic activity.

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+]i in 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.

Our findings suggest that tissue anoxia might be a mechanism for prolonged aura in FHM1. Reduced Ca(2+) signals during normal network activity in FHM1 as compared to WT mice may explain impaired neurovascular responses in the mutant, and these alterations could contribute to brain frailty in FHM1 patients. Ann Neurol 2016;80:219-232.

Neural activity regulates local increases in cerebral blood flow (ΔCBF) and the cortical metabolic rate of oxygen (ΔCMRO2) that constitutes the basis of BOLD functional neuroimaging signals. Glutamate signaling plays a key role in brain vascular and metabolic control; however, the modulatory effect of GABA is incompletely understood. Here we performed in vivo studies in mice to investigate how THIP (which tonically activates extrasynaptic GABAARs) and Zolpidem (a positive allosteric modulator of synaptic GABAARs) impact stimulation-induced ΔCBF, ΔCMRO2, local field potentials (LFPs), and fluorescent cytosolic Ca(2+) transients in neurons and astrocytes. Low concentrations of THIP increased ΔCBF and ΔCMRO2 at low stimulation frequencies. These responses were coupled to increased synaptic activity as indicated by LFP responses, and to Ca(2+) activities in neurons and astrocytes. Intermediate and high concentrations of THIP suppressed ΔCBF and ΔCMRO2 at high stimulation frequencies. Zolpidem had similar but less-pronounced effects, with similar dependence on drug concentration and stimulation frequency. Our present findings suggest that slight increases in both synaptic and extrasynaptic GABAAR activity might selectively gate and amplify transient low-frequency somatosensory inputs, filter out high-frequency inputs, and enhance vascular and metabolic responses that are likely to be reflected in BOLD functional neuroimaging signals.

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.

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.

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 GABA 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 Ca , 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 Ca increase in astrocytes loaded with Oregon Green BAPTA. Finally, chelating Ca 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.