Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

No abstract

Summary The spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localised spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings astrocyte responses were global, suppressed by neurotransmitter clearance and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

Jackson-Weaver O, Osmond JM, Riddle MA, Naik JS, Gonzalez Bosc LV, Walker BR, Kanagy NL. Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca 2ϩ -activated K ϩ channels and smooth muscle Ca 2ϩ sparks. Am J Physiol Heart Circ Physiol 304: H1446 -H1454, 2013. First published March 22, 2013 doi:10.1152/ajpheart.00506.2012We have previously shown that hydrogen sulfide (H2S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H2S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca 2ϩ -activated potassium channels (BKCa). Ca 2ϩ sparks are ryanodine receptor (RyR)-mediated Ca 2ϩ -release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H2S activates Ca 2ϩ sparks in small mesenteric arteries. Ca 2ϩ sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca 2ϩ indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H2S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H2S donor NaHS (10 mol/l) increased Ca 2ϩ sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine ␥-lyase (CSE)-derived H2S with ␤-cyano-L-alanine (BCA) also reduced VSMC Ca 2ϩ spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC E m in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K ϩ currents in freshly dispersed mesenteric ECs. These results suggest that H2S increases Ca 2ϩ spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.cytochrome P-450 epoxygenase; sodium hydrosulfide; membrane potential HYDROGEN SULFIDE (H 2 S) is a recently described vasodilator produced in the vasculature by the enzymes cystathionine ␥-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). H 2 S has been proposed to cause vasodilation through a variety of mechanisms (1,3,26,33) and genetic knockout of the CSE gene causes hypertension (32). We previously reported that inhibiting CSE or disrupting the endothelium enhances myogenic tone in small mesenteric arteries from rats and that exogenous H 2 S hyperpolarizes vascular smooth muscle cell (VSMC) membrane potential (E m ) and dilates arteries through activation of large-conductance Ca 2ϩ -activated K (4) and ...

Rationale Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H2S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine γ-lyase (CSE) and acts by hyperpolarizing VSM. Objective Determine whether IH decreases endothelial H2S production to increase myogenic tone in small mesenteric arteries. Methods and Results Myogenic tone was greater in mesenteric arteries from IH than Shamfrom sham rat arteries, and VSM membrane potential was depolarized in IH in comparison with Shamsham arteries. Endothelium inactivation or scavenging of H2S enhanced myogenic tone in Shamsham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in Shamsham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H2S dilated and hyperpolarized Shamsham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in Shamsham than IH. CSE immunofluorescence was less in the endothelium of IH than in Shamsham mesenteric arteries. Endogenouse H2S dilation was reduced in IH arteries. Conclusions IH appears to decrease endothelial CSE expression to reduce H2S production, depolarize VSM, and enhance myogenic tone. H2S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BKCa channels.