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.

Astrocytes are found throughout the brain where they make extensive contacts with neurons and synapses. Astrocytes are known to display intracellular Ca²⁺ signals and release signaling molecules such as d-serine into the extracellular space. However, the role(s) of astrocyte Ca²⁺ signals in hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, remains unclear. Here, we explored a recently discovered novel TRPA1 channel-mediated transmembrane Ca²⁺ flux pathway in astrocytes. Specifically, we determined whether block or genetic deletion of TRPA1 channels affected LTP of Schaffer collateral to CA1 pyramidal neuron synapses. Using pharmacology, TRPA1^−/− mice, imaging, electrophysiology, and d-serine biosensors, our data indicate that astrocyte TRPA1 channels contribute to basal Ca²⁺ levels and are required for constitutive d-serine release into the extracellular space, which contributes to NMDA receptor-dependent LTP. The findings have broad relevance for the study of astrocyte–neuron interactions by demonstrating how TRPA1 channel-mediated fluxes contribute to astrocyte basal Ca²⁺ levels and neuronal function via constitutive d-serine release.

The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1°C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by threeAquificaegenera—Thermocrinis, Hydrogenobaculum, andSulfurihydrogenibium—and members of theAlpha-,Beta-, andGammaproteobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, and candidate division OP5. The in situ chemical affinities were calculated for 41 potential metabolic reactions using measured environmental parameters and a range of hydrogen and oxygen concentrations. Reactions that use oxygen, ferric iron, sulfur, and nitrate as electron acceptors were predicted to be the most energetically favorable, while reactions using sulfate were expected to be less favorable. Samples were screened for genes used in ammonia oxidation (amoA, bacterial gene only), the reductive tricarboxylic acid (rTCA) cycle (aclB), the Calvin cycle (cbbM), sulfate reduction (dsrAB), nitrogen fixation (nifH), nitrite reduction (nirK), and sulfide oxidation (soxEF1) by PCR. Genes for carbon fixation by the rTCA cycle and nitrogen fixation were detected. AllaclBsequences were phylogenetically related and spatially correlated toSulfurihydrogenibium16S rRNA gene sequences using qPCR (R²= 0.99). This result supports the recent finding of citrate cleavage by enzymes other than ATP citrate lyase in the rTCA cycle of theAquificaceaefamily. We briefly consider potential biochemical mechanisms that may allowSulfurihydrogenibiumandThermocrinisto codominate some hydrothermal environments.

We 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 Ca²⁺-activated potassium channels (BK_Ca). Ca²⁺ sparks are ryanodine receptor (RyR)-mediated Ca²⁺-release events that activate BK_Ca channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca²⁺ sparks in small mesenteric arteries. Ca²⁺ sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca²⁺ indicator fluo-4. VSMC membrane potential ( E_m) 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, BK_Ca blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca²⁺ sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H₂S with β-cyano-l-alanine (BCA) also reduced VSMC Ca²⁺ 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 E _m 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⁺ currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca²⁺ spark activity in mesenteric artery VSMC through activation of endothelial BK_Ca channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

A peer-reviewed open access journal published by the National Institute of Environmental Health Sciences

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 sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H₂S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H₂S dilated and hyperpolarized sham 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 sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham 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 BK_Ca channels.