Abstract
Astrocytes elicit bidirectional control of microvascular diameter in acutely isolated brain slices through vasoconstriction and vasodilation pathways that can be differentially recruited via the free Ca2+ concentration in endfeet and/or the metabolic status of the tissue. However, the Ca2+-level hypothesis has not been tested using direct manipulation. To overcome this, we used Ca2+-clamp whole-cell patching of peri-arteriole astrocytes to change astrocyte-free Ca2+ to different concentrations and examined the vascular response. We discovered that clamping Ca2+ at the approximate resting value (100 nM) had no impact on arteriole diameter in a pre-constricted arteriole. However, a moderate elevation to 250 nM elicited sustained vasodilation that was blocked by the COX-1 antagonist SC-560 (500 nM). The vasodilation to 250 nM Ca2+ was sensitive to the metabolic state, as it converted to vasoconstriction when oxygen tension was dramatically elevated. In normal oxygen, clamping astrocyte Ca2+ well above the resting level (750 nM) produced sustained vasoconstriction, which converted to vasodilation in the 20-HETE blocker HET0016 (1 μM). This response was fully blocked by the addition of SC-560 (500 nM), showing that 20-HETE-induced vasoconstriction dominated the dilatory action of COX-1. These data demonstrate that direct changes in astrocyte free Ca2+ can control multiple arteriole tone states through different mediators.
Highlights
Ca2+-dependent signaling in astrocytic endfeet contributes to the regulation of arteriole diameter [1,2,3]
Using a whole-cell patch technique designed to buffer free Ca2+ to a particular target concentration using BAPTA plus CaCl2 [18], we tested the hypothesis that clamping astrocyte Ca2+ near resting physiological levels would have little impact on existing arteriole tone
Neocortical brain slices were bulk-loaded with a Rhod-2/AM Ca2+ indicator, arterioles were pre-constricted with U-46619 (100 nM), and the tissue was equilibrated in physiological levels of O2 (30%) before the start of the astrocyte patching experiment
Summary
Ca2+-dependent signaling in astrocytic endfeet contributes to the regulation of arteriole diameter [1,2,3]. While different transmitters and G-protein-coupled receptors drive distinct cell pathways that cause different vascular responses, it is notable that within the neurovascular unit, even the same transmitter and receptor system can elicit vasoconstriction or vasodilation depending on the physiological context or the current state of the microenvironment. These include the level of free Ca2+ in astrocyte endfeet [4], the degree of arteriole tone [5,6], and the metabolic status of the tissue [7,8]. An alternative method for testing this hypothesis is to directly ‘clamp’ astrocyte free Ca2+ at different concentrations via a patch pipette, though this method has not yet been performed to either support or refute the hypothesis
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