Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration (Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot Ca(2+)](i) signals. Neuronally evoked increases in astrocytic Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.