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 Ca2+ concentration ([Ca2+](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca2+](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 [Ca2+](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca2+](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca2+ indicator and confocal microscopy. Increases in endfoot [Ca2+](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca2+](i) and vascular diameter. Neuronal activity- evoked elevation of endfoot [Ca2+](i) was reduced by inhibition of inositol 1,4,5- trisphosphate ( InsP(3)) receptor Ca2+ release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca2+ uptake. To probe the Ca2+ 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 [Ca2+](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)- induced [Ca2+](i) increases were sensitive to depletion of the intracellular Ca2+ store, but not to ryanodine, suggesting that Ca2+-induced Ca2+ release from ryanodine receptors does not contribute to the generation of endfoot [Ca2+](i) signals. Neuronally evoked increases in astrocytic [Ca2+](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca2+](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca2+ release processes within the endfeet were evident, as were localized regions of Ca2+ release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca2+](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 Ca2+ release signals that modulate arteriolar diameter.