Project Abstract Astrocytes comprise the major cell type in the brain and regulate numerous functions including neural development, clearance of neurotransmitters, and regulation of blood flow. Recent evidence indicates that astrocytes also play a direct role in regulating synaptic transmission by modulating neuronal activity through the secretion of `gliotransmitters' such as glutamate, ATP, and D-serine. Astrocytes are intimately associated with pre- and post-synaptic neuronal membranes in an anatomical unit referred to as the tripartite synapse. This close association allows astrocytes to sense neurotransmitters released by neurons, and conversely communicate with neighboring neurons through the action of gliotransmitters. Previous studies have suggested that a major mechanism mediating release of gliotransmitters is vesicular exocytosis evoked by elevations in intracellular [Ca2+]i. However, the molecules and pathways involved in generating Ca2+ elevations in astrocytes remain poorly understood. Our preliminary evidence indicates that store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a major mechanism for neurotransmitter-evoked Ca2+ signals in astrocytes. We further find that ablation of CRAC channel expression or pharmacological blockade suppresses gliotransmitter release. Based on this evidence, we hypothesize that CRAC channels are essential regulators of astrocyte gliotransmitter exocytosis and the bidirectional communication between neurons and astrocytes at the tripartite synapse. We propose the three specific aims to test this hypothesis: 1) Define the molecular composition of CRAC channels in astrocytes and their contribution for the generation of complex astrocyte Ca2+ signals. 2) Determine the role of CRAC channels for secretion of gliotransmitters, and 3) Determine the role of CRAC channels for astrocyte modulation of synaptic transmission. We will approach these questions using a multidisciplinary approach that combines genetic knockouts of CRAC channel proteins with in-depth molecular and biochemical assays, whole-cell and slice electrophysiological recordings, Ca2+ imaging using wide-field and spinning disk confocal microscopy, and gliotransmission assays. Collectively, results from these studies will advance our understanding of the physiological role of CRAC channels for regulating Ca2+ homeostasis and gliotransmission in astrocytes and aid the quest for developing new therapies for pathological diseases affecting synaptic function.