Microglia are the tissue resident macrophage-like cells in the central nervous system that regulate numerous physiological functions ranging from phagocytosis, detection of brain injuries, and synaptic pruning. Growing evidence indicates that microglia also play powerful roles in regulating neuronal excitability and synaptic transmission by secreting a variety of neuroactive factors especially proinflammatory cytokines. These cytokines evoke a vast array of effects on neurons and glia including alterations in Ca2+ signaling and synaptic transmission, which are implicated in brain pathologies such as neuropathic pain. In particular, following nerve injury, microglia rapidly produce TNFa, IL1b , and the growth factor, BDNF, which enhance the strength of excitatory synaptic transmission in nociceptive circuits of the spinal cord to induce neuropathic pain. However, the checkpoints that regulate the production and release of inflammatory mediators from microglia are not well-understood. Our preliminary results indicate that store-operated Orai1 channels are a major mechanism for purinergic-evoked Ca2+ signals in microglia and their opening stimulates the induction and release of TNFa and IL1b. Based on this evidence, we hypothesize that Orai1 channels are essential regulators of microglia-mediated neuroinflammation in the context of neuropathic pain. We propose three specific aims to test this hypothesis: 1) Define the role of Orai1 channels for ATP-evoked Ca2+ signals and the inflammatory output of microglia, 2) Determine the effects of Orai1 channel-mediated secretion of proinflammatory mediators on synaptic transmission in the dorsal horn of the spinal cord following nerve injury, and 3) examine the in vivo relevance of Orai1 channel-mediated inflammatory cytokine production from microglia in a model of neuropathic pain. We will approach these questions using a newly created microglia-specific inducible Orai1 KO mouse and Ca2+ imaging, slice electrophysiology, and behavioral analysis. Results from these studies will advance our understanding of the physiological role of Orai1 channels for regulating microglia-mediated neuroinflammation and aid the quest for developing new microglial-targeted therapies for pathological diseases affecting brain function.