Project Summary Microglia, the immune cells of the brain, perform essential homeostatic functions including debris clearance by phagocytosis and maintenance of neurons by release of growth factors and other molecules. However, during Alzheimer’s disease (AD), microglia accumulate near amyloid plaques where they acquire an activated phenotype, lose their homeostatic phenotype, and take on an activated phenotype that can induce neurotoxicity. Intracellular calcium in microglia regulates their transformation from this homeostatic resting state to an activated immune-effector state. Microglia intracellular calcium orchestrates functions such as phagocytosis, proliferation, migration, generation of neurotrophic factors, cytokine production and release, and formation of neurotoxic reactive oxygen species. Intracellular calcium dysregulation appears in activated, non- homeostatic microglia near amyloid β (Aβ) plaques in mouse models of AD. Our overall hypothesis that dysregulated intracellular calcium shifts microglia phenotype in AD. Our preliminary data suggest that blocking L-type voltage dependent calcium channels (L-VDCCs) can shift microglia towards a more homeostatic phenotype. The proposed experiments test the hypothesis that L-VDCCs can drive microglia calcium dysregulation during AD pathology. We will test this by using pharmacological antagonism of L-VDCCs combined with genetic ablation of microglia-specific L-VDCCs in the presence of AD pathology to define the role of L-VDCCs on microglia phenotype and function. For the training aspect of this proposal, we will use 1) in vivo multiphoton imaging to assess microglia function and calcium dynamics and 2) single-nuclei RNA sequencing to define the cell-specific effects of L-VDCC antagonism in vivo. If our hypothesis is correct, L- VDCC function will be a mechanistic link microglia calcium dysregulation and microglia functional phenotype during AD. This finding would be relevant to numerous age-associated neurodegenerative disorders where changes in microglia phenotype are observed.