PROJECT SUMMARY Stress is a major risk factor for neuropsychiatric disorders including anxiety and depression. Stress increases the propensity of lateral habenula (LHb) neurons to emit high-frequency bursts of action potentials. This cellular adaptation contributes to stress-related behavioral abnormalities, including deficits in reward processing and cognition. Mechanisms by which stress increases the burst-firing of LHb neurons are poorly understood. Microglia play critical roles in maintaining homeostatic levels of neuronal activity. Preliminary single-cell RNA sequencing and in situ hybridization data collected in support of this application suggest that microglia but not neurons in the LHb express β2 adrenergic receptors (β2ARs). Stressors that increase burst-firing of LHb neurons enhanced norepinephrine (NE) signaling in the LHb of freely moving mice. NE acted directly on LHb microglia via β2ARs to attenuate ATP-evoked calcium signaling in these cells, which is known to promote homeostatic interactions between microglia and activated neurons. Finally, lesioning microglia precipitated a stress-like increase in burst-firing of LHb neurons. These exciting preliminary data suggest that stress enhances NE signaling in the LHb, which acts via β2ARs to disrupt microglial surveillance of local neuronal activity, thereby promoting burst-firing of LHb neurons. Here, I will investigate the role of β2AR-mediated NE signaling in microglia in regulating stress-induced cellular adaptations in the LHb and associated behavioral abnormalities. In Aim 1, I will use high-resolution spatial transcriptomics to characterize the effects of stress on gene programs in microglia and other cell types in the LHb of wild-type (WT) mice and mice in which β2ARs have been conditionally knocked out from microglia (microgliaβ2AR-cKO mice). I predict that stress will shift gene programs in LHb microglia from homeostatic to pro-inflammatory mode in WT but not microgliaβ2AR-cKO mice. In Aim 2, I will use miniscope- based single-cell fluorescence imaging to characterize the effects of stress on calcium signaling in LHb microglia from WT and microgliaβ2AR-cKO mice. I predict that stress will attenuate calcium signaling in LHb microglia in WT but not microgliaβ2AR-cKO mice. Whole-cell recordings will also be used to characterize the effects of stress on burst-firing of LHb neurons in WT and microgliaβ2AR-cKO mice. I further predict that stress will increase burst- firing of LHb neurons in WT but not microgliaβ2AR-cKO mice. In Aim 3, I will characterize stress-induced behavioral abnormalities in WT and brain-wide microgliaβ2AR-cKO mice. To restrict β2AR deletion to microglia in the LHb, I will administer tamoxifen directly into the LHb of our inducible microgliaβ2AR-cKO mice. I predict that stress will precipitate deficits in reward processing and cognition in WT mice and that these behavioral deficits will be attenuated in brain-wide microgliaβ2AR-cKO, and also in LHb-restricted m...