Project Summary/Abstract Dietary excess rapidly induces the inflammatory activation and accumulation of a heterogeneous population of myeloid cells, broadly termed microglia, in the mediobasal hypothalamus (MBH), a critical brain region involved in the regulation of energy and glucose homeostasis. We showed that microglial activation in this context is sufficient to stimulate food intake and body weight gain, however the metabolic factors initiating this response remain to be elucidated. Diet is a major factor affecting the composition of the gut microbiota, and high-fat diet (HFD) consumption induces unfavorable alterations in the type and proportion of commensal microorganisms in the gut. These changes influence the inflammatory and metabolic properties of the host and may also impact microglial function in the MBH. To this end, microglia in germ-free (GF) mice are hyperactivated in the MBH compared to other brain regions such as cortex. This microglial activation is already manifested during neonatal period and colonizing the gut of pregnant GF dams at embryonic day 16 (E16) restored postnatal microglial homeostasis in the MBH of their pups. Moreover, we found that supplementation of short chain fatty acids (SCFAs), metabolites produced by bacterial fermentation of non-digestible carbohydrates, is sufficient to reduce microglial activation in the MBH and body weight gain in HFD- fed mice. Based on substantial work, both published and preliminary, we propose here to test the hypothesis that MBH microglia, which reside close to fenestrated blood vessels, are uniquely regulated by specific microbial metabolites such as SCFAs. Specifically, we aim to A) determine the cellular and molecular mechanisms by which SCFA-FFAR2 (free fatty acid receptor 2) signaling regulates microglial homeostasis in the MBH, B) determine whether SCFA-AhR (aryl hydrocarbon receptor) interactions regulate microglial homeostasis in the MBH, and C) determine whether microbial metabolites regulate postnatal immunological imprinting of MBH microglia. Completing these aims has the potential to reveal unprecedented mechanistic insights of how microbial metabolites modulate identity and function of MBH microglia engaged in regulating metabolic function, providing novel therapeutic targets for the prevention and treatment of metabolic diseases.