PROJECT SUMMARY This R01 proposal tests the central hypothesis that gonadal hormones present during a critical window of perinatal brain organization result in distinct sex-specific microglial and mitochondrial phenotypes that increase male vulnerability to perinatal immune stimuli. There is a strong male bias in the incidence of many early-onset neurodevelopmental disorders. Although neurodevelopmental disorders are quite heterogeneous, growing evidence demonstrates immune and mitochondrial abnormalities in a subset of these patients. Microglia, the innate immune cells of the brain, have emerged as key effectors of this neuroimmune communication. We find that microglia isolated from mice exposed to an early-life immune challenge show significant downregulation of genes encoding mitochondrial electron transport genes, specifically in males. Additionally, we find that this early-life immune challenge results in male-biased social behavior deficits, and specifically induces dysfunction in other aspects of male microglial mitochondrial functionality. To determine what could be mediating these early-onset sex differences, we first used the knowledge that there is a surge of gonadal hormones that occurs only in males during a perinatal critical period of brain development. Injecting female mouse pups during this perinatal critical period with estradiol, the aromatized form of the androgen testosterone that directly masculinizes the male brain, results in a male-typical phenotype. We have found that masculinized females subjected to early-life immune challenge display similar deficits in social behavior as did immune-challenged males, with similar microglial mitochondrial alterations as were observed in immune-challenged males. We have also demonstrated that estrogen receptors known to influence mitochondrial function are differentially expressed on microglia between males and females. Using this knowledge, we can alter estrogen receptor signaling in a cell-specific manner to study the importance of microglial estrogen receptors to male-biased neurodevelopmental disorder vulnerabilities. In this proposal, we will assess whether microglia-specific alterations in mitochondrial morphology, gene expression, and bioenergetic function are dependent upon microglia-specific estrogen or androgen receptor signaling, as well as assess whether early-life immune challenge impacts social behavior through aberrant microglial phagocytosis of synapses, as well as the impact of the perinatal gonadal hormone surge and microglial estrogen/androgen receptors on these processes. This proposal will be the first to examine a mechanistic relationship between perinatal gonadal hormone exposure through microglial estrogen and androgen receptors and the establishment of sex-biased neurodevelopment that induce male-biased vulnerabilities to neurodevelopmental disorders.