Microglia Regulation of Sexually Dimorphic Amygdala Development

Abstract

Sex differences in the brain are established early in development and generate lasting changes in brain and behavior, a process known as sexual differentiation. Sexual differentiation of the amygdala produces a highly conserved sex difference in juvenile rough and tumble play behavior; however, the mechanisms underlying this sex difference are unknown. Here, we report that microglia, resident immune cells of the brain, actively shape the sexual differentiation of the amygdala. We found that microglia are more phagocytic in the amygdala of males from postnatal day 0 and 4, during which they also have a higher endocannabinoid (eCB) tone. Administering a masculinizing dose of testosterone to increase the eCB tone in females, or cannabinoid receptor agonists to female pups increased the number of phagocytic microglia and correspondingly decreased the number of newborn cells. Given these data, we hypothesized that microglia control the number of postnatally-born cells in the developing rat amygdala by phagocytosing newborn cells in an endocannabinoid-dependent manner. We found that these phagocytic microglia engulf newly proliferated cells, which are enriched for complement proteins. To directly implicate microglia phagocytosis, we used a function-blocking antibody against the complement receptor 3 (CR3) to prevent phagocytosis. Anti-CR3 antibody treatment increased the number of BrdU+ cells only in males, demonstrating that newborn cells can survive if phagocytosis is prevented. Moreover, administration of cannabinoid receptor antagonists to male pups occluded the effects of phagocytic blockade, suggesting that newborn cell phagocytosis was dependent on the developing ECB tone. Finally, to understand how these early life events manifest changes in the composition of the amygdala, we used a fate mapping approach to phenotype postnatally-born cells at the juvenile age. Our analysis found that the majority of newborn cells differentiated into astrocytes, which were overall higher in density in the posterodorsal region of the medial amygdala, an amygdalar nucleus essential to the integration of social stimuli. Together, these data indicate that sex differences in the local environment of the developing amygdala instruct microglia to actively phagocytose newborn cells as a means to sculpt later life architecture of the amygdala and produce sex differences in social play.