Project Summary Social play, or rough-and-tumble play, is a characteristic pattern of social behavior exhibited by most mammals during adolescence. While the function of play is debated, converging evidence finds it vital for appropriate social behavior, cognition, and reproductive success in adulthood. Importantly, there is a sex difference in expression of play. Males exhibit greater intensity and frequency of play than females; however, there is individual variability in that some females play at the level of high-playing males while some males play at the level of low-playing females. In a recent experiment, we aimed to use the power of these individual differences to gain novel insight into sex-dependent and sex-independent transcriptional signatures associated with play. We scored play once daily for 4 days, designating rats in the top third per sex as “high-playing” and those in the bottom third as “low- playing”, collected tissue from the medial amygdala (MeA; site of play masculinization) for RNA-sequencing (RNA-seq), and used Weighted Gene Co-Expression Network Analysis (WGCNA) to identify 22 gene co- expression modules, or networks of coordinately regulated genes. Notably, all but one of the 12 modules (for p<0.05) associated with play were sex-specific in expression, indicating a sex difference in the transcriptomic profile associated with play in males compared to females. Building on previous theories that play’s function is to shape circuitry enabling expression of adult behavior, our central hypothesis posits that the transcriptional networks in the MeA underlying expression of play are sex-specific because juvenile play regulates expression of sex-typical adult social behaviors. Using two representative sex-specific, play-associated modules, we will test this hypothesis across 3 Specific Aims (SAs). SA1 determines whether module expression causally regulates playfulness in a sex-specific manner. In this aim, we will test the effect of overexpressing module regulators from our representative sex-specific modules in the MeA and determine whether this affects play in the predicted sex only. SA2 correlates expression of juvenile play with expression of sex-typical adult social behavior, determining if individual differences in playfulness track to differences in expression of territorial aggression (males) and maternal behavior (females). Finally, SA3 determines whether module expression causally regulates expression of sex-typical adult social behavior. As in SA1, we will induce expression of our representative sex-specific modules. We will then determine whether this is sufficient to increase expression of two sex-typical adult social behaviors, maternal behavior and territorial aggression as in SA2, in the absence of play. My sponsor is Dr. Margaret McCarthy, a leader in the field of brain sex differences, and my co-sponsor is Dr. Seth Ament, an expert in the functional genomics of social behavior. With their guidance, as well as ...