Project Summary Healthy behavioral development in children relies on social connectedness. Social isolation and loneliness during childhood can lead deleterious behavioral phenotypes that continue into adulthood, such as aggression towards oneself and others. However, the basic principles of juvenile aggression are unknown, in part, because traditional laboratory animals do not show aggression as juveniles and functional studies in human children remain challenging. To address this deficit, this research aims to uncover basic neurobiological mechanisms of how social isolation leads to juvenile aggression in tadpoles that can be violently aggressive or socially gregarious based on social rearing conditions. We combine this novel research organism and behavioral paradigm with advanced neurogenetic tools to interrogate the neuronal substrates of aggression induced by social isolation. We focus this research on microRNAs because microRNAs are responsible for the sociality switches across a wide range of taxa and have promising therapeutic potential through application of microRNA mimics or inhibitors that can change gene expression programs without altering DNA sequences. Recent data from our lab shows that tadpole aggression is associated with increased activity in the amygdala, endorphin signaling, and a decrease in forkhead box protein 2 (FOXP2) expression, a gene linked to social behavior in many taxa. Based on this robust preliminary data, we propose to test the hypothesis that microRNAs alter FOXP2 expression in endorphin-sensitive neurons to induce aggressive behavior in juveniles susceptible to social isolation. We will first determine how social isolation shifts the microRNA landscape of the juvenile brain using single cell sequencing and testing how microRNA expression changes in FOXP2- and endorphin-sensitive neurons. Then, we will examine how microRNAs in the amygdala tune the behavioral responses of juveniles based on social rearing conditions. Finally, as connectivity of the human amygdala is reduced in aggressive humans, we will functionally test the role of miR-9 in altering amygdala connectivity and predisposing tadpoles to be gregarious or aggressive. Together, the proposed experiments will systematically dissect the mechanisms by which microRNAs regulate FOXP2 and the µ opioid receptor to regulate juvenile sociality and aggression in a research organism with experimental tractability and a robust behavioral output that is difficult to achieve in other research organisms. As the molecular factors and overall brain organization of social-motor behaviors are conserved across vertebrates, this research will identify generalizable principles of juvenile aggression. There is a pressing need for this research because there are currently no established models for studying the neural mechanisms of juvenile aggression. This work is important to public health because the COVID-19 pandemic isolated children from their peers and led to an incre...