PROJECT SUMMARY New experiences elicit distinct patterns of brain activity, leading to the changes in gene expression, neuronal properties, and connectivity that underlie brain plasticity. In humans, the period of enhanced plasticity during brain development is particularly protracted compared to other species. However, the mechanisms and extent to which human neurons have changed to support increased plasticity remain unknown. Furthermore, although prolonged developmental plasticity may support increased cognitive capabilities and behavioral flexibility, it may also increase vulnerability to neurodevelopmental disorders. Neuronal plasticity depends on activity-regulated changes in gene expression that are controlled by activity-responsive genomic regulatory elements. Although we and others have identified regulatory elements as prominent substrates of human-specific evolutionary change, recent atlases of postmortem human and non-human primate brains overlook such dynamic stimulus- responsive regulatory elements. Without training on context-dependent data, current computational models that infer regulatory function based on sequence fail to predict activity-dependent regulatory elements. We hypothesize that there have been genetic changes in human divergent activity-regulated elements (hDAREs) and that we can discover these human-specific genetic underpinnings of plasticity using genome-wide approaches. We will use experimental and computational methods to predict and compare the activity-regulated responses of human neurons versus neurons from rhesus macaque and chimpanzee. We have developed innovative model systems that will allow us to stimulate physiological activity states in previously inaccessible primate neurons, machine learning models to predict regulatory function based on sequence, and massively parallel reporter assays and CRISPRi assays that will allow us to assess the function of candidate hDAREs. Through the successful completion of these studies, we will determine which genomic elements and genetic changes underlie activity-dependent responses in human neurons and the extent to which changes in these elements represent a major substrate of evolutionary selection in the human lineage. This will lay the groundwork for further phenotypic characterization of cellular plasticity mechanisms in the developing human brain. Additionally, these datasets will provide a valuable resource for dissecting genetic mechanisms of neurodevelopmental disorders.