PROJECT SUMMARY/ABSTRACT The balance between response execution and response inhibition (i.e., going vs. not-going or stopping) plays a fundamental role in regulating normal behavior, and it is disrupted in many psychiatric diseases associated with impulsivity, including ADHD, OCD, schizophrenia, and substance abuse. Action control is regulated by a number of brain structures, and prefrontal cortex (PFC) plays a particularly prominent role in shaping go vs. no- go or stop decisions. However, the mechanisms of how PFC neurons control this response execution vs. inhibition balance are currently unknown. The projects in this proposal will address this issue by testing a number of hypotheses related to the dynamic nature of PFC neuron ensembles in behavior control. The overarching hypothesis to be tested is that separate ensembles of PFC neurons, distributed across multiple PFC subregions are defined by the intersection between 1) diverging connectivity with downstream targets, and 2) selective activation with precise temporal dynamics during either action initiation or action suppression. We will study PFC ensemble contributions in rats performing a novel Go/NoGo task designed to specifically extract information related to action decisions. In Aim 1 we will use new calcium integrator tools to identify task-activated ensembles of neurons, map their efferent connectivity, and optogenetically manipulate them, thereby demonstrating a causal role for neuron populations defined by temporal co-activation anatomical features in regulating action control. In Aim 2, we will identify the specific temporal dynamics of action-specific ensembles through large-scale cellular neurophysiological recording across the PFC and will identify how anatomically-defined ensembles are differentially activated using optogenetics-paired ensemble neurophysiology. The results from these studies will provide key evidence supporting or refuting the hypothesis that PFC neuron ensembles, aligned into groups via temporally correlated activity and anatomical connectivity, regulate decisions to initiate or withhold behaviors. The results from these studies will also provide a launchpad for future work investigating additional anatomical, molecular, and genetic identities of neural ensembles related to response selection, both within PFC and in other associated structures. In addition to significantly advancing our understanding of executive control, these and future studies will identify novel treatments for mental diseases involving impulsivity and other aspects of disrupted response selection based on the intersection of circuitry, molecular identity, and physiology.