PROJECT SUMMARY/ABSTRACT Deep sequencing technologies have revolutionized our understanding of gene expression in the brain. In particular, single cell RNA sequencing of many brain regions has revealed an incredible diversity of transcriptomically-defined neuronal cell types. This is true not only for anatomically defined brain regions such as the frontal cortex but also for more discrete neuronal populations such as those confined to a particular cortical layer. This diversity of transcriptomically specified neuronal cell types is a major step forward for a central goal of contemporary neuroscience: understanding how neural circuits composed of specific neuronal cell types regulate behavior. Nevertheless, this diversity in neuronal cell types immediately raises the question as to how the functional output of a brain region (or discrete neuronal population) is parcellated among the many transcriptomically-defined neuronal cell types that comprise that region. Despite the enormous work that has gone into defining these neuronal cell types, their functional relevance remains unclear in most, if not all, instances. Our proposed project seeks to fill this knowledge gap by focusing on the neurons that comprise the principal component of the bed nucleus of the stria terminalis (BNSTpr). Our genetically-targeted single nucleus RNA sequencing of BNSTpr neurons has identified many transcriptomically-defined neuronal cell types, one of which is defined by its unique expression of the neuropeptide tachykinin 1 (Tac1). We have developed intersectional genetic strategies to interrogate functionally and anatomically the relevance of the Tac1-expressing BNSTpr neuronal cell type in generating behavioral output of the BNSTpr. In parallel, our genetic strategies enable us to interrogate the contribution of the complementary Tac1 non-expressing BNSTpr neuronal cell types to behavioral output of this region. In Aim 1, we will determine the activity of Tac1-expressing and non-expressing BNSTpr neuronal cell types in freely moving mice using a genetically encoded calcium indicator in engineered mouse strains. In Aim 2, we will use intersectional optogenetic actuators to determine the necessity and sufficiency of Tac1-expressing and non-expressing BNSTpr neuronal cell types in behavior. In Aim 3, we will match the connectivity of the Tac1-expressing neuronal cell type with functional output of its projection targets; in addition, we will engineer a targeted deletion of Tacr1, the cognate receptor for Tac1, in these projection targets and test whether Tac1 signaling through Tacr1 in these projection targets is essential for behavioral output. Together, our project will determine the specific contribution of a particular transcriptomically-defined neuronal cell type to behavior in contrast to the behavioral output of the region within which it resides. Health relatedness: The BNSTpr is an integrative center linking sensory input to motor output for social behaviors, and...