PROJECT SUMMARY The lateral septum (LS) is an enigmatic brain region that contributes to a multitude of behaviors and has been implicated in a number of psychiatric disorders including anxiety. Over many decades, researchers have attempted to ascertain how this one brain region gives rise to such functional diversity. However, the results of these studies have often been contradictory. Early studies of the LS divided it into distinct anatomical subregions, suggesting a putative role for its subregions in mediating different functions. Recent research has revisited this earlier work and postulates that there are functional subunits within the LS, which may be selecting for different behavioral outcomes. This proposal seeks to address this theory by characterizing the anatomical organization, neural encoding properties, and function of two LS projection populations. In particular, I will focus on two target regions that receive input from the lateral septum and exemplify the functional diversity of the LS: the ventral tegmental area (VTA) known for its role in reward representation and the bed nucleus of the stria terminalis (BNST) known for its role in cue encoding. In Aim 1, I will perform projection-specific cellular resolution calcium imaging during a series of operant behavioral paradigms to evaluate the role of each projection population in reward-related behavior. I hypothesize that each projection population will encode distinct task features, contributing to the functional diversity of the LS. Thus, I predict that the LS-VTA will encode reward-related information and the LS-BNST will encode reward cues. In Aim 2, I will use projection-specific, closed-loop optogenetic manipulations to determine the causal role of the LS-VTA and LS-BNST projection populations in different trained reward-related behaviors. I will quantify the effects of inhibition of these two populations on different behavioral outcomes on an operant assay, which will provide insight into how these LS subpopulations are functionally distinct. The results from this proposal will provide a more complete view of how the LS functions, specifically in reward- related behaviors. It will further our understanding of a brain region that has evaded explanation for many years and may prove to be a crucial target for the treatment of various psychiatric disorders.