Neuropeptidergic modulation of neuronal signaling facilitates context-dependent differences in neuronal circuit activity, and can thus generate profound differences in behavior. We study Cart (Cocaine- and Amphetamine-Regulated Transcript), a neuropeptide with proposed roles in the regulation of several behaviors, including arousal, anxiety, appetite, and reward. In mammals, a single Cart gene exhibits complex expression in many brain regions. In contrast, Cart is encoded in zebrafish by seven separate genes, each with a relatively simple expression pattern. The genetic tractability of zebrafish, along with its optical transparency and wide range of experimentally validated behavioral assays, provides an unparalleled system to link functions of Cart in specific brain regions with specific behaviors. First, we are pinpointing regions of Cart expression in zebrafish larvae via colocalization with markers of conserved neurotransmitter and peptidergic systems. Second, we are working to elucidate the development and targeting of Cart neurons using transgenic larvae that express GFP driven by cis-regulatory sequences of each Cart gene. Third, we are working to analyze the behavioral functions of each zebrafish Cart gene. Specifically, we have generated fish with loss-of-function mutations in four Cart genes, and transgenic lines that enable inducible overexpression of four Cart genes. Analogous reagents for the other Carts are in progress. We and others have established behavioral assays that are especially suited for analyzing behaviors relevant to Cart function, including arousal, sensory responsiveness, anxiety, and appetite. Hierarchical clustering of behavioral outcomes will elucidate the roles of each Cart gene in regulating specific behaviors. Finally, to explore the significant difference in Cart peptide family representation between species, we are using computational tools to model patterns of gene gain and loss in all neuropeptide families across a panel of vertebrate species.