PROJECT SUMMARY Bacteria, such as those found in the vertebrate gut microbiome, have been shown to have widespread effects on both the enteric (ENS) and central nervous systems (CNS) in numerous animals. Reciprocally, the ENS has been shown to have an impact on the progression of microbiome establishment in the gut. However, despite the profound influence of bacteria on neuronal development and physiology, the diversity of the vertebrate gut microbiome and the complexity of the ENS make the mechanisms underlying these phenomena difficult to study. Despite the importance of the topic and vertebrate models, these systems involve microbiomes that are both spatially and compositionally complex, often with thousands of bacterial species that can very between individual hosts. Therefore there is a role for simple, exquisitely manipulable model systems in which to study the effects of bacterial symbionts on neuronal development and function. To address the dearth of such models for the mechanisms by which bacteria influence host neurobiology, we propose to use the symbiosis between the Hawaiian bobtail squid (Euprymna scolopes) and its luminescent bacterial symbiont Vibrio fischeri as a tractable system in which to study how bacteria drive host neuronal development. The squid-vibrio system is a well-established model for the effects of bacterial products on host physiology and development, with outcomes that are often generalizable to other animal systems. This proposal aims to develop the squid-vibrio system into a new model for the study of the effects of microbes on host neurobiology. In our first aim we will define the neuronal landscape of the light organ, including neuronal position and identity and the effect of symbiosis and bacterial products on both. To achieve this aim, we will develop new methods for dual labeling of RNA and proteins in our system and adapt neuronal tracing techniques for imaging in the light organ. In our second aim we propose to study the role of neuropeptide signaling on transducing microbial signals from V. fischeri to tissues on the outside of the symbiotic organ. Specifically, we will determine the location and dynamics of FMRFamide signaling in the light organ over the first three days of symbiosis and will examine the effects of FMRFamide signaling on bacterially-mediated post-embryonic development. The data arising from this proposal will not only lay the foundation for a powerful new model system, but will also generate new resources and techniques for the community and provide new insights as to the role of the peripheral nervous system in potentiating microbially-mediated development.