PROJECT SUMMARY/ABSTRACT Fluid intake is precisely regulated to match physiological need. This is imperative to maintain fluid homeostasis and ultimately for survival, although the underlying mechanisms remain poorly understood. Critically, the body needs to be able to sense the osmolarity of ingested fluids, as different osmolarities can have opposing impacts on physiological need and behavioral responses. It has been recently demonstrated that the gastrointestinal tract is the locale that detects and communicates fluid osmolarity in real-time to central thirst circuits, but the specific mechanisms underlying this osmosensation are completely unknown. The aim of this proposal is to identify the key cell types and afferent neural pathways that detect osmolarity in the gut and relay this information to the brain to control thirst. The hypothesis proposed here is that this happens in two steps. First, specialized chemosensory cells in the gut, called enteroendocrine cells, detect the osmolarity of the gut lumen. These cells then communicate with the brain by activating adjacent vagal sensory neurons. Aim 1 will determine the role of enteroendocrine cells in thirst and osmosensation, whereas Aim 2 will focus on the vagus nerve. These experiments will be done using genetic and virally mediated tools to target and manipulate, for the first time, specific enteroendocrine and vagal cell types in awake, behaving mice. The effect manipulating these cell types has on fluid intake will be measured, and the corresponding activity of thirst neurons in the subfornical organ that track gut osmolarity will be monitored using calcium imaging in vivo. These data will reveal fundamental mechanisms that allow the body to appropriately respond to ingested substances and maintain fluid homeostasis. Ultimately, these experiments will advance our understanding of basic physiologic processes in the gut and how fluid and salt are handled by the body; dysregulation of these processes contributes to conditions like hypertension and gastrointestinal disorders.