Project Summary Improved understanding of the neurobiological systems involved in excessive caloric consumption is critical for developing novel prevention and treatment strategies for obesity. Traditionally the field has focused on hypothalamic and brainstem substrates that control `homeostatic' food intake that occurs in response to energy deficits. In addition to studying these classic feeding centers, it is critical to also identify the systems through which higher-order brain regions regulate reward-driven food seeking and consumption based on learned, incentive, and hedonic cognitive factors. This project investigates the hippocampus (HPC) as a critical brain substrate integrating memory processes and feeding-related signals to regulate conditioned food- motivated behavior, including appetitive responses linked with excessive caloric intake and obesity. Our focus is on two HPC subregions that intersect feeding behavior and memory: the ventral HPC CA1 (CA1v) and the dorsal CA3 (CA3d). Our findings from the previous funding cycle identify a role for CA1v projections targeting the medial prefrontal cortex (mPFC), lateral hypothalamic area (LHA), and lateral septum (LS) as pathways functionally relevant to feeding behavior 1-3. Aim 1 experiments will advance these findings to identify the role of three HPC projection pathways (CA1v -> mPFC, LHA, LS) in HPC-dependent associative learning tasks that are relevant to excessive caloric intake and are based on categorically separate food-associated stimuli, including [1] interoceptive energy status cues, [2] external contextual cues, and [3] social-based olfactory cues. In addition to the appetitive associative memory processes described above, HPC-dependent meal- related episodic memory (recalling who, what, when, and where surrounding a meal) powerfully influences feeding behavior 4-8. Results from the previous funding cycle identified a neural pathway through which gastrointestinal (GI) vagus afferent nerve (VAN) signaling, traditionally studied in the context of meal size control, promotes HPC-dependent memory 9. Our preliminary results support the hypotheses that [1] the stomach-derived hormone ghrelin acts via GI VAN signaling to promote meal-related episodic memory, and [2] medial septum (MS) cholinergic signaling is a relay connecting GI VAN signaling and HPC function. These hypotheses are investigated in Aim 2 experiments using an innovative combination of state-of-the-art methodologies, including in vivo fiber photometry-based imaging of novel fluorescent genetically-encoded sensors for acetylcholine (ACh) 10,11 and stomach distention-dependent electrical VAN stimulation. The extent to which these ventral and dorsal HPC pathways converge through shared collateral projections, and/or common downstream targets is examined in Aim 3 experiments that utilize neural pathway tracing approaches to [1] map the collateral and 2nd-order projections of CA1v projections to mPFC, LHA, and LS, and [2] i...