PROJECT SUMMARY The brain serotonin (5-HT) system has been a target for multiple weight loss therapies. Compounds that elevate 5-HT content reduce food intake and body weight. Our previous and current findings show that 5-HT acts on two postsynaptic 5-HT receptors, Htr2c and Htr1b in the arcuate nucleus of the hypothalamus (ARH) to suppress food intake. Within the ARH, 5-HT reciprocally inhibits the orexigenic AgRP neurons (via Htr1b) while activating the anorectic POMC neurons (via Htr2c). Moreover, we have demonstrated that Htr2c and Htr1b in these neurons are necessary for the anorectic effects of 5-HT agents including the once-popular diet pill Fen/Phen. However, despite these significant findings, the precise source of the presynaptic 5-HT inputs to ARH neurons remains largely elusive. This has posed a considerable challenge due to the heterogeneity of 5-HT neurons in the midbrain dorsal raphe nucleus (DRN), comprising multiple subpopulations with distinct projection patterns and physiological functions. Consequently, our current proposal aims to unravel the neural circuit mechanism by which 5-HT regulates satiety. To accomplish this, we propose a multidisciplinary approach to isolate the 5-HT neurons that innervate the ARH (5-HTARH neurons). Our central hypothesis posits that these neurons provide direct synaptic inputs to the ARH and play a critical role in feeding regulation. To investigate this, we will use in vivo calcium imaging to determine the activity patterns of 5-HTARH neurons during hunger and satiety in behaving mice [Aim 1a]. Furthermore, we will manipulate the activity of these neurons in live mice by either stimulating or inhibiting them, aiming to uncover their specific contributions to food intake [Aim 1b]. Finally, we have developed a new intersectional genetic approach that enables us to selectively deplete 5-HT inputs to the ARH in adult mice and evaluate their physiological impact on energy homeostasis [Aim 2].