Sleep, wakefulness, and arousal regulate key behavioral and physiological processes and are critical for the survival of an organism. Sleep is a quiescent state and prevents animals from engaging in other goal-directed behaviors like feeding, mating, and escaping predators. An understanding of how these behaviors are co-regulated will help treat and manage sleep and eating disorders. The recent identification of shared genes and circuits in sleep and feeding in Drosophila pave way for understanding the mechanistic basis of how sleep is balanced with hunger and feeding motivation. Here we will combine the unique cell-specific neurogenetic manipulability of the Drosophila model system with an array of behavioral and novel neurophysiological approaches to investigate the role of mushroom body inputs specifically dopamine and octopamine in linking wakefulness to hunger-induced arousal and sleep suppression. In pursuit of identifying and characterizing aminergic regulation of sleep control networks in Drosophila, we have proposed three specific aims. In Aim 1, we will identify how protein hunger is encoded in the dopamine neurons that innervate the sleep- and wake-promoting microcircuits of the mushroom body. Specifically, we will use test how the activity of dopamine neurons and dopamine release is altered by nutrient-specific deprivation and sleep deprivation. In Aim 2, we will map connectivity between hunger-sensitive dopamine neurons and downstream sleep-regulating Kenyon cells and output neurons. By reciprocal activation and blocking of functionally upstream and downstream neurons, we will map how the sleep and hunger information flow occurs within the identified neurons. Finally, in Aim 3 we will test if the activity of the sleep-regulating octopamine neurons is influenced by altered nutrient signaling. Additionally, we will characterize the nature of synaptic connections and key molecular players that mediate interactions between octopamine neurons and the mushroom body network. The combination of genetic, behavioral, and physiological approaches some of which we have successfully validated and implemented will help address questions of great relevance to mammalian sleep and feeding co-regulation. These include the role of dopamine in modulating the activity of sleep-regulating and hunger-sensitive neurons and synaptic plasticity mechanisms underlying the sleep-wake and feeding switch. Through this competitive renewal application, we will continue building on our existing work characterizing sleep, wake, and hunger-based arousal in regulating the mushroom body network. The project will involve significant mentorship of undergraduate and post-baccalaureate student researchers to expand student opportunities and institutional capacity at California State University-East Bay.