ABSTRACT Sleep disorders are pervasive, contribute to morbidity in several psychiatric disorders, and cause an annual economic burden of $100 billion. However, despite its importance for health, the mechanisms that regulate sleep are poorly understood. We are taking a new approach to this problem by exploiting useful features of zebrafish to answer an important and basic question: What genetic and neuronal mechanisms regulate sleep? Sleep is regulated by a homeostatic process that reflects internal cues of sleep need and a circadian process that is entrained by environmental cues and restricts sleep to the appropriate time. Sleep is also directly and rapidly regulated by a phenomenon known as masking, in which light induces wake and dark induces sleep in diurnal animals. Factors that regulate the homeostatic process have been identified, including our recent finding that the serotonergic raphe promote sleep homeostasis in zebrafish and mice. We also showed that melatonin is essential for circadian regulation of sleep in zebrafish, and identified a pathway in the brain that regulates masking. Here we build upon these discoveries to elucidate mechanisms that underlie homeostatic, circadian, and light-dependent regulation of sleep. We will investigate these mechanisms using zebrafish, a diurnal vertebrate with several advantages that complement rodent models, using a combination of genetic, optogenetic, and chemogenetic perturbations coupled with high-throughput behavioral assays and whole-brain neuronal activity monitoring with single cell resolution. In Project 1, we will identify raphe subsystems that promote sleep homeostasis, and identify genetic and neuronal circuits that act upstream and downstream of these subsystems in sleep control. In Project 2, we will identify melatonin receptors that mediate the sleep-promoting function of melatonin, and also perform a screen to identify neurons through which melatonin implements circadian regulation of sleep. Project 3 builds on our recent discovery that the hypothalamic neuropeptide prokineticin 2 suppresses both light- and dark-induced masking behavior. Similar to Project 1, we will identify genetic and neuronal circuits that act upstream and downstream of prokineticin 2 to regulate masking. In Project 4, we will validate a large number of human sleep disorder candidate genes that were identified by genome-wide association studies. We will do so by leveraging zebrafish to efficiently and inexpensively generate and test many mutant lines for sleep phenotypes. We will determine the mechanisms through which validated candidate genes regulate sleep, and integrate these genes into the pathways identified in Projects 1-3. The homeostatic (Project 1), circadian (Project 2) and light-dependent (Project 3) mechanisms that regulate sleep, as well as the sleep disorder genes identified in humans and validated in zebrafish (Project 4), are likely to be integrated at multiple levels to produce either sleep or wa...