ABSTRACT Sleep is regulated by homeostatic and circadian mechanisms. Several factors mediate homeostatic control of sleep and mechanisms that regulate the circadian clock are well understood, but little is known about how the circadian clock regulates sleep. Melatonin (MT) is a good candidate link between the circadian clock and sleep because the circadian clock regulates its production, it can induce sleep in several diurnal species, including humans, and its loss is associated with reduced sleep in humans. MT may play an ancient role in regulating rest/activity states, as it does so in animals such as nematodes and zooplankton. However, the role of MT in vertebrate sleep is controversial for two reasons. First, most nocturnal lab mouse strains do not synthesize MT, yet they exhibit circadian control of sleep. However, MT synthesis peaks at night in both diurnal and nocturnal animals, and MT does not induce sleep in nocturnal animals, suggesting that MT only regulates sleep in diurnal animals. Second, a large number of studies in vertebrates have failed to show a consistent role for MT in sleep, suggesting that it is not a central sleep regulator, but rather has species-specific roles. However, these studies used pinealectomy (Px), a crude, imprecise and invasive procedure that does not address the function of MT in a clean manner. Thus, it is likely that distinct effects of Px result from differences in the Px procedure in different species and in different labs. The pineal gland also likely has functions in addition to MT production, so Px may cause effects that obscure MT-dependent phenotypes. These confounds are avoided by using genetics to create MT-deficient animals. We have addressed this question using zebrafish, a diurnal vertebrate that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep. We found that circadian regulation of sleep is abolished in zebrafish that lack MT due to mutation of arylalkylamine N-acetyltransferase (aanat2). This finding suggests that MT mediates the circadian regulation of sleep in a diurnal vertebrate and provides a basis to explore genetic and neurological mechanisms through which the circadian system and MT regulate sleep. In Specific Aim 1 we use genetics to determine which MT receptors are required for circadian regulation of sleep and for sleep induced by exogenous MT. In Specific Aim 2 we identify neurons that are activated or inhibited in response to exogenous MT and in MT-deficient animals using cfos in situ hybridization and whole-brain GCaMP6s calcium imaging. In Specific Aim 3 we use genetics and mass spectrometry to test the hypothesis that MT promotes sleep by stimulating adenosine signaling. If correct, this would suggest a simple mechanism that integrates homeostatic and circadian control of sleep. This project will help to reveal how MT, and thus the circadian clock, regulates sleep in a diurnal vertebrate. Sleep disorders are common, have poor therapeu...