Role of Prokineticin 2 in Regulating Sleep in Zebrafish

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Light affects sleep indirectly by entraining the circadian clock, but also directly and rapidly via a phenomenon known as the masking or direct effect of light on behavior. Masking refers to the observation that light exposure at night induces sleep in nocturnal animals and suppresses sleep in diurnal animals, including humans, whereas dark exposure during the day inhibits sleep in nocturnal animals. While masking is widespread in the animal kingdom, the mechanisms that mediate masking are largely unknown. Understanding mechanisms that regulate sleep is important because over 10% of Americans suffer from chronic sleep disorders that have poor therapeutic options and cause an annual economic burden of $100 billion. Understanding how masking in particular affects sleep is important because at least some of the sleep disruption observed in modern societies results from masking due to widespread exposure to artificial light at night. Our preliminary experiments using zebrafish suggest that the neuropeptide prokineticin 2 (Prok2) regulates masking. To our knowledge, this is the first gene shown to regulate masking beyond the initial step of light detection by melanopsin in the retina, and thus it provides a foothold to explore the genetic and neurological mechanisms through which light directly affects sleep. Although a similar phenotype was observed in rodents, a role in masking was not explored, and the developmental defects of prok2 mutant mice confound sleep studies. The zebrafish is a vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep. While the zebrafish has some limitations as a sleep model, its amenability to genetic, optogenetic and pharmacological approaches, as well as its transparency and relatively simple yet conserved vertebrate brain, and the lack of developmental defects in prok2 mutant animals, provide advantages for sleep studies that we exploit in this proposal. The zebrafish is particularly appropriate for studying mechanisms that may underlie masking in humans because it is a diurnal vertebrate. In Specific Aim 1 we use gain- and loss-of-function genetics, as well as optogenetic and chemogenetic tools, to test the hypotheses that Prok2 and prok2-expressing neurons regulate masking. We also test whether our findings for Prok2 apply to other peptides that have been proposed to regulate behavior in a manner similar to Prok2. In Specific Aim 2, we explore potential mechanisms through which Prok2 regulates masking, such as by regulating the activity of the noradrenergic locus coeruleus (Aim 2A), the expression of the sleep-promoting neuropeptide galanin in the hypothalamus (Aim 2B) or other sleep regulatory pathways. These studies will for the first time identify genes and neurons in the brain that regulate masking, and thus provide a basis to study the genetic and neurological mechanisms that underlie this phenomenon. Because Prok2 acts via G-protein coupled receptors, which are amenable to small molecule modulation, this work may eventually lead to novel therapies for sleep disorders and for neuropsychiatric disorders that are exacerbated by disrupted sleep.