Several lines of evidence suggest that epidermal growth factor receptor (EGFR) signaling may play an evolutionarily conserved and important role in regulating sleep. Invertebrate genetic studies have demonstrated that EGFR signaling is required for sleep. Vertebrate studies, however, have been inconclusive. To address this, I found that EGFR signaling is both necessary and sufficient to promote sleep in zebrafish, a diurnal vertebrate. Furthermore, I identified effectors of EGFR-regulated sleep in worms and zebrafish. However, several fundamental questions remain regarding how EGFR regulates sleep at the molecular and neuronal levels, what factors mediate EGFR-driven sleep, and in what contexts EGFR is required for sleep. The objectives of this proposal seek to answer these particular questions. Zebrafish are well suited to address these questions because they exhibit behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep, and unlike nocturnal rodents that are commonly used for sleep research, zebrafish are diurnal like humans. The optical transparency and relatively simple vertebrate brain of zebrafish larvae facilitate noninvasive studies of neuronal function in intact animals. The small size of zebrafish larvae allows for large-scale sleep/wake behavioral assays that generate statistically robust data and enable rapid testing of many genetic perturbations on behavior. We will exploit these advantageous features to further explore the EGFR-mediated sleep regulation. In Specific Aim 1, we will test the hypothesis that egfr-expressing neurons are necessary and sufficient to promote sleep. We will use high-throughput optogenetic and chemical-genetic assays to stimulate, inhibit and ablate these neurons noninvasively in freely behaving zebrafish larvae. In Specific Aim 2, I will test the hypothesis that cellular stress-induced sleep in zebrafish requires EGFR signaling and egfr-expressing neurons by stimulating, inhibiting and ablating them using optogenetic and chemogenetic approaches. In Specific Aim 3, we will test the hypothesis that EGFR signaling promotes sleep by inducing the secretion of CSF sleep promoting factors by activating EGFR signaling, then extracting CSF and characterizing potential sleep promoting CSF factors using mass spectrometry. During the R00 phase, I will perform gain- and loss-of-function genetic studies of CSF candidate sleep-promoting factors to determine whether they are necessary and sufficient to promote sleep in zebrafish. These experiments may reveal novel genetic and neurological mechanisms through which EGFR regulates sleep. Investigating these questions is important because mutated EGFR signaling components are implicated in several human disorders, including disrupted rest-activity rhythms. By investigating this poorly understood sleep regulatory pathway and identifying new mechanism, this project may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neurological disorders, including depression, schizophrenia and autism, and may be causal in some cases, this project may also lead to improved therapies for some neurological disorders.