Project 4: Project Summary/Abstract Sleep is critical for proper synaptic connections and brain development. Our group previously established that sleep disruptions in zebrafish, like in other species, prevent normal structural synapse plasticity. Conversely, proper sleep and melatonin hypnotic/circadian treatment can improve these synaptic defects. Animal models of ASD, like ASD patients, suffer from sleep disruptions during development and display synaptic and behavioral deficiencies. Here, we hypothesize that sleep disruptions during development are causal and/or aggravating factors of ASD synaptic and behavioral defects, and that sleep interventions could alleviate these issues. While human (Projects 1 & 2) and mouse (Project 3) approaches permit exquisite studies of social interactions, repetitive behaviors, and associated cortical synaptic defects, zebrafish is a transparent vertebrate popular in developmental biology allowing whole brain and body investigation. Importantly, ASD risk genes like Fmr1, Shank3, and Cntnap2 are pan-neuronal, and their loss likely impacts the entire central nervous system during sleep. We have recently developed fluorescence-based polysomnography (fPSG) in zebrafish, a novel, non-invasive method allowing whole-brain and whole-body imaging with single cell resolution during sleep. Using fPSG, we have shown that zebrafish have sleep brain dynamics analogous to mammals, including a state we coined slow bursting sleep (SBS) which shares many commonalities with Non-REM slow wave sleep (SWS). Our preliminary data indicates that SBS is fragmented in developing Fmr1 zebrafish mutants. Further, studies from other groups have shown that based on actimetry, sleep/wake pattern is also disrupted in zebrafish cntnapt2ab and shank3ab mutants. However, their brain activity during sleep has not yet been investigated. Thus, in Aim 1, we will apply fPSG to these three genotypes (fmr1, shank3ab, and cntnap2ab mutants) and controls to fully characterize their sleep neural and muscular dynamics during development. Next, we will apply the same pharmacological interventions (H1R antihistamine, GABAA agonist, and hypocretin/orexin receptors antagonist) used in human (Project 2) and mouse (Project 3), to improve sleep onset latency and sleep/SBS consolidation in these ASD risk gene mutants. Then, in Aim 2, we will investigate the respective beneficial effects of these NREM/SWS/SBS-sleep interventions on structural synapse density using longitudinal imaging of telencephalic, hypothalamic and spinal cord circuits expressing synaptic proteins fused to fluorescent markers such as PSD95-eGFP, Synaptophysin-eGFP or Gephyrin-eGFP. In parallel, treated fish will be assessed for improvement in repetitive and social behaviors like in mouse (Project 3) and human (Project 2). Complementing the latter, the transparency of the zebrafish model will reveal how sleep dynamics are disrupted throughout the entire brain and how sleep interventions can also ...