Whole Brain and Body Characterization of Sleep Disturbances and Interventions in Fmr1, Shank3 and Cntnap2 Knockout Zebrafish

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. While human (Projects 1 & 2) 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, genes associated with autism like Fmr1, Shank3, and Cntnap2 are pan-neuronal, and their loss likely impacts the entire central nervous system during sleep. Using fluorescence-based polysomnography (fPSG) to capture whole-brain and whole-body imaging with single cell resolution during sleep, we have shown that zebrafish have sleep brain dynamics analogous to mammals. Similarities include 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. 

In this project 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), to improve sleep onset latency and sleep/SBS consolidation in these autism risk gene mutants. Then, 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. The transparency of the zebrafish model will reveal how sleep dynamics are disrupted throughout the entire brain and how sleep interventions can also be beneficial for synaptic normalization throughout the CNS, further establishing the causal/aggravating role of disrupted sleep in the development of autistic traits.