Sleep is tightly regulated by the circadian clock and homeostatic mechanisms. Although the sleep/wake cycle is known to be associated with structural and physiological synaptic changes that benefit the brain, the function of sleep is still debated. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate various functions including feeding, reward, sleep, and wake. Continuous imaging of single neuronal circuits in live animals is vital to understanding the role of sleep in regulating synaptic dynamics, and the transparency of the zebrafish model enables time-lapse imaging of single synapses during both day and night. Here, we use the gephyrin (Gphnb) protein, a central inhibitory synapse organizer, as a fluorescent post-synaptic marker of inhibitory synapses. Double labeling showed that Gphnb-tagRFP and collybistin-EGFP clusters co-localized in dendritic inhibitory synapses. Using a transgenic hcrt:Gphnb-EGFP zebrafish, we showed that the number of inhibitory synapses in the dendrites of Hcrt neurons was increased during development. To determine the effect of sleep on the inhibitory synapses, we performed two-photon live imaging of Gphnb-EGFP in Hcrt neurons during day and night, under light/dark and constant light and dark conditions, and following sleep deprivation (SD). We found that synapse number increased during the night under light/dark conditions but that these changes were eliminated under constant light or dark conditions. SD reduced synapse number during the night, and the number increased during post-deprivation daytime sleep rebound. These results suggest that rhythmic structural plasticity of inhibitory synapses in Hcrt dendrites is independent of the circadian clock and is modulated by consolidated wake and sleep.
Keywords: Gephyrin; Live imaging; Plasticity; Sleep; Synapse; Zebrafish.