Zebrafish sleep displays distinct sub-states.

Sleep is an essential and evolutionarily-conserved behavior. While mammals and several other species have been shown to exhibit well-defined sleep sub-states, some of which have been ascribed specific functions, it remains unclear to what extent such differentiation exists across the animal kingdom. Here we show, using long-term behavioral data combined with Hidden Markov Modeling, that larval zebrafish display distinct deep and light sleep sub-states. Although both states occur primarily at night, fish respond differently to sleep deprivation and arousing stimuli depending on which sleep sub-state they are in. Moreover, the proportions of deep and light sleep are selectively altered by genetic and pharmacological manipulations of melatonin, serotonin, and norepinephrine signaling, offering new insights into how these neuromodulators shape sleep architecture. These results support zebrafish as a tractable model for dissecting the regulation and function of sleep sub-states. More broadly, they demonstrate that structured, multi-state sleep is a conserved feature of vertebrate behavior.