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, 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 two behaviorally defined sleep-related sub-states: a low-activity state consistent with light sleep and a quiescent state consistent with deep sleep. These are likely analogous to light and deep NREM sleep in mammals. Although both states occur primarily at night, arousal responses differ by sleep sub-state, and sleep deprivation induces deep sleep-dominated rebound. 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.