Eye movement kinematics reveal novel circadian organization of sleep substates

In most non-mammalian model organisms, sleep is operationally defined as persistent locomotor quiescence (e.g., ≥1 min) associated with decreased arousal^1-3. In contrast to the long-established subdivision of mammalian sleep by eye movements^4-9, the existence of sleep-associated eye movements and, more broadly, discrete sleep substates in non-mammalian organisms remains actively debated^10-14. Here we present the first systematic investigation of fish eye movements during naturally occurring sleep across the full circadian cycle, under light-dark cycles as well as constant light and constant darkness. Across Danio species (Danio rerio, Danio nigrofasciatus and Danio aesculapii), we identify four discrete, conserved sleep substates with circadian organization: three sleep states with distinct eye-movement kinematics (QEM-1, QEM-2 and QEM-3) and one sleep state with no eye movements (QNEM). QNEM predominates at night, QEM-2 increases toward morning, and unexpectedly, QEM-1 occurs almost exclusively during the day. QEM-1 fulfills multiple criteria for sleep in zebrafish, including elevated arousal thresholds, partial loss of postural control, homeostatic rebound after deprivation, noradrenergic suppression, and brain-wide neural dynamics that encode state progression. Altogether, these findings uncover a previously unrecognized sleep architecture in larval fish, in which multiple substates with distinct eye-movement kinematics are conserved across Danio species and gated by circadian time and ambient light.