Behavioral arousal organizes visual cortical population micro-states across the sleep-wake cycle

Abstract

Brain states are conventionally defined as discrete categories, such as wakefulness, NREM sleep and REM sleep. Yet, whether these labels fully capture the organization of cortical population dynamics remains unclear. Using large-field-of-view two-photon calcium imaging of layer II/III pyramidal neurons in mouse primary visual cortex (V1), we show that population activity organizes into recurring micro-states that span conventional sleep-wake boundaries. A Hidden Markov Model fitted independently of behavioral state labels identified temporally stable micro-states with structured transition pathways. Micro-state organization was more strongly aligned with a continuous behavioral arousal axis, indexed by pupil diameter and orofacial motion, than with discrete NREM-wake categories, a relationship confirmed by mediation analysis. At the circuit level, two spatially intermingled but functionally opposing neuronal ensembles tracked this arousal axis in opposite directions. These findings establish behavioral arousal, rather than discrete wake-sleep state, as the primary organizing dimension of V1 population dynamics.