Spontaneous and stimulus-driven arousal produce distinct acetylcholine dynamics across sensory and prefrontal cortex.

Acetylcholine (ACh) release from the basal forebrain has traditionally been viewed as a slow, spatially diffuse signal regulating cortical arousal across sleep and wakefulness1-3. Recent characterizations with higher resolution optical sensors have revealed rapid, local cholinergic modulation supporting dynamic changes in sensory processing, associative learning, and behavioral state4-7. However, sensory events that recruit cortical ACh often also change arousal and evoke movements, making it difficult to determine whether ACh transients reflect sensory features of environmental stimuli or the behavioral state changes that accompany sensory stimulation. To separate these contributions, we performed optic fiber recordings of a genetically encoded ACh fluorescent sensor in the auditory, visual, and prefrontal cortex of awake, head-fixed mice while monitoring pupil size and facial movements. Across cortical areas, ACh release tracked spontaneous fluctuations in arousal state, as indexed by pupil dilation and orofacial movements. Sensory stimuli also evoked rapid ACh transients, with sounds producing larger and more widespread responses than visual stimuli. Because sounds also elicited time-locked pupil dilations and facial movements, we used multivariate modeling to estimate the relative contributions of stimulus features, arousal, and behavior to cortical ACh dynamics. We identified a regional dissociation: sound-evoked ACh release in prefrontal cortex was largely explained by arousal- and movement-related variables, whereas auditory cortical ACh release retained a stronger relationship to stimulus features. These findings show that cortical ACh signaling reflects both shared arousal state and area-specific sensory processing and demonstrate that sound is especially effective at recruiting rapid, widespread cholinergic modulation across cortex.