n anatomical hotspot for striatal dopamine-acetylcholine interactions during reward and movement.

Dopamine (DA) and acetylcholine (ACh) are key neuromodulators that regulate striatal circuits underlying movement and reinforcement learning. Evidence suggests that DA and ACh systems interact, but where and how interactions are expressed across striatal regions in behaviorally relevant release dynamics remains unknown. We applied micro-fiber arrays to simultaneously measure striatum-wide DA and ACh in behaving mice, revealing an anatomical organization in which DA-ACh anti-correlations were concentrated in a hotspot in the anterior dorsolateral striatum (aDLS). Anti-correlations resulted from temporally coincident pairs of spontaneous and event-locked transient peak and dip events occurring in a DA→ACh sequence. The aDLS localized hotspot was consistently expressed within distinct signals associated with unpredicted rewards, learned and extinguished Pavlovian cues, and locomotion initiation and invigoration phases, for which we revealed novel, opposing DA-ACh dynamics. Optogenetic activation of DA neurons selectively suppressed spontaneous ACh release within the aDLS hotspot, and ex vivo recordings revealed enhanced D2-mediated ACh inhibition in aDLS relative to ventral regions, suggesting a mechanistic basis for this spatial specificity. These findings demonstrate that DA-ACh interactions during behavior are spatially organized, rather than uniformly conserved, and shape behaviorally relevant dynamics to regulate region-specific functions in learning and movement control.