Dorsolateral striatal acetylcholine reorganizes neural ensembles to anticipate threat.

Adaptive behavior requires flexible encoding of emotional valence. Although striatal acetylcholine (ACh) signaling is critical for reinforcement learning, its contribution to aversive learning has remained poorly defined. Here, we demonstrate that ACh release in the dorsolateral striatum (DLS) is selectively biased toward negative valence. Using fiber photometry with a genetically encoded ACh sensor, we found that ACh release robustly increased during threat prediction but decreased in anticipation of rewarding outcomes, revealing a bidirectional and valence-specific signature. Optogenetically stimulating ACh release at cue onset accelerated threat learning, impaired extinction, and shifted behavioral responding toward persistent threat expectancy. Concurrent single-cell calcium imaging and optogenetic manipulation revealed that elevated ACh release dynamically reorganized DLS ensemble activity, increasing both excited and inhibited neurons and producing large-scale state-space divergence during threat cues. During extinction, optogenetically sustained ACh release preserved the organization of threat-predictive DLS ensemble activity despite the absence of shock. These findings identify DLS ACh as a valence-specific neuromodulatory signal that reconfigures striatal network dynamics, primes ensembles for impending threat, and biases learning toward threat persistence.