Outcome-Locked Cholinergic Signaling Suppresses Prefrontal Encoding of Stimulus Associations

Abstract

Acetylcholine (ACh) is thought to control arousal, attention, and learning by slowly modulating cortical excitability and plasticity. Recent studies, however, discovered that cholinergic neurons emit precisely timed signals about the aversive outcome at millisecond precision. To investigate the functional relevance of such phasic cholinergic signaling, we manipulated and monitored cholinergic terminals in the mPFC while male mice associated a neutral conditioned stimulus (CS) with mildly aversive eyelid shock (US) over a short temporal gap. Optogenetic inhibition of cholinergic terminals during the US promoted the formation of the CS–US association. On the contrary, optogenetic excitation of cholinergic terminals during the US blocked the association formation. The bidirectional behavioral effects paralleled the corresponding change in the expression of an activity-regulated gene, c-Fos in the mPFC. In contrast, optogenetic inhibition of cholinergic terminals during the CS impaired associative learning, whereas their excitation had marginal effects. In parallel, photometric recording from cholinergic terminals in the mPFC revealed strong innate phasic responses to the US. With subsequent CS–US pairings, cholinergic terminals weakened the responses to the US while developing strong responses to the CS. The across-session changes in the CS- and US-evoked terminal responses were correlated with associative memory strength. These findings suggest that phasic cholinergic signaling in the mPFC exerts opposite effects on aversive associative learning depending on whether it is emitted by the outcome or the cue.