Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer’s disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator β-PIX. Acetylcholine induced β-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-β-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade’s therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217)