Cholinergic--dopaminergic interplay underlies prediction error broadcasting

Neuromodulatory systems, notably basal forebrain cholinergic and midbrain dopaminergic pathways, critically influence reinforcement learning 1-3. However, whether and how they cooperate or compete to jointly control associative learning functions remains unresolved. Here we demonstrate that basal forebrain cholinergic and midbrain dopaminergic projection systems form a coordinated and cross-regulating architecture for encoding prediction errors. Using dual-cell-type optogenetic tagging and real-time neurotransmitter measurements in mice performing a psychometric operant learning task, we simultaneously monitored cholinergic and dopaminergic activity during learning. Dopamine and acetylcholine jointly encoded reward prediction errors synergistically following reward and reward-predicting stimuli. In contrast, aversive outcomes elicited opposite responses in cholinergic neurons and approximately half of dopaminergic neurons. Activity in these two populations exhibited negative trial-by-trial correlations, revealing antagonistic dynamics. Consistently, channelrhodopsin-assisted circuit mapping uncovered a disynaptic inhibitory pathway from cholinergic to dopaminergic neurons. Chemogenetic suppression of cholinergic activity disrupted dopaminergic prediction error signaling, reduced punishment-induced suppression of dopamine release, and impaired learning. These results demonstrate that prediction error signaling is jointly implemented by coordinated interactions between major neuromodulatory systems, challenging the prevailing view of their functional independence and revealing coordinated cross-system interactions as an organizing principle of reinforcement learning, with implications for neuropsychiatric disease 4-6.