Cholinergic neurons in the pedunculopontine nucleus guide reversal learning by signaling the changing reward contingency.

Cognitive flexibility enables effective switching between mental processes to generate appropriate responses. Cholinergic neurons (CNs) within the pedunculopontine nucleus (PPN) are associated with many functions, but their contribution to cognitive flexibility remains poorly understood. Here we measure PPN cholinergic activities using calcium indicators during the attentional set-shifting task. We find that PPN CNs exhibit increasing activities correlated with rewards during each stage and error trials in reversal stages, indicating sensitivity to rule switching. Inhibition of PPN cholinergic activity selectively impairs reversal learning, which improves with PPN CN activation. Activation of PPN CNs projecting to the substantia nigra pars compacta, mediodorsal thalamus, and parafascicular nucleus in a time-locked manner with reward improves reversal learning. Therefore, PPN CNs may encode not only reward signals but also the information of changing reward contingency that contributes to guiding reversal learning through output projections to multiple nuclei that participate in flexibility.