Cholinergic activity was monitored while mice performed a probabilistic Pavlovian conditioning task. Mice were headfixed and listened to auditory stimuli of different frequency, which signaled either likely reward and unlikely punishment, or likely punishment and unlikely reward. Cholinergic activity was measured in two ways. First, bulk calcium imaging of cholinergic neurons was performed by fiber photometry. Second, extracellular electrophysiology recordings were performed and optogenetic tagging was used to identify cholinergic neurons in task-performing mice. This dataset contains fiber photometry data, electrophysiology data and behavioral data of mice.

Behavioral training was performed as previously described (Hegedus et al., 2021, Training protocol for probabilistic Pavlovian conditioning in mice using an open-source head-fixed setup). Briefly, mice were habituated to a headfixation apparatus and were trained to listen to tones and collect water rewards in a headfixed setup. Two tones of different frequency were associated with likely reward / unlikely punishment or unlikely reward/likely punishment (12 kHz tone predicted 80% water reward, 10% air-puff punishment, 10% omission; 4 kHz tone predicted 25% water reward, 65% air-puff punishment, 10% omission).

Bulk calcium imaging of cholinergic neurons was performed by fiber photometry (dual fiber photometry setup, Doric Neuroscience) and visualized online with Doric Studio Software. Two LED light sources (465 nm, 405 nm) were channeled into fluorescent Mini Cubes (iFMC4, Doric Neuroscience). Light was amplitude-modulated by the command voltage of the two-channel LED driver (LEDD_2, Doric Neuroscience, the 465 nm wavelength light was modulated at 208 Hz and 405 nm wavelength was modulated at 572 Hz). Light was channeled into 400 µm diameter patch cord fibers and was connected to optical fiber implants during training sessions. The same optical fibers were used to collect the bilateral emitted fluorescence signal, which were detected with 500-550 nm fluorescent detectors integrated in the Mini Cubes. Emitted signals were sampled at 12 kHz, decoded in silico and saved in a *.csv format.

Chronic extracellular electrophysiology recordings were performed using tetrode electrodes, as described in Hegedus et al, 2021, Differential recruitment of ventral pallidal e-types by behaviorally salient stimuli during Pavlovian conditioning. We performed optogenetic tagging of cholinergic neurons in ChAT-Cre mice, as detailed in Hangya et al., 2015, Central Cholinergic Neurons Are Rapidly Recruited by Reinforcement Feedback.

Associated code located at https://github.com/hangyabalazs/cholinergic_Pavlovian_analysis.