Learning of whisker-guided object location discrimination and associated motor actions

A.Two-location discrimination task design with trial outcomes. B. Single trial structure with example licking, whisking, GRAB-ACh signal traces. C. Learning curves of 8 mice, mean ± SEM sessions to expert. Blue circles, unbalanced go and no-go trials. Gold dash, expert threshold. Gray dash, chance level. D. Mean performance for three early and expert sessions, per mouse. 3 mice with 1 no-whisker session, 4 mice with 3 no-whisker sessions averaged. E. Average whisking amplitude aligned to pole in cue. Mean and SEM. 3 expert sessions / mouse. F. Same aligned to pole out cue. G. Grand mean trial averaged whisking amplitude by trial outcome. Mean and SEM. 3 expert sessions / mouse. H. Same for licking rates.

Acetylcholine release in S1 varies with trial outcome

A. GRAB-ACh3.0 AAV expression in S1 barrel cortex, 3 weeks post injection. Cartoon generated by BioRender.com. B. Phasic increase of acetylcholine release after pole in cue in most trials. C. Acetylcholine induced fluorescence changes sorted by trial types, one expert example session. D. Grand average, 8 mice, 3 expert sessions each. E. Comparison of grand average acetylcholine dynamics within the C2 column (solid) and in the surround (dashed). 8 mice, 3 expert sessions each. Traces are vertically offset for display.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Whisking drives acetylcholine release in S1

A. Top, grand mean acetylcholine fluorescence change aligned to pole in cue. Bottom, average whisking amplitude. 8 mice, 3 expert sessions each. B. Same to pole out cue. C. Whisking amplitude sorted by mean of 500ms post pole in cue. No lick trials pooled from 3 expert sessions, 1 mouse. D. Same, for acetylcholine fluorescence change sorted by whisking amplitude. E. Grand mean of acetylcholine fluorescence change vs. whisking amplitude of 500ms after pole in cue. No lick trials from 3 early and 3 expert sessions, 8 mice. F. Grand mean of acetylcholine fluorescence change vs. whisking amplitude during 1s before pole in cue for expert sessions. Trials with no licks within 1.5s after trial start, 3 expert sessions, 8 mice.

Licking strongly drives acetylcholine release in S1

A. Acetylcholine fluorescence change across trials, sorted by number of licks in trial, aligned to first lick. One example expert session. B. Lick pattern across trials, sorted by number of licks in trial, aligned to first lick. Same example session as A. C. Pooled inter-lick interval from 3 early (gray) and 3 expert (black) sessions each, 8 mice. D. Acetylcholine fluorescence change aligned to first lick for trials with different numbers of licks. Same session as A. E. Mean acetylcholine fluorescence change in 1 second following first lick binned by number of licks in that period. Circles, mean 3 expert sessions / mouse. Linear fit from 1-7 licks. F. Duration, onset to trough, of the first acetylcholine transient, binned by number of licks within 2 seconds of first lick. Circles, mean 3 expert sessions / mouse. Linear fit from 1-10 licks.

Reward delivery does not drive acetylcholine release in sensory cortex

A.Top: Acetylcholine fluorescence change aligned to first lick from Hit (blue) and False Alarm (green) trials. Mean and SEM. 3 expert sessions, 8 mice for all panels. Bottom: Significance test between Hit and False Alarm acetylcholine over time (p-value, paired t-test, corrected for multiple comparisons). B. Mean number of licks in the answer period on Hit and False Alarm trials. C. Distribution of lick counts in the answer period histogram for Hit (blue) and False Alarm (green) trials. D.Top: Mean acetylcholine fluorescence change in answer period, binned by licks in answer period for Hit (blue) and False Alarm (green). Bottom: Significance of difference between Hit and False Alarm trials, binned by licks (p-value, paired t-test, corrected for multiple comparisons). E-H. Same as A-D, but in auditory cortex .

Learning selectively potentiates acetylcholine release to first licks in S1

A. Grand mean acetylcholine fluorescence change aligned to first lick in early (gray) and expert (black) sessions, 3 sessions per condition, 8 mice for all panels except Paired comparison of average acetylcholine fluorescence change within one second after first lick between early (gray) and expert (black) sessions for each individual mouse is shown in inset figure. B. Bands SEM. B. Relationship between correct answer rate and acetylcholine fluorescence change within 1 second following first lick. Shade indicates mouse identity. All 51 single-whisker imaged sessions, Fit equation r = 0.05328*x-0.0123. C. Mean lick rates for trial types in early (top) and expert (bottom) sessions. D. Mean lick numbers per trial. E. Peak lick rate. F. Grand mean ± SEM acetylcholine fluorescence change in no lick trials. G. Same for mean ± SEM average whisking amplitude H. Mean acetylcholine fluorescence change in 1 second following first lick binned by number of licks in that period. Linear fits from 1-7 licks. I. Acetylcholine fluorescence change from whisking, subsequent lick, and first lick for early (gray) and expert (black) sessions. Red, grand mean ± SEM.

General linear model of acetylcholine fluorescence dynamics from sensory, motor, and reward predictors

A. Sample acetylcholine fluorescence trace. Object model fit to predict fluorescence from below predictors. B. Sample true fluorescence (black), corresponding predicted fluorescence trace using the predictors in A (red), and licks (blue). C. Different predictors’ β coefficient for early (black) and expert (red) sessions. 3 sessions for each condition, 7 mice.

Pole out cue induced whisking in all trial types

A. Average Hit rate (blue) and False Alarm rate (green) of 8 mice across training sessions. Individual mouse Hit rate in light blue, False Alarm rate in light green. B. Average whisking amplitude aligned to pole out cue by trial outcome, 3 expert sessions / mouse.

Technical details of targeting and imaging

A.Intrinsic signal imaging result showing C2 barrel column location through the cranial window. B.Left: Raw fluorescence trace of an example session. The colored area is excluded from analysis due to non-stationary photodynamcis. Right: Zoom of same. C. Acetylcholine fluorescence changes averaged by trial types, same session as Figure 2C.

Whisking drives acetylcholine release in S1

A. Mean whisking amplitude of 1s post pole in cue. B. Mean acetylcholine change vs. mean whisking amplitude over 1s following pole in cue. C. Whisking amplitude heatmap sorted by the mean whisking amplitude of 0-500ms post pole withdrawal cue. No lick trials pooled from 3 expert sessions, one mouse. D. Acetylcholine release signal heatmap sorted by the same criteria as C. E. Mean acetylcholine release signal of 500ms post pole withdrawal cue vs. mean whisking amplitude of 500ms post pole withdrawal cue. No lick trials pooled from 3 early and 3 expert sessions, 8 mice.

First lick time and first lick related acetylcholine release comparison between Hit and False Alarm trials

A. Mean acetylcholine fluorescence change aligned to first lick from Hit trials (left) and False Alarm trials (right) for each mouse (n=8 mice), somatosensory task. B. First lick time distribution for Hit (blue) and False Alarm (green) trials. 3 expert sessions, 8 mice. C. Mean acetylcholine fluorescence change aligned to first lick from Hit trials (left) and False Alarm trials (right) for each mouse (n=6 mice), Auditory task.

First lick time and first lick related acetylcholine release comparison between early and expert sessions

A. First lick time distribution for early (gray) and expert (black) sessions. 3 sessions per condition, 8 mice. B. Left, Hit trials average acetylcholine fluorescence change for early (light blue) and expert (blue) sessions. Right, same for False Alarm trials. 3 sessions per condition, 8 mice.