Fig. 2: ACh release components for conditioned cues evolve bi-directionally over distinct anatomical gradients during Pavlovian learning.
a Schematic of dual-cue delay Pavlovian conditioning task. b Normalized mean lick spout contacts aligned to light (top) and tone (bottom) cue onset for 10 consecutive sessions from the start of training for one representative mouse. c Mean lick index on light (top) and tone (bottom) cue trials (n = 20 trials for each cue) for each day after the start of Pavlovian training for each mouse (n = 8). Error bars, S.E.M. d Mean percent time spent licking during the 3 s light (top) and tone (bottom) cue interval prior to reward for each mouse for sessions pre and post learning. Red points are means (± S.E.M.) across all mice (n = 8). For both light and tone, W8 = 36, **p = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. e Same as (d) for licking during ITI periods (W8 = −28, *p = 0.0273, one-tailed Wilcoxon matched-pairs signed rank test). f Top: Mean ∆F/F aligned to the light cue onset for 2 representative fibers for trials pre (blue) and post (orange) learning. Left example, significant early peak change; right, no change. Shaded regions, S.E.M. Red dots in insets indicate the fiber locations in the coronal plane. Bottom: Light-cue-aligned ∆F/F for all trials included in the triggered averages at top. g Left: percent of all fibers with significant increases or decreases, no change, or no significant component from pre to post learning for the early peak ∆F/F at light cue onset. Right: histogram of number of fibers per mouse with significant early peak changes. Each bar is the fiber count for one mouse for each condition indicated by colors at left. h Mean ∆F/F aligned to the light cue onset as in (f) for a representative fiber with a significant change in the dip with learning. i Same as (g) for dip ∆F/F changes from pre to post learning. j Maps (axial, top; sagittal, bottom) showing spatially weighted means across locations of differences with learning (post-pre ∆F/F) for the mean early peak ∆F/F at light cue onset. Lines indicate the axes of maximal variation and arrows indicate the direction of peak increases from smallest to largest changes. White contours indicate regions with changes in the highest 10th percentile. k Same as (j) for the dip component. l Model coefficients indicating the relative magnitude and direction of the variation in mean ∆F/F differences with learning for the peak and dip components for light and tone cues across each striatal axis (AP: anterior-posterior, ML: medial-lateral, DV: dorsal-ventral). The coefficients represent fixed effects derived from a linear mixed-effects model, with individual recording sites as the unit of analysis and mouse identity included as a random effect. The sign of the coefficient indicates the direction of the largest differences (see arrows). Error bars, S.E.M. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001, two-tailed Wald t-test on model coefficients followed by bonferroni post hoc analysis on model coefficients. Significant interaction terms (black) indicate difference in coefficients between peak and dip for a given axis. m Same as (l) but comparing spatial coefficients for tone and light for all three components. The exact p-values and additional statistical details can be found in Supplementary Table 1. Water drop schematic in (a) adapted from SciDraw (scidraw.io), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). Source (https://doi.org/10.5281/zenodo.3925935). Brain schematics in (f) and (h) were adapted from the Allen Mouse Brain Common Coordinate Framework (CCFv3) (https://atlas.brain-map.org/). Source data are provided as a Source data file.
