Extended Data Fig. 9: Classification of Purkinje cell subpopulations during PAC.
a, Strategy to label Oprd1+ Pn neuron outputs. b, Cerebellar sections showing projections from Oprd1+ Pn neurons. Scale bars: 50 μm (top), 20 μm (bottom). c, Cerebellar sections showing projections in lobule VI, Crus I, and Crus II. Scale bars, 200 μm. d, Boxplot of the number of Purkinje cells detected in each mouse during the Pre (grey), Cond (green), and Post (blue) phases of PAC, and the number of cross-day-aligned neurons (red). n = 6 mice. e, Activity of cross-day-aligned Purkinje cells in cluster 1 (n = 58 neurons from 6 mice) and cluster 2 (n = 218 neurons from 6 mice) during the first border crossing, first crossing back, and the last border crossing on the post-test day. Neurons are ordered according to their cluster and mean Ca2+ activity during the first border crossing. f, Averaged activity of Purkinje cells within each cluster during first border crossing (red), first crossing back (green), and last border crossing (blue) on the post-test day at the level of individual neurons. g, Similar to (f), for individual mice. h, Averaged activity of Purkinje cells in cluster 2 during first border crossing at the level of individual neurons in the Pre (grey), Cond (green), and Post (blue) phases of PAC (F(2,651) = 10.28, P = 4 × 10−5; n = 218 neurons). i, Similar to (h), but at the level of individual mice (F(2,15) = 7.13, P = 0.006; n = 6 mice). j, Ca2+ spike frequency of cluster 1 Purkinje cells during first border crossing (F(2,171) = 13.43, P = 3.8 × 10−6). k, similar to (j), for cluster 2 Purkinje cells (two-sided Wilcoxon signed-rank test; n = 218 neurons). l, Cumulative histograms of Ca2+ spike amplitudes from cluster 1 Purkinje cells. Inset: Average amplitude of spikes >3 z-score (P = 0.0001, n = 75, 93, 178 in Pre, Cond, Post). m, similar to (l), but for cluster 2 Purkinje cells. n, Averaged waveforms of extracted Ca2+ spikes with amplitudes exceeding 3 z-scored ΔF/F for Purkinje cells in cluster 1 (left) and cluster 2 (right) during the Pre, Cond, and Post phases of PAC. Traces are aligned by the time point of their peak. o, Scatterplot of the latency preceding first border crossing against averaged signal of Purkinje cells in cluster 1 during day 3 (Pre), day 5, day 6 (Cond), and day 7 (Post) of PAC for each mouse. Data points were fit by linear regression. p, Similar to (o), for Purkinje cells in cluster 2. q, Purkinje cell activity during first border crossing (left), first crossing back (middle), and last border crossing (right) during the post-test. Neurons are ordered by mean activity during the first border crossing. r, s, Activity of Purkinje cells averaged for individual neurons (r) (F(2,2079) = 11.91, P = 1.3 × 10−6; n = 694) and individual mice (s) (F(2,15) = 13.1, P = 0.0004; n = 6). t, Control for Fig. 4l and (r) with randomized border crossing time. One-way ANOVA with Tukey post-hoc test was used in (f-m) and (r-t). In boxplots, horizontal lines represent median; boxes, quartiles; whiskers, most extreme data points ≤ interquartile range from box edges; and single points, data from individual cells or mice. Shaded area in (n) represents mean ± SEM.
