Fig. 4: Oprd1+ Pn neurons and cerebellar Purkinje cells participate in PAC-induced analgesia.
a, The strategy to characterize gene expression in the Pn using single-cell transcriptomics. Scale bar, 1 mm. D, dorsal; L, lateral. b, Pn neurons in low-dimensional uniform manifold approximation and projection (UMAP) space, colour coded by cluster. E, excitatory; I, inhibitory. c, Opioid and cluster-specific gene expression. d, Euler diagram depicting Oprm1 and Oprd1 expression in Pn excitatory neurons. e, Fluorescence in situ hybridization verifying Oprd1 expression by excitatory Pn neurons. Scale bar, 50 µm. f, The strategy to optogenetically manipulate the activity of Oprd1+ neurons in the Pn. Scale bar, 2 mm. g, The latency preceding first paw licking (left; P = 0.01), rearing (middle; P = 0.07) and jumping (right; P = 0.01) during the post-test with photoinhibition of eYFP control versus NpHR mice. n = 8 (eYFP control) and 9 (NpHR) mice. h, The strategy to monitor Purkinje cell activity using a miniscope. Scale bar, 2 mm. i, The mean projection of a Ca2+ video with 89 Purkinje cells. In total, 40 cross-day-aligned Purkinje cells were classified into clusters 1 or 2. Scale bar, 100 μm. j, Ca2+ trace of 5 Purkinje cells in each cluster. k, Purkinje cell activity during the first border crossing. For each cluster, neurons are ordered by mean Ca2+ activity during the last day of conditioning. l,m, Cluster 1 Purkinje cell activity averaged for individual neurons (l; F2,171 = 23.63, P = 8.6 × 10−10; n = 58) and individual mice (m; F2,15 = 13.94, P = 0.0003; n = 6). Statistical analysis was performed using two-sided Wilcoxon rank-sum tests (g) and one-way ANOVA with Tukey post hoc test (l and m). For the box plots, the centre lines show the median values, the box limits show the quartiles, and the whiskers show the most extreme datapoints ≤interquartile range from the box edges. *P < 0.01 and ***P < 0.001.
