Extended Data Fig. 8: Oprd1+ Pn neurons mediate PAC-induced analgesia.
a, UMAP plot of Pn neuron clusters with data from two single-cell RNA-seq platforms. 10x: 10x Chromium 3′ v3; SS: SMART-seq v4. b, UMAPs illustrating single-cell expression patterns of neuronal genes. c, Euler diagram showing coexpression of excitatory neuron marker genes and Oprd1 in Pn neurons. d, Strategy to confirm the connection between rACC projection neurons and Oprd1+ Pn neurons using an AAV1-Cre virus that exhibits anterograde transsynaptic spread properties. e, Fluorescence in situ hybridization (FISH) experiments show that the eGFP+ neurons are also Oprd1+. Scale bar, 50 μm. f, Boxplot of the overlay ratio of eGFP and DAPI signals. g, Strategy to label neurons that have a monosynaptic connection with Oprd1+ neurons in the Pn in a retrograde manner using G-deleted mutant rabies virus (RVdG). h, Left top: Coronal section from the Pn of Oprd1Cre mice injected with AAV helpers (red) and RVdG (green). Arrows indicate examples of co-infected starter cells (yellow). Left bottom panels show a magnified view of the dashed box shown in the left top panel. Scale bars, 50 μm (left top) and 20 μm (left bottom). Right: Representative images of GFP-labelled neurons in the ACC. Cg1, cingulate area 1; Cg2, cingulate area 2. Scale bar, 50 μm. i, Strategy and experimental timeline to optogenetically manipulate the activity of Oprd1+ neurons in the Pn during the conditioning phase of PAC. j, Boxplot of the latency preceding first border crossing during the post-test after photoinhibition of eYFP control (green) versus NpHR (blue) mice during the conditioning phase (P = 0.04; n = 10 for each group). k, Boxplots of the latency preceding first paw licking (left; P = 0.06), rearing (middle), and jumping (right; P = 0.05) on the post-test day after photoinhibition of eYFP control (green) versus NpHR (blue) mice during the conditioning phase of PAC. n = 10 for each group. l, Strategy to express inhibitory opsin specifically in the Oprd1+ Pn neurons that receive rACC inputs using an AAV1 virus. m, Representative coronal section of the Pn showing Oprd1+ neurons that express the inhibitory opsin eNpHR. Scale bar: 100 µm. n, Boxplot of the latency for mice to cross back to chamber 1 for the first time (left), and the proportion of time mice spent in chamber 2 (right) with photoinhibition of eYFP control (green) versus NpHR (red) mice on the post-test day. n = 8 in eYFP and 6 in NpHR groups. o, Boxplots of the latency preceding first paw licking (left; P = 0.02), rearing (middle), and jumping (right) on the post-test day with photoinhibition of eYFP control (green) versus NpHR (blue) mice. n = 8 in eYFP and 6 in NpHR groups. p, Experimental timeline for the PAC assay (left) with both µ- and δ-opioid receptor antagonists injection during the post-test to examine which opioid receptor contributes to PAC-induced pain relief. q, Boxplots of the latency preceding first paw licking (left), rearing (middle), and jumping (right) on the post-test day for mice injected with saline, naltrindole (δ-opioid receptor antagonist), or β-funaltrexamine (µ-opioid receptor antagonist). Note that injection of either naltrindole or β-funaltrexamine abolished PAC-induced analgesia. n = 10 for each group. r, Boxplot of the pain threshold of Oprd1Cre mice with eYFP (cyan) or NpHR (brown) injection in the von Frey assay with or without light stimulation (F(1,30) = 7.988, P = 0.008; n = 8 in eYFP control and 9 in NpHR groups). s, Boxplot of the latency preceding paw withdrawal on a 48 °C plate (P = 0.003; left) or 52 °C plate (P = 0.04; right). n = 8 in eYFP control and 9 in NpHR groups. t, Photoinhibition of Oprd1+ Pn neurons does not significantly affect locomotion in the open-field test (F(1,28) = 0.356, P = 0.55; n = 8 in eYFP and 9 in NpHR groups). u, Photoinhibition of Oprd1+ Pn neurons does not affect the latency of mice to fall during the rotarod test (F(1,30) = 0.03, P = 0.984; n = 8 in eYFP and 9 in NpHR groups). Two-sided Wilcoxon rank-sum test was used in (j), (k), (o) and (s); two-way ANOVA with Tukey post-hoc test in (n), (r), (t), and (u). In boxplots, horizontal lines represent median; boxes, quartiles; whiskers, most extreme data points ≤ interquartile range from box edges; and single points, data from individual mice. Data in (t) are mean ± SEM.
