Extended Data Fig. 5: Ca²⁺ dynamics of rACC→Pn neurons during thermal and mechanical pain.

a, Strategy to measure innocuous mechanical, noxious mechanical, or noxious heat responses while monitoring the Ca²⁺ activity of rACC→Pn neurons. b, Average silhouette width plotted for cluster numbers k from 1 to 10. Dashed line indicates the number of clusters corresponding to the maximal silhouette width. c, Averaged response (10 trials) of individual neurons upon each stimulation. Dashed lines (red) indicate the time point of stimulation (n = 335, 238, and 284 neurons for innocuous mechanical, noxious mechanical and noxious heat, respectively). Red and blue bars represent the cluster to which each neuron belongs (cluster 1 or cluster 2, respectively). d, Average activity of neurons in response to innocuous mechanical, noxious mechanical and noxious heat for cluster 1 (left) and 2 (right). e, Average activity of individual neurons in response to innocuous mechanical, noxious mechanical and noxious heat in cluster 1 (left; one-way ANOVA, Tukey post-hoc test, F_(2,286) = 20.26, P = 1.1 × 10⁻⁸; n = 124, 92, and 73 neurons for innocuous mechanical, noxious mechanical and noxious heat, respectively) and 2 (right; one-way ANOVA, Tukey post-hoc test, F_(2,565) = 16.73, P = 5.1 × 10⁻⁶; n = 211, 146, and 211 neurons for innocuous mechanical, noxious mechanical and noxious heat, respectively). Note that innocuous mechanical stimuli induced an increase in the activity of neurons in cluster 1, but a decrease in the activity of neurons in cluster 2. f, Average Ca²⁺ activity of rACC→Pn neurons during innocuous mechanical (red), noxious mechanical (green), and noxious heat (blue; n = 5 mice). g, Similar to (c), with cross-session-aligned neurons (n = 118 neurons). h, Similar to (d), with cross-session-aligned neurons. i, Similar to (e), with cross-session-aligned neurons (cluster 1; left; one-way ANOVA, Tukey post-hoc test, F_(2,67) = 3.9, P = 0.02; n = 25, 20, and 25 neurons for innocuous mechanical, noxious mechanical and noxious heat; cluster 2; right; one-way ANOVA, Tukey post-hoc test, F_(2,281) = 3.2, P = 0.04; n = 93, 98, and 93 neurons for innocuous mechanical, noxious mechanical and noxious heat). j, Similar to (f), with cross-session-aligned neurons. k, Heatmap of rACC→Pn neuron activity relative to licking (left) and rearing (right) behaviours in the post-test of PAC. l, Average Ca²⁺ activity of rACC→Pn neurons during licking (red) and rearing (green). n = 6 mice. 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 (d, f, h, j, l) represents mean ± SEM.