Extended Data Fig. 4: rACC IT neurons are not activated during pain relief expectation.

a, Strategy and experimental timeline to record IT neuron activity in the rACC during PAC using in vivo Ca²⁺ imaging. b, Maximal projection of a Ca²⁺ video with 37 IT neurons. Scale bar, 50 μm. c, Z-scored activity traces (coloured traces) of the 10 example IT neurons outlined in (b). d, Boxplot of the number of detected IT neurons during Pre (grey), Cond (green), and Post (blue) phases, and the number of cross-day-aligned neurons (red; n = 3 mice). e, Activity of cross-day-aligned IT neurons (n = 133 cells from 3 mice) during first border crossing in Pre (left), Cond (middle) and Post (right) phases of PAC. Neurons are ordered according to their mean Ca²⁺ activity for each day. f, Averaged activity of IT neurons during first border crossing at the level of individual neurons in Pre (grey), Cond (green) and Post (blue) phases of PAC (one-way ANOVA, Tukey post-hoc test, F_(2,396) = 2.19, P = 0.113; n = 133 neurons). g, Similar to (f), averaged for individual mice. h, Fisher information calculated between first crossing and crossing back for Ca²⁺ traces, averaged for individual neurons (two-sided Wilcoxon matched-pairs signed-rank test, P = 0.03; n = 133 neurons). i, Similar to (h), averaged for individual mice. n = 3 mice. j, Activity of cross-day-aligned IT neurons (n = 218 from 3 mice) during first border crossing (left), first crossing back (middle), and last border crossing (right) on post-test day. Neurons are ordered according to their mean Ca²⁺ activity for each condition. k, Averaged activity of IT neurons during first border crossing (red), first crossing back (green), and last border crossing (blue) at the level of individual cells on the post-test day (one-way ANOVA, Tukey post-hoc test, F_(2,651) = 5.41, P = 0.004; n = 218 neurons). l, Similar to (k), for individual mice. n = 3 mice. m, Average firing rate of IT neurons during first border crossing in Pre (cyan), Cond (green), and Post (blue) phases of PAC. n = 3 mice. n, Average firing rate of rACC→Pn neurons during first border crossing (red), first crossing back (green), and last crossing (blue) on the post-test day. n = 3 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 neurons or mice.