Extended Data Fig. 1: Validation of an animal model for placebo analgesia investigation.
a, Experimental timeline to examine whether PAC-induced preference for chamber 2 is due to pain relief expectation. b, Latency for mice to cross back to chamber 1 for the first time (left; P = 1) and the proportion of time mice spent in chamber 2 (right; P = 0.91) while setting both chambers at 30 °C on the post-test day. n = 10 mice in each group. c, Experimental timeline to examine the timescale of PAC-induced analgesia (top). Comparison of the chamber preference and nociceptive behaviours of mice 1 (Day 7), 3 (Day 10), and 7 (Day 14) days after the conditioning phase of PAC (bottom). n = 10 in each group. d, Experimental timeline for the PAC assay (left) with naloxone injection during the post-test to test the dependency of PAC-induced pain relief on the endogenous opioid system. e, Latency for mice to cross back to chamber 1 for the first time (left; F(1,37) = 1.54, P = 0.66) and the proportion of time mice spent in chamber 2 (right; P = 0.22) after saline (red) or naloxone (purple) injection on the post-test day. n = 10 mice in each group. f, Latency preceding first paw licking (left; P = 0.007), rearing (middle; P = 0.07), and jumping (right; P = 0.007) behaviour after reaching chamber 2 on the post-test day for saline- or naloxone-injected mice. n = 10 mice in each group. g, Experimental timeline for the PAC assay (left) with naloxone injection during the conditioning phase to test the dependency of PAC-associated learning on the endogenous opioid system. h, Latency for mice to cross back to chamber 1 for the first time (left; P = 0.07) and the proportion of time mice spent in chamber 2 (right; P = 0.02) after saline (yellow) or naloxone (blue) injection on the post-test day. n = 10 mice in each group. i, Latency preceding first paw licking (left; P = 0.91), rearing (middle; P = 0.01), and jumping (right; P = 0.14) behaviour after reaching chamber 2 on the post-test day for saline- or naloxone-injected mice. n= 10 mice in each group. j, Experimental timeline to examine chemical pain sensitivity of mice during the post-test. k, Boxplots of the proportion of time mice spent in chamber 2 (left) and the duration of attending behaviours during the whole formalin test (right) in Ctrl and Cond groups. n = 4, 6 mice in Ctrl and Cond. l, Similar to (k), but for the first 3 min after formalin injection (P = 0.005 for time spent in chamber 2 and P = 0.03 for attending duration). n = 4, 6 mice in Ctrl and Cond. m, Experimental timeline to examine mechanical, thermal, and chemical pain sensitivity of mice after the conditioning phase of PAC. n, Boxplots of the mechanical (left), thermal (middle), and chemical (right) pain sensitivity after the conditioning phase of PAC, measured with von Frey, Hargreaves, and formalin injection, respectively. n = 10 mice in each group. o, Experimental timeline and strategy to examine the nocifensive behaviours of mice after confining them in either chamber 1 or chamber 2 by blocking the opening between chambers. p, Latency preceding first paw licking (left), rearing (middle), and jumping (right) behaviours of mice confined in chamber 1 (purple) and chamber 2 (red) on the post-test day. n = 5 mice in each group. q, Experimental timeline for the PAC with female mice. r, Boxplots of the latency for female mice to cross back to chamber 1 for the first time (left) and the proportion of time mice spent in chamber 2 (right) in Ctrl and Cond groups. n = 10 mice in each group. s, Latency preceding first paw licking (left; P = 0.01), rearing (middle), and jumping (right; P = 0.07) behaviour after reaching chamber 2 on the post-test day for female mice in Ctrl and Cond groups. n = 10 mice in each group. Two-sided Wilcoxon rank-sum test was used in (b), (f), (i), (k), (l), and (s); two-way ANOVA, Tukey post-hoc test in (e) and (h). 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.
