Extended Data Fig. 4: Fibre photometry recording.
From: Deconstruction of a spino-brain–spinal cord circuit that drives chronic pain
a, Representative average traces of non-withdrawal and withdrawal trails Pre-SNI (left) and Post-SNI (right) in OPRM1+ RVMSC neurons. Scale bar: 1% ΔF/F, 3 sec. b, Quantification of AUC of a. n = 6, Two-sided Paired t-test (Normal non-withdrawal vs. withdrawal: P = 0.006; SNI non-withdrawal vs. withdrawal: P = 0.0017). c, Quantification of OPRM1+ RVMSC neurons respond to mechanical (von Frey) and thermal (Hargreaves and Plantar cold) stimuli in normal and SNI mice (n = 7–8). Two-sided Mann–Whitney test (Von Frey normal vs. SNI: P = 0.0003; Hargreaves normal vs. SNI: P = 0.62; Plantar cold normal vs. SNI: P = 0.0041). Related to Fig. 1h. d,e, Representative traces (d) and quantification (e) of mechanical (von Frey) and thermal (Hargreaves) stimulation evoked calcium response in OPRM1+ RVMSC neurons of before, 2, 7, and 21 days after CFA injection. n = 6, Non-parametric ANOVA (Friedman test), post-hoc pairwise comparisons were conducted using Dunn’s multiple comparison test (von Frey Pre vs. 2D post CFA: P = 0.0048; von Frey Pre vs. 7D post CFA: P > 0.9999; von Frey Pre vs. 21D post CFA: P > 0.9999; von Frey 2D post CFA vs. 7D post CFA: P = 0.1521; von Frey 2D post CFA vs. 21D post CFA: P = 0.0834; von Frey 7D post CFA vs. 21D post CFA: P > 0.9999; Hargreaves Pre vs. 2D post CFA: P = 0.4418; Hargreaves Pre vs. 7D post CFA: P > 0.9999; Hargreaves Pre vs. 21D post CFA: P > 0.9999; Hargreaves 2D post CFA vs. 7D post CFA: P = 0.7051; Hargreaves 2D post CFA vs. 21D post CFA: P = 0.2650; Hargreaves 7D post CFA vs. 21D post CFA: P > 0.9999). Scale bar: 1% ΔF/F, 2 sec. f, Representative traces of calcium response (left, upper panel) and movement speed (left, lower panel), and correlation of calcium response vs. movement speed (right) in normal mice. Dash line indicates the time of movement start. Dot indicates the average speed calcium signal during each movement event. Red line: linear fitting of dots. Pearson’s r = −0.1453 or 0.9176 for locomotion and escape, respectively. Scale bar: 50 % of normalized movement bout, 1% ΔF/F, 5 cm/s. Related to Fig. 1j. Locomotion: spontaneous movement without stimulus; Escape: escape response after von Frey stimulus. g, Quantification of f and Fig. 1j. n = 6, one-way ANOVA, post-hoc pairwise comparisons were conducted using Tukey’s multiple comparisons test (Locomotion BL vs. escape BL: P = 0.0169; Locomotion BL vs. Locomotion SNI: P = 0.9686; Locomotion BL vs. escape SNI: P = 0.0083; Escape BL vs. Locomotion SNI: P = 0.0026; Escape BL vs. Escape SNI: P = 0.0351; Locomotion SNI vs. Escape SNI: P = 0.0039). h, Representative traces (left) and quantification (right, n = 7) of calcium response in OPRM1+ RVMSC neurons evoked by von Frey stimulation or occurring spontaneously following SNI. Scale bar: 1% ΔF/F, 3 sec. Two-sided Paired t-test, P = 0.1553. i, Experimental paradigm for j and k. j, Representative image from six independently performed experiments shows fibre track and jGCaMP7s expression in RVM-projecting vGlut2+ lSuC neurons. Scale bar: 500 μm (lSuC), 200 μm (RVM). k, Representative traces (left) and quantification (right) of calcium response in RVM-projecting vGlut2+ lSuC neurons evoked by von Frey stimulation before and after SNI. Scale bar: 1% ΔF/F, 3 sec. n = 6, two-sided paired t-test, P = 0.0069. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are presented as mean ± s.e.m.
