Fig. 3: mMORp-hM4Di spinal cord expression and chemogenetic-induced analgesia.
a AAV1-mMORp-hM4Di-mCherry injection schema within the lumbar spinal cord in C57BL/6J mice to inhibit dorsal horn MOR+ cells and not MOR+ nociceptors or descending brain stem circuits. b mMORp-hM4Di-mCherry expression and spread across the L4 spinal cord; scale bar = 100 μm. c Location map and quantification of mMORp-hM4Di-mCherry+ cells across the Rexed laminae in the dorsal and ventral horns (N = 3 mice). d Experimental timeline for viral injections and chemogenetic behavioral testing. e Mechanical sensory thresholds (von Frey Up-Down testing) in mMORp-hM4Di-mCherry injected mice (N = 9 mice) compared with hSyn-mCherry injected controls (N = 7 mice) at baseline and 30 min following systemic CNO administration (3 mg/kg; Two-way ANOVA + Bonferroni: main effect: P = 0.011 [viral treatment × CNO treatment]; multiple comparisons: basal v. CNO, P = 0.990 [mCherry], P = 0.006 [hM4Di]). Average response changes per group shown as thick gray (mCherry) or red (hM4Di) lines. Individual mice are shown as thin gray and red lines. f Nocifensive behaviors observed on an inescapable 52.5 °C hot plate over a 30-sec trial for the same animals (N = 9 hM4Di-mCherry, N = 7 mCherry): latency (sec) to hind paw withdrawal (two-way ANOVA + Bonferroni: main effect: P = 0.085 [viral treatment], P = 0.162 [CNO treatment]; multiple comparisons: basal v. CNO, P > 0.999 [mCherry], P = 0.067 [hM4Di]), hind paw licking duration (two way ANOVA + Bonferroni; main effects: P = 0.008 [viral treatment × CNO treatment], P = 0.008 [viral treatment]; multiple comparisons: basal v. CNO, P > 0.999 [mCherry], P = 0.0007 [hM4Di]), and total jumping bouts (two way ANOVA + Bonferroni; main effects: P = 0.004 [viral treatment × CNO treatment], P = 0.002 [viral treatment], P = 0.006 [CNO treatment]; multiple comparisons: basal v. CNO, P = 0.0006 [mCherry], P > 0.999 [hM4Di]). g Time course (left) and cumulative global scoring (right) of nocifensive behaviors (licking, biting, jumping, etc.) observed in the formalin injection test during the first and second phases of testing in mMORp-hM4Di-mCherry (N = 9) and control (N = 7) animals at basal and post-CNO time points (Time course: two-way ANOVA + Bonferroni; main effects: P = 0.006 [time bin × nocifensive behaivors], P < 0.0001 [time bin], P = 0.001 [nocifensive behaviors]; multiple comparisons: mCherry v. hM4Di, P = 0.014 [5 min], P = 0.008 [20 min], P = 0.0002 [25 min], P = 0.48 [30 min]. Global scoring: two-way ANOVA + Bonferroni; main effects: P = 0.006 [stage × nocifensive behaviors], P < 0.0001 [stage], P = 0.001 [nocifensive behaviors]; multiple comparisons: mCherry v. hM4Di, P = 0.222 [1st stage], P < 0.0001 [2nd stage]). All data are presented as means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
