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. 2024 Apr 26;10(17):eadj9581. doi: 10.1126/sciadv.adj9581

Fig. 2. LC activity is required for systemic morphine antinociception.

Fig. 2.

(A) c-Fos expression after injection. Scale bars, 150 μm. (B) Percentage of LC neurons that colocalize with c-Fos (five to eight images per mouse; n = 6 saline, n = 6 morphine, n = 4 naloxone; one-way ANOVA with Tukey’s multiple comparisons test, P < 0.0001, F2,13 = 20.97). (C) Injection of GCaMP8s and fiber implantation for fiber photometry. (D) Average normalized Ca2+ response to morphine or saline at time 0 (n = 10 mice). (E) Area under the curve (AUC) from 0 to 20 min (two-sided paired t test: t = 2.652, n = 10 pairs). (F) Left: Slice recordings from LC neurons with light-evoked uncaging. Scale bar, 150 μm. Right: Representative traces of CNV-Y-DAMGO uncaging (purple arrows, 50 ms; 365-nm LED, 84 mW) in current (top) and voltage clamp (bottom). (G to J) Response to CNV-Y-DAMGO uncaging (n = 16 cells). (G) Change in membrane potential. (H) Latency to spike after uncaging. (I) Tonic firing rate in the 10 s before and after uncaging (P < 0.0001, two-sided Wilcoxon matched-pairs signed rank test). (J) Outward current amplitude. (K) f-I curves before and after DAMGO bath application (n = 7 cells; two-sided Wilcoxon matched-pairs signed rank test at each current level). (L) Quantification of neuronal ablation by AAV-DIO-Casp3 (scale bars, 500 μm; n = 11 Dbh-cre, n = 15 control; two-sided Mann-Whitney test). (M) Left: Hot plate withdrawal latencies after morphine. Right: Response to 10 hind paw pin pricks (n = 11 Dbh-cre, n = 15 control; two-way repeated-measures ANOVA with Sidak’s multiple comparisons test; hot plate: P = 0.0006, F1,24 = 15.67; pin: P = 0.0006, F1,24 = 15.52. (N) Representative image of zsGreen expression in the LC (scale bar, 150 μm) and quantification of viral coverage (n = 12 Dbh-cre, n = 13 control; two-sided Mann-Whitney test). (O) Same as (M) for Kir2.1 silencing (n = 12 Dbh-cre, n = 13 control; two-way repeated-measures ANOVA with Sidak’s multiple comparisons test, hot plate: P = 0.001, F1,23 = 14.30; pin: P = 0.0004, F1,23 = 17.20). Data reported as means ± SEM.