Extended Data Fig. 5. Neuronal and behavioral effects of CRISPR/Cas9-mediated cell-type specific Chrnb2 and/or Chrna7 deletion in S1.

a, Experimental design for selective knockout (KO) of Chrnb2 (β2) or Chrna7 (α7) in S1 VIP INs and expression of hM3D(Gq) in aNB→S1 projections (left), two-photon images of VIP INs expressing GCaMP6s and Cas9-mCherry (middle), and Ca²⁺ activity before and 20 min post-CNO (right). VIP INs increase activity upon activation of aNB→S1 projections, which is abolished with β2 KO. (non-KO/KO cells/mice; n = 53/117/three, 73/96/four, 61/106/four; KO vs. non-KO, P = 0.87, 0.0054, 0.23). b, Experimental design for β2 or α7 KO in VIP (left), images of Cas9-EGFP⁺ VIP and GCaMP6s⁺ PNs (middle), and PN Ca²⁺ activity before and after SNI (n = 208, 253, 253, 189 cells from three mice per group). SNI-induced PN hyperactivation is abolished after VIP β2 KO (D14, wake, F_{(2, 1458)} = 6.91, P = 0.0005; NREM, F_{(2, 1458)} = 6.91, P < 0.0001). c, VIP β2 or α7 KO has no effects on baseline nociceptive thresholds (n = 11, 5, 5 mice; punctate, P = 0.17, 0.40, 0.78). d, VIP α7 KO partially reduces SNI-induced allodynia (n = 6, 5, 5 mice; punctate, P = 0.24, 0.44, 0.017; dynamic, 0.035, 0.031, 0.0087; cold, 0.015, 0.10, 0.067; hot, 0.042, 0.27, 0.20). e,f,g, Experimental design for β2 and/or α7 KO in SST, PV, or PNs in S1 (e), which has no effects on baseline nociceptive thresholds (f; n = 9, 9, 5, 5, 4, 4, 5 mice; punctate, P = 0.99, 0.13, 0.37, 0.75, 0.43, 0.83, 0.25), and SNI-induced allodynia (g; n = 9, 9, 5, 5, 4, 4, 5; SST-α7 KO, punctate, F_{(8, 96)} = 0.09, P = 0.99; dynamic, F_{(3, 36)} = 0.79, P = 0.51; cold, F_{(3, 36)} = 0.02, P = 0.99; hot, F_{(3, 36)} = 0.31, P = 0.81). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant; by two-sided Wilcoxon (a, c, f), Kolmogorov-Smirnov (a), two-way ANOVA followed by Bonferroni’s test (vs. SNI; b, d, g). Detailed statistics in Supplementary Table 1.