Figure 3.

Increased GABA^CeA neuronal excitability is required for pain and anxiety-like behaviors. (A) Schematic of Chemogenetic viruses injected into the CeA of C57 mice. (B,C) Images (B) and statistics data (C) showing that GABA immunofluorescence were colocalized with mCherry-labeled neurons within the CeA (n = 6 slices from three mice). Scale bar, 20 μm. (D,E) Sample traces (D) and statistical data (E) of GABA^CeA neurons in C57 mice with CeA infusion of AAV-VGAT1-hM4Di-mCherry or AAV-VGAT1–mCherry before and after CNO intraperitoneal injection. (mCherry, n = 3 cells from three mice; hM4Di-mCherry, n = 4 cells from three mice; F_{(1, 5)} = 15.59, p = 0.0109). (F) Schematic diagram of the chemogenetic experimental procedure. (G) Chemogenetic inhibition of GABA^CeA neurons reversed CFA-induced mechanical hyperalgesia in the hM4Di-mCherry mice. (n = 7 mice per group; F_{(1, 12)} = 38.29, p < 0.0001). (H,J) Heatmaps showing the locomotion traces of CFA-mCherry-saline mice, CFA-mCherry-CNO mice and CFA-hM4Di-CNO mice in the open field test (OFT) and elevated plus maze test (EPMT). (I,K) Chemogenetic silencing of GABA^CeA alleviated CFA-induced anxiety-like behaviors in CFA-hM4Di-CNO mice in the OFT and EPMT (I, CFA-mCherry-Saline, n = 8 mice; CFA-mCherry-CNO, n = 10 mice; CFA-hM4Di-CNO, n = 11 mice in the OFT; entries into the center in the OFT, F_{(2, 26)} = 7.889, p = 0.0021; time in the center in the OFT, F_{(2, 26)} = 5.303, p = 0.0117; total distances in the OFT, F_{(2, 26)} = 2.169, p = 0.1346; K, CFA-mCherry-Saline, n = 9 mice; CFA-mCherry-CNO, n = 8 mice; CFA-hM4Di-CNO, n = 9 mice in EPMT; entries into the open arms in the EPMT, F_{(2, 23)} = 13.36, p = 0.0001; time in the open arms in the EPMT, F_{(2, 23)} = 16.77, p < 0.0001). All data are shown as the mean ± SEM, n.s., not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001. Two-way repeated-measures ANOVA with Bonferroni post-hoc analysis for (E,G); one-way ANOVA with Bonferroni post-hoc analysis for (I,K).