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. 2023 Jan 12;15:1006125. doi: 10.3389/fnmol.2022.1006125

Figure 7.

Figure 7

Genetic manipulations of ASIC1a in the CeA influence inflammatory pain hypersensitivity and anxiety-like behaviors. (A) Schematic of the genetic knockdown experiment in C57 mice. (B) Schematic illustration of the viral vectors for AAV-NC-Ctrl-GFP and AAV-ASIC1a-shRNA-GFP. (C) Representative images and quantification of western blots showing that AAV-ASIC1a-shRNA injection reduced ASIC1a expression in the CeA (NC-Ctrl, n = 6 mice; shRNA, n = 5 mice; t9 = 4.408, p = 0.0017). (D,E) Representative images of AAV-ASIC1a-shRNA-GFP expression in the CeA of C57 mice. Scale bar, 200 μm; GFP-labeled neurons within the CeA were colocalized with GABA immunofluorescence. Scale bar, 20 μm. (F) Genetic knockdown of ASIC1a in the CeA produced analgesic effects (CFA-NC, n = 14 mice; CFA-shRNA, n = 16 mice; F(1, 28) = 40.26, p < 0.0001). (G,J) Heatmaps showing the locations of CFA-NC mice and CFA-shRNA mice in the open field test (OFT) and elevated plus maze test (EPMT). (H,I,K) Genetic knockdown of ASIC1a in the CeA significantly attenuated anxiety-like behaviors (H, CFA-NC, n = 10 mice; CFA-shRNA, n = 13 mice in OFT; entries in center in OFT, t21 = 2.187, p = 0.0402; time in center in OFT, t21 = 3.226, p = 0.004; I, total distances in OFT, t21 = 0.3884, p = 0.7017; K, CFA-NC, n = 14 mice; CFA-shRNA, n = 15 mice in EPMT; entries in the open arms in EPMT, t27 = 2.661, p = 0.013; time in the open arms in EPMT, t27 = 2.392, p = 0.024). (L,M) Sample traces (L) and statistical data (M) for action potential firing recorded from GABACeA in CFA 1D mice treated with AAV-NC-Ctrl or AAV-ASIC1a-shRNA (n = 17 cells from four mice per group; F(1,32) = 17.86, p = 0.0002). (N) Summary of the rheobases of action potentials from CFA 1D mice treated with AAV-NC-Ctrl or AAV-ASIC1a-shRNA (n = 17 cells from four mice per group; t32 = 5.069, p < 0.0001). (O) The resting membrane potential (Vrest) showing no difference between CFA-NC mice and CFA-shRNA mice (n = 17 cells from four mice per group; t32 = 1.511, p = 0.1406). 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 (F,M); unpaired t-test for (C,H,I,K,N,O).