Fig. 5

Facial pre-injection of BoNT/A inhibits the overexcitation of CaMKII-positive neurons in the vlPAG of CRS mice. a Representative images of the c-Fos expression in the ipsilateral vlPAG to WIM injection side among subgroups of naïve and CRS-induced depression mice 3 weeks post-saline or BoNT/A (10 U/kg) pre-injection. Bottom, magnified view of images indicated in the white rectangle of upper images. Scale bar, 500 μm. b Quantificational analysis of c-Fos numbers among subgroups of naïve mice, saline and BoNT/A pre-injected CRS mice. n = 3 mice per subgroup, One-way ANOVA test and Dunnett’s multiple comparisons test comparing each subgroup with the subgroup of CRS mice pre-treated with saline, F _{(2, 16)} = 4.967, P = 0.0210. The P-value of Dunnett’s multiple comparisons: Naïve + Saline vs. CRS + Saline: P = 0.0274, CRS + Saline vs. CRS + BoNT/A: P = 0.0321. c c-Fos co-labelled with CaMKII-positive neurons. Bottom left, magnified view of the image indicated in white rectangle. Scale bar, 200 μm; 50 μm (left bottom). d Proportion of c-Fos and CaMKII double-labelled neurons in c-Fos-labelled neurons. n = 3 mice per group, Dunnett’s multiple comparisons test comparing each subgroup with the subgroup of CRS mice who were pre-treated with saline, F _{(2, 16)} = 15.39, P = 0.0002. The P-value of Dunnett’s multiple comparisons: Naïve + Saline vs. CRS + Saline: P = 0.0002, CRS + Saline vs. CRS + BoNT/A: P = 0.0013. e Representative images of c-Fos co-labelled with GAD67. Scale bar, 500 μm. f Proportion of c-Fos and Gad67 double-labelled neurons in c-Fos-labelled neurons. n = 3 mice per group, Dunnett’s multiple comparisons test comparing each subgroup with the subgroup of CRS mice pre-treated with saline, F _{(2, 10)} = 0.7974, P = 0.4772. The P-value of Dunnett’s multiple comparisons: Naïve + Saline vs. CRS + Saline: P = 0.9102, CRS + Saline vs. CRS + BoNT/A: P = 0.5721. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., non-significance. Note: Naïve subgroup: naïve mice treated with saline