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. 2023 Apr 17;14:2198. doi: 10.1038/s41467-023-37878-y

Fig. 8. Semaphorin-4A is essential for the depressive-like behaviors induced by the activation of the BLA–ACC pathway.

Fig. 8

a Graphical representation of the experimental design for shRNA validation in vitro. HEK-293 cells were transfected with plasmids expressing the mouse Sema4a exon 15 (green), and 1 out of 3 shRNAs tested for knockdown efficiency (red; with 1 scrambled shRNA as control). b Representative fluorescence images showing that the pAAV-mCherry-mU6-Sema4A-sh791 plasmid was the most efficient. Scale bar =50 µm. c Graphical representation of the bilateral virus injection in the ACC for in vivo validation of the selected sh791, inserted into an AAV vector. d qPCR analysis showing a downregulation of Sema4a expression level in the ACC of mice injected with Sema4A-sh791 (Uscramble: n = 5 mice; 1.22 ± 0.37; sh-Sema4A: n = 5 mice; 0.39 ± 0.07; P = 0.0079). e Graphical representation of the experimental design, including bilateral virus delivery in the BLA (AAV5-CaMKIIa-ChR2(H134R)-EYFP) and the ACC (rAAV-mCherry-scrambleUsh or rAAV-mCherry-Sema4A-sh791), cannula implantation, optogenetic stimulation and behavioral testing. f Representative images of mCherry+ cells in the ACC after the injection of UScramble (upper panel) or sh-Sema4A (lower panel). Scale bars =100 µm. g–i Effect of Sema4a knockdown on optogenetically induced emotional deficits (UScramble-Ctrl: n = 18 mice; UScramble-Stim: n = 15 mice; sh-Sema4A-Ctrl: n = 18 mice; sh-Sema4A-Stim: n = 11 mice). Grooming time in the ST was decreased by repeated activation of the BLA–ACC pathway (g: F(1,58) = 4.623; P = 0.036) but was not affected by knocking down Sema4a in the ACC (g: F(1,58)=1.694; P = 0.20). Knocking down Sema4a in the ACC counteracted the increased immobility time observed in stimulated animals in the FST (h: F(1,58)=8.032; P = 0.006; post hoc: Uscramble-Ctrl<Uscramble-Stim; P < 0.05; sh-Sema4A-Stim<Uscramble-Stim; P = 0.05). Knocking down Sema4a in the ACC normalized the emotionality z-score (i: F(1,58)=4.39; P = 0.041; post hoc: Uscramble-Ctrl>Uscramble-Stim; p < 0.01; UScramble-Stim<sh-Sema4A-Ctrl; P < 0.01 UScramble-Stim<sh-Sema4A-Stim; P < 0.05; sh-Sema4A-Ctrl=sh-Sema4A-Stim; p > 0.05). Data are mean ± SEM. *P < 0.05; **P < 0.01; one-tailed Mann–Whitney test (Sema4A quantification), two-way ANOVA (Stimulation ×  KnockDown; ST, FST and z-score). Sagittal mouse brain cartoons (c, e) were created with Biorender.com. Source data are provided as a Source Data file.