Fig. 9: Chemogenetic inhibition of PL neurons projecting to the dBNST, BLA, or NAcc blocks the anti-depressive effects of RS5.

a Experimental design. AAV8-hSyn-hM4D(Gi)-mCherry injection into the PL and a retrograde Cre vector injection into the dBNST, BLA, or NAcc. Mice were then subjected to CRST, followed by RS5 treatment with Veh or CNO injection. b–f mCherry expression by the injected vector in the PL (b). Diagram for the PL→dBNST circuit (c) labeled with hM4D(Gi) expression. Immunofluorescence staining of c-Fos (red) and GAD67 (green) in the dBNST (d) of the indicated groups. Quantification of RS5-induced c-Fos expression in the dBNST, PL (e), and PVN (f) after CNO injection (Exp #1) (n = 4 per group). DAPI, blue. g–m Basal CORT levels (g), the % time of social interaction in the SIT (h), the % sucrose-preference in the SPT (i), and immobility time in the TST (j) and FST (k) for the indicated groups (Exp #2). K-means clustering of individuals in the SIT × SPT × [TST × FST] matrix (l) and the % composition of each group in the clusters (m) (n = 8 animals per group). n–s Diagrams for the PL→BLA (n) and PL→NAcc (o) circuits labeled with hM4D(Gi) expression. RS5-induced c-Fos expression in the BLA (p) and NAcc (r) after CNO or Veh injection for the PL→BLA (p) and PL→NAcc (r) groups. Quantification of RS5-induced c-Fos expression levels in BLA, PL, and NAcc (q) and NAcc, PL, and BLA (s) after CNO injection for the indicated groups (Exp #1) (n = 3–4 animals per group). t–x Basal CORT levels (t), the % time of social interaction in the SIT (u), the % sucrose-preference in the SPT (v), and immobility time in the TST (w) and FST (x) for the indicated groups (Exp #2) (n = 10 animals per group). Data were mean ± SEM. *, the difference compared to control; #, the difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (Two-sided Student’s t-test; one-way ANOVA followed by Newman–Keuls post hoc test). n.s. not significant. See Supplementary Data 4 for statistical details.