Fig. 2: Chemogenetic inhibition of the vSub-to-dBNST circuit blocked repeated mild stress effects.

a–e Experimental design (a). Diagram for the possible circuits projected to the dBNST (b). The retrograde tracer CTB488 was injected into the dBNST (c). Diagram for the location of vSub (d). Photomicrographs showing CTB488 localization in vSub neurons (e), with higher magnification of the box (e1). The details of this tracking experiment are presented in Supplementary Fig. 3. f–k Experimental design (f). Mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the vSub and a retrograde AAV-WGA-Cre vector into the dBNST (g) were subjected to CRST, followed by repeated 5-min restraint (RS5), with CNO or vehicle (Veh) administration. Photomicrographs showing mCherry expression in the dBNST (h), and c-Fos expression in CRF- or AVP-positive cells (i) in the PVN of the indicated groups. Quantification of c-Fos expression in dBNST (j) and vSub (k). l–o Basal serum GC levels (l). Immobility time in the TST (m) and FST (n), as well as the social interaction (% time) with a target versus an empty in the SIT (o), for the indicated groups. (n = 6 animals per group). p Experimental design. Mice received an injection of AAV-hSyn-hM3D(Gq) or AAV-hSyn-hM4D(Gi) into the dBNST, followed by a retrograde Cre vector into PVN. They were then subjected to CRST. q–t Quantification of c-Fos expression in the PVN after CNO injection of the indicated groups (q and s, hM3D(Gq); r and t, hM4D(Gi)). u–x Basal serum GC levels (u), the immobility time in the TST (v) and FST (w) and social interaction (% time) in the SIT (x). Data are mean ± s.e.m. *,**, difference between indicated groups. *P < 0.05, **P < 0.01 (Student’s t-test; one-way ANOVA followed by Newman–Keuls post-hoc test). See Supplementary Data 4 for statistical details.