To confirm the role of the vSub in mediating the MSDB cholinergic activation to induce depression-like behaviors and exclude the possibility of an indirect role of a non-cholinergic MSDB projection, we infused CNO into the vSub of ChATcre-hM3Dq mice through a cannula, and these mice also showed depression-like behaviors as tested by the SPT and TST (Fig. 5E–G) but had no effect on locomotor activity in the OFT (Fig. S5B). The gene discussed is BLOC1S4; the disease is depressive symptom measurement.