(A) Time course of mPFC-BNST chemogenetic strategy in C57BL/6J mice before EtOH and TMT. (B) Images of AAV-hM4Di-mCherry expression in mPFC cell bodies, left, and BNST terminals, right (H2O mCherry n = 7, H2O hM4Di-mCherry n = 7. EtOH mCherry n = 8, EtOH hM4Di-mCherry n = 7). mCherry was enhanced with a GFP immunostain. Slice physiology validation of the DREADD strategy, in mPFC neurons, as represented by (C), hyperpolarization of resting membrane potential after CNO bath application. Inset scale bar indicates 2 mV height and 30 s time. (D) Latency to action potential threshold before and after CNO with 100 pA current ramp steps. Scale bar indicates 20 pA height and 100 ms time. (E) EtOH drinking (g/kg/24 hr) across 6 weeks with mPFC-BNST hM4Di (red) or mCherry (light red). EtOH intake (g/kg) across (F) 1 hr, (G) 4 hr, and (H) 24 hr after i.p. saline (circles) or 3 mg/kg CNO (diamonds). (I) Sample TMT heatmaps of EtOH mPFC-BNST mCherry (left) and EtOH mPFC-BNST hM4Di (right) mice. In the TMT test, (J) distance traveled (cm), (K) TMT contact, (L), far corners (sec), and (M), burying (sec). In the elevated plus maze, duration in the (N), open arms (sec), and (O), closed arms (sec). *p<0.05.