Figure 3: Chemogenetic Inhibition of CeA^DYN Attenuates Stress-Enhanced Alcohol Drinking.

(A) For targeted expression of an inhibitory DREADD, Pdyn-IRES-Cre mice were bilaterally infused with AAV8-hSyn-DIO-hM4Di-mCherry or AAV8-hSyn-DIO-mCherry into the CeA 2-weeks prior to start of Baseline drinking; vehicle or CNO (3 mg/kg) was injected 30-min prior to the 2^nd and 4^th drinking sessions during Test 3 in a balanced cross-over design (denoted by green arrows). (B) Viral expression was localized to the CeA as indicated by visualization of mCherry fluorescence. (C) Average alcohol intake in mice expressing hM4Di-contaning virus (N= 10-11/group). In vehicle-treated mice, alcohol consumption was greater in CIE-alone and CIE+FSS groups compared to CTL and FSS-alone groups which did not differ (* ps< 0.05). Also, intake following vehicle injection was greater in CIE+FSS mice compared to CIE-alone mice (^ p< 0.005). Activation of the inhibitory DREADD expressed in CeA^DYN neurons via CNO injection resulted in a significant reduction in alcohol intake in the CIE+FSS group compared to those mice receiving vehicle (# p< 0.001). (D) Average alcohol intake in mice treated with control virus (N= 6-7/group). Vehicle injection resulted in the expected greater alcohol intake in CIE-alone and CIE+FSS groups compared to the other groups (* ps< 0.05), and intake was greater in the CIE+FSS mice compared to CIE-alone mice (^ p< 0.005). CNO injection did not alter alcohol drinking in any group relative to when those mice received vehicle injection. (E) Change from respective vehicle alcohol intake across treatment and virus groups. CNO injection significantly reduced alcohol intake (relative to vehicle) in CIE+FSS mice that harbored active vs. control virus (* p< 0.001) and this reduction was greater in CIE+FSS mice compared to when CNO was injected in CIE-alone, FSS-alone, and CTL groups that were treated with active virus (# ps< 0.01). All values are mean ± s.e.m.