Fig. 5
Unilateral inhibition of CRFCeA-BNST terminals during alcohol withdrawal. a Timeline of the experiment. b Representative images of the areas of injection (CeA) and of optogenetic inhibition (BNST). Scale bar = 200 μm. c Black circles: effect of optogenetic inactivation of CRFCeA-BNST terminals in animals injected with AAV-DIO-eNpHR 3.0-eYFP. There was a significant time × laser interaction (F2,22 = 5.31, p < 0.05, two-way ANOVA) with a reduction of alcohol intake when the green laser was turned ON (p < 0.01, vs. laser OFF, Newman Keuls post hoc test). Animals escalated their alcohol intake compared with baseline pre-CIE (BSL, F6,66 = 11.43, p < 0.0001, one-way ANOVA) and exhibited escalation only on laser OFF days (p < 0.001, Newman Keuls post hoc test) and not after the inhibition of CRFCeA-BNST terminals. ##p < 0.01, vs. baseline (BSL); ***p < 0.001, vs. laser OFF. White circles: laser activation had no effect in AAV-DIO-eYFP-injected rats. Control rats exhibited the escalation of intake (F2,14 = 7.39, p < 0.01, one-way ANOVA) on both laser ON and OFF days (both p < 0.01, vs. baseline, Newman Keuls post hoc test). ##p < 0.01, vs. BSL. d CRFCeA-BNST terminal inhibition significantly decreased abnormal gait (Mann–Whitney U = 6.00, p < 0.05), body tremors (U = 10.50, p < 0.05), and overall withdrawal severity (U = 1.5, p < 0.01) (inset). *p < 0.05, **p < 0.01, vs. laser OFF. e Optogenetic inhibition of CRFCeA-BNST terminals did not affect saccharin self-administration (t11 = 0.12, p > 0.05, paired t-test). f Exposure to the green light decreased action potential firing. g Inhibition of CRFCeA-BNST terminals significantly reduced firing frequency (t8 = 4.321, p < 0.01, paired t-test) in the BNST. h (Top) Timeline of the current-clamp recording experiments in the BNST. (Bottom) Representative traces for the experimental conditions. i R121919 significantly reduced the firing rate of BNST neurons (F2,16 = 11.63, p < 0.001, one-way ANOVA) and occluded the effect of the optogenetic inhibition of CRFCeA-BNST terminals (**p < 0.001, vs. BSL, Newman Keuls post hoc test). The data are expressed as mean ± SEM
