Sections from naive, prolonged nondependent, and prolonged dependent animals (Set II, brain tissue collected on day 96) were processed for Ki-67 and DCX IHC to quantify proliferation and immature neurons. Proliferation was robustly decreased by prolonged nondependent and dependent conditions (F2,14 = 63.8, p < 0.001, Fig 5a) compared with controls. Notably, alcohol-induced reduction in proliferation in Set II animals were nearly identical to those in Set I animals (Fig. 4c vs. 5a) despite 28 days of no further access to alcohol self-administration and continued exposure to air control (prolonged nondependent animals) or alcohol vapors (prolonged dependent animals). Likewise, alcohol-induced reduction in the total number of DCX-IR cells of prolonged nondependent animals of Set II was similar to nondependent animals of Set I compared to naïve controls (Fig. 4d vs. 5b), thus showing no signs of reversal after cessation of alcohol. On the other hand, prolonged dependence showed a greater decrease in total DCX-IR cells compared to nondependent drinking (F2,33 = 27.02, p < 0.01, Fig. 5b), the decrease in total number of DCX-IR cells was due to decreases in
