Figure 1. Graphical representation of the adolescent intermittent ethanol (AIE) exposure paradigm.

(A) Rats were treated from postnatal day (P) 25 to P55 with either a single daily dose of ethanol (AIE; 5.0 g/kg 20% ethanol w/v, i.g.) or a comparable volume of water (CON) on a two-day on/two-day off administration schedule. Blood ethanol concentrations (BEC) were measured one hr after ethanol exposure on P38 and P54. Twenty-four hours (P56; CON = 8, AIE = 8), 25 days (P80; CON = 8, AIE = 8), and 165 days (P220; CON = 7, AIE = 7) following the conclusion of AIE, rats were sacrificed for immunohistochemistry and Western blot analysis (P80). A subset of CON- and AIE-treated animals was treated with lipopolysaccharide (LPS; 1.0 mg kg, i.p., CON = 8, AIE = 8) or saline (SAL; CON = 8, AIE = 8) on P70 and sacrificed on P80. An additional subset of CON- and AIE-treated animals was exposed to voluntary wheel running from P24 to P80 and sacrificed on P80 (No Exercise: CON = 8, AIE = 8; Exercise: CON = 9, AIE = 10). Body weights were assessed at the initiation of AIE (P25), the conclusion of AIE (P55), and the conclusion of each experiment, depending on the endpoint. Across experiments, we observed that all subjects evidence dramatic weight gains (main effect of Age: Exercise study, F_[2,62] = 5152.9, p < 0.01; LPS study, F_[2,54] = 4203.5, p < 0.01; Aging study: P56, F_[1,14] = 3926.4, p < 0.01; P80, F_[2,28] = 4434.8, p < 0.01; P220, F_[2,24] = 1553.9, p < 0.01). Further, there were no differences in body weights across treatments and conditions (Exercise study: main effect of Treatment - F_[1,31] = 0.9, p > 0.05; main effect of exercise - F_[1,31] = 1.1, p > 0.05; LPS study: main effect of Treatment - F_[1,28] = 1.8, p > 0.05; main effect of LPS - F_[1,28] = 0.01, p > 0.05; Aging study: P56 - main effect of Treatment: F_[1,14] = 1.4, p > 0.05; P80 - main effect of Treatment: F_[1,14] = 0.03, p > 0.05) with the exception of the P220 study in which we observed that AIE-treated animals weighed approximately 10% less than CON subjects at P220 (CON = 622 ± 18 g, AIE = 553 ± 17 g [one-way ANOVA: F_[1,12] = 7.9, p < 0.05]). (B) Representative photomicrographs based on the atlas of Paxinos and Watson (1998) defining the regions of interest assessed for serotonin terminal field immunoreactivity. PrL: prelimbic cortex; HTH: hypothalamus; AMY: amygdala. (C) Representative photomicrograph depicting the brain regions dissected for Western blot analysis. The frontal cortex and midbrain were dissected and used for Western blot analysis of serotonergic protein expression. (all p’s < 0.05) that did not differ as a function of treatment during AIE exposure (repeated measures ANOVAs: all p’s > 0.9; see Figure 1A). However, AIE-treated animals weighed approximately 10% less than CON subjects at P220 (CON = 622 ± 18 g, AIE = 553 ± 17 g [one-way ANOVA: p < 0.05]).