Alcohol Dependence and Withdrawal in Mice

Alcohol dependence and withdrawal were induced using a liquid diet in 3 cycles of 5 days on 6.7% v/v alcohol, 2 days forced abstinence, as previously described (Figure 1; Bornebusch et al., 2021 (link)). After acclimation, mice were switched to liquid diets over 2 days (liquid diet with 3% alcohol + 3 g chow/mouse; then liquid diet with 4.5% alcohol + 1.5 g chow/mouse). Liquid diets with and without alcohol were isocaloric and were based on commercial rodent diets: the control diet consisted of 150.2 calories from fat, 659.9 from carbohydrates, and 190.2 from protein (per liter diet); the alcohol diet consisted of 150.5 calories from fat, 303.9 from carbohydrates, 189.9 from protein, and 356.3 from alcohol (per liter diet). After the third alcohol exposure cycle, alcohol was withheld for 3 weeks, and withdrawal symptoms were assessed using several assays described below. Finally, mice were re-exposed to alcohol for 5 days, then euthanized and blood and brains were collected as described below. For feasibility and internal replication, mice were tested in two consecutive cohorts balanced for strain and treatments (i.e., randomized block design). Mice were randomly assigned to one of two groups in each strain, control, and alcohol-exposed (n = 12 per group/strain). The experimenter was blinded to experimental group for all behavioral tests.