Abstinence following alcohol drinking produces depression-like behavior and reduced hippocampal neurogenesis in mice

Abstract

Alcoholism and depression show high degrees of comorbidity. Clinical evidence also indicates that depression that emerges during abstinence from chronic alcohol use has a greater negative impact on relapse than pre-existing depression. Although no single neurobiological mechanism can account for the behavioral pathologies associated with these devastating disorders, converging evidence suggests that aspects of both alcoholism and depression are linked to reductions in hippocampal neurogenesis. Here, we report results from a novel preclinical behavioral model showing that abstinence from voluntary alcohol drinking leads to the emergence of depression-like behavior and reductions in neurogenesis. C57BL/6J mice were allowed to self-administer ethanol (10% v/v) vs H(2)O in the home cage for 28 days. Alcohol was then removed for 1 or 14 days, and mice were tested in the forced swim test to measure depression-like behavior. After 14 days, but not 1 day of abstinence from alcohol drinking, mice showed a significant increase in depression-like behavior. The significant increase in depression-like behavior during abstinence was associated with a reduction in proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) immunoreactivity in the dentate gyrus of the hippocampus indicating that both the number of proliferating neural progenitor cells (NPC) and immature neurons were reduced, respectively. The number of NPCs that were labeled with bromo-deoxyuridine (BrdU) at the beginning of alcohol exposure was not altered indicating that survival of NPCs is not linked to abstinence-induced depression. Chronic treatment (14 days) with the antidepressant desipramine during abstinence prevented both the emergence of depression-like behavior and the reduction in hippocampal neurogenesis indicating that abstinence-induced depression is associated with structural plasticity in the hippocampus. Overall, the results of this study support the conclusion that profound functional (i.e. behavioral) and structural changes occur during abstinence from alcohol use and suggest that antidepressant treatment may alleviate some of these pathological neurobehavioral adaptations.

Figure 1

Timeline of experimental procedure. Each stage of the experiment is labeled as a separate section in the arrow. The duration of each stage (number of days) is shown under the arrow. (a) After 7 days of adaptation to the lab, mice received daily injections of BrdU (300 mg/kg, i.p.) for 3 days. The following day, the two-bottle drinking procedure began. Mice voluntarily self-administered alcohol vs water in the home cage for 28 days. On the 28th day, alcohol bottles were removed from the cage. One day later or 14 days later, locomotor activity and forced swim tests were performed. The following day, mice were killed, and brains were removed. (b) After 7 days of adaptation to the lab, mice began 28 days of a two-bottle drinking procedure. On the 28th day, alcohol bottles were removed and mice received their first injection of either vehicle or desipramine (15 mg/kg, i.p.). Injections were given once daily for 14 days. One day after the last injection, locomotor activity and forced swim tests were performed. One day later, mice were killed, and brains were removed.

Figure 2

Abstinence from alcohol drinking increases depression-like behavior. Mean immobility (sec) in the forced swim test (FST) increased as a function of days of abstinence from alcohol drinking as compared with water-drinking controls. Data are plotted as mean ± SEM from n = 12 mice in each condition. Please note that the y axis origin is 50 s. *Significantly different from water-drinking control, p<0.05 (Dunnett's t-test).

Figure 3

No changes in survival of NPCs. (a). The mean number of BrdU-labeled cells that survive is unchanged in the dentate gyrus following voluntary drinking and 1- or 14-day abstinence. (b) Representative images showing BrdU-labeled cells in the dentate gyrus from control, 1 day of abstinence, and 14 days of abstinence. (c) Triple-label immunofluorescence was used to evaluate differentiation of surviving NPCs. A representative dentate gyrus is shown. GFAP-labeled glial cells are shown in blue, NeuN-labeled neuronal nuclei are shown in red, BrdU-labeled cells are shown in green. (d) An orthogonal view of a representative cell showing colocalization of BrdU and NeuN. The number of surviving BrdU-labeled cells that colabeled with the neuronal marker NeuN was unchanged in ethanol-treated brains. Data are plotted as mean ± SEM from n = 9–12 mice in each condition.

Figure 4

Abstinence from alcohol reduces adult hippocampal neurogenesis. PCNA and DCX immunohistochemistry. (a) Relative change in PCNA immunoreactivity (IR) in the dentate gyrus showing a reduction in NPC proliferation as a function of 0, 1, or 14 days of abstinence from alcohol drinking. (b) Relative decrease in DCX IR showing that the number of new neurons in the dentate gyrus is reduced as a function of abstinence from alcohol drinking. (c and d) Representative images of dentate gyrus from control, 0 days, 2 days, and 14 days of abstinence for PCNA IR (c) and DCX IR (d). Arrow heads point to representative PCNA-labeled cells. Mean data were obtained from averaging results from four tissue sections per mouse and plotted as percent change from water-drinking control. *Significantly different from water-drinking control, p<0.05 (Dunnett's t-test).

Figure 5

Desipramine treatment prevents abstinence-induced changes in depression-like behavior and neurogenesis. (a) Abstinence-induced increase in immobility (sec) in the forced swim test (FST) was completely blocked by treatment with desipramine during 14 days of abstinence (n = 12 per group). Please note that the y axis origin is 50 s. *Significantly different from water-drinking mice that received vehicle (desipramine, 0 mg/kg), p = 0.007, and significantly different from ethanol- 15 mg/kg, p = 0.008 (Tukey test). (b) Desipramine treatment (14 days) prevented the abstinence-induced decrease in PCNA immunoreactivity in the dentate gyrus (n = 12 per group). *Significantly different from water-drinking mice that received vehicle (desipramine, 0 mg/kg), p = 0.012, (Tukey test). (c) Abstinence-induced reduction in DCX density was completely blocked by desipramine during 14 days of abstinence (n = 12 per group). *Significantly different from water-drinking mice that received vehicle (desipramine, 0 mg/kg), p = 0.004, and significantly different from ethanol- 15 mg/kg, p = 0.005 (Tukey test). Mean ± SEM data for PCNA and DCX IR were obtained by averaging results from four tissue sections per mouse and plotted as percent change from water- 0 mg/kg controls. (d and e) Representative images of dentate gyrus of PCNA IR (d) and DCX IR (e) from water and alcohol exposed mice that received Desipramine (0 or 15 mg/kg) during the 14-day abstinence period. Arrow heads point to representative PCNA-labeled cells.