Sex-Specific Regulation of Depression, Anxiety-Like Behaviors and Alcohol Drinking in Mice Lacking ENT1

Abstract

OBJECTIVES: Adenosine signaling has been implicated in the pathophysiology of several psychiatric disorders including alcoholism, depression, and anxiety. Adenosine levels are controlled in part by transport across the cell membrane by equilibrative nucleoside transporters (ENTs). Recent evidence showed that a polymorphism in the gene encoding ENT1 is associated with comorbid depression and alcoholism in women. We have previously shown that deletion of ENT1 reduces ethanol intoxication and elevates alcohol intake in mice. Interestingly, ENT1 null mice display decreased anxiety-like behavior compared to wild-type littermates. However, our behavioral studies were performed only in male mice. Here, we extend our research to include female mice, and test the effect of ENT1 knockout on other behavioral correlates of alcohol drinking, including depressive and compulsive behavior, in mice. METHODS: To assess depression-like behavior, we used a forced swim test modified for mice. We examined anxiety-like behavior and locomotor activity in open field chambers, and perseverant behavior using the marble-burying test. Finally, we investigated alcohol consumption and preference in female mice using a two-bottle choice paradigm. RESULTS: ENT1 null mice of both sexes showed reduced immobility time in the forced swim test and increased time in the center of the open field compared to wild-type littermates. ENT1 null mice of both sexes showed similar locomotor activity levels and habituation to the open field chambers. Female ENT1 null mice displayed increased marble-burying compared to female wild-types, but no genotype difference was evident in males. Female ENT1 null mice showed increased ethanol consumption and preference compared to female wild-types. CONCLUSIONS: Our findings suggest that ENT1 contributes to several important behaviors involved in psychiatric disorders. Inhibition of ENT1 may be beneficial in treating depression and anxiety, while enhancement of ENT1 function may reduce compulsive behavior and drinking, particularly in females.

Figure 1

Antidepressant-like phenotype of ENT1 null mice. Female (left) and male (right) ENT1 null mice (KO; n = 12/sex) displayed a significant decrease in immobility time in the forced swim test compared to wild-type (WT) littermates of the same sex (n=12/sex). Bars represent mean ± SEM. *P < 0.05 by two-tailed t-test.

Figure 2

Anxiolytic effect of ENT1 deletion in mice. ENT1 null mice of both sexes (n=12–15/sex) spent significantly more time in the center of the open field in comparison to their wild-type littermates (n=12-15/sex) (a). No differences in basal locomotor activity were observed between genotypes (b). Significant decreases in both measures between days indicated that the mice habituated normally to the open field chambers. Bars represent mean ± SEM

Figure 3

Sex difference in ENT1 regulation of perseverant behavior in mice. Female ENT1 null mice (n=12) buried more marbles than female wild-types (n=12), whereas no difference in marble-burying was evident in males (n=12/genotype). Bars represent mean ± SEM. *P < 0.05 by Tukey post-hoc test.

Figure 4

Two-bottle choice ethanol intake in female ENT1 null vs. wild-type mice. Female ENT1 null mice (n=9) showed increases in both ethanol consumption (a) and ethanol preference (b), compared to female wild-types (n=10). Volumetric analysis indicated that ENT1 null mice drank significantly more ethanol than wild-types (c), but that water consumption was similar between genotypes (d). Body weight was significantly lower in ENT1 null vs. wild-type mice (e). Bars represent mean ± SEM. *P < 0.05 by two-tailed t-test.