Genotypic and sex differences in anxiety-like behavior and alcohol-induced anxiolysis in High Drinking in the Dark selected mice

Abstract

Alcohol use disorders and anxiety disorders are highly comorbid in humans. In rodent lines selected for alcohol drinking, differences in anxiety-like behavior are also seen. The High Drinking in the Dark (HDID) lines of mice are selectively bred for drinking to intoxication during limited access to alcohol, and these mice represent a genetic model of risk for binge-like drinking. The present studies investigated whether these selected lines differ from control (HS) mice in basal anxiety behavior or in anxiolytic response to alcohol. We also assessed the genetic correlation between alcohol drinking in the dark (DID) and basal anxiety-like behavior using existing inbred strain data. Mice of both sexes and HDID replicates (HDID-1 and HDID-2) were tested on an elevated zero maze immediately following a DID test. In general, HDID mice showed more time spent in the open arms after drinking alcohol than HS mice, and open-arm time was significantly correlated with blood alcohol concentration. HDID-1 male mice also showed less anxiety-like behavior at baseline (water-drinking controls). In a separate experiment, HDID-1 and HS mice were tested for anxiolytic dose-response to acute alcohol injections. Both genotypes showed increasing time spent in the open arms with increasing alcohol doses, and HDID-1 and female mice had greater open-arm time across all doses. HDID-1 control males showed lower anxiety-like behavior than the HS control males. Inbred strain data analysis also showed no significant genetic relationship between alcohol DID and anxiety. These findings suggest that HDID selection has not produced systematic changes in anxiety-like behavior or sensitivity to alcohol-induced anxiolysis, though there is a tendency in the male mice of the first replicate toward reduced basal anxiety-like behavior. Therefore, anxiety state and sensitivity to alcohol's anxiolytic effects do not appear to contribute significantly to the high drinking behavior of the HDID mice.

Introduction

Alcohol use disorders (AUDs) and anxiety disorders have been shown to have a high degree of comorbidity (for review, see Kushner et al., 2000, Smith and Randall, 2012). Two broad hypotheses that are not mutually exclusive might account for this. Alcohol has significant anxiolytic effects (e.g., Gilman, Ramchandani, Davis, Bjork, & Hommer, 2008), and there is evidence that higher basal anxiety may promote greater alcohol intake which can lead to abuse (Bolton, Cox, Clara, & Sareen, 2006). Another possibility is that anxiety disorders and AUDs may share some of the same underlying genetic risk factors. Family and twin studies have suggested possible common transmission of both anxiety disorders and AUDs, which may represent shared genetic risk (e.g., Merikangas et al., 1994, Tambs et al., 1997).

There is also evidence from the animal literature to suggest a relationship between anxiety and alcohol consumption. Rats classified as anxious by performance on an elevated plus maze (EPM) voluntarily drink more alcohol in a subsequent test than those classified as non-anxious (Spanagel et al., 1995). Similarly, some rodent lines selected for high vs. low alcohol preference also show innate differences in basal anxiety and sensitivity to alcohol-induced anxiolysis (Colombo et al., 1995, Stewart et al., 1993). These anxiety-related behaviors appear to be correlated responses to selection for high alcohol intake in these lines. However, this relationship is not seen for all rodent lines selected for high vs. low drinking, with some lines showing an opposite relationship or no relationship with drinking (Can et al., 2012, Sandbak et al., 1998). Another way of examining this relationship is by testing animals selected for anxiety-like behavior for alcohol drinking. Rats selectively bred for high (HAB) or low (LAB) anxiety-related behaviors on an EPM show differences in alcohol preference in a 2-bottle choice test (Henniger, Spanagel, Wigger, Landgraf, & Hölter, 2002). LAB rats have a greater alcohol preference than the HAB rats, but the anxiolytic effect of an injection of alcohol is greater in the HAB rats. Consequently, the possible genetic relationship between alcohol intake and anxiety appears to be complex for both alcohol- and anxiety-related selection phenotypes.

With the exception of the study by Can and colleagues using mice, most of the previous work involves rat lines, and all previous studies have used animals either selected for, or tested on, 2-bottle choice alcohol preference drinking. In the present experiments, we sought to extend these findings to another model animal species and a test of binge-like drinking by determining the relationship between anxiety-like behavior and alcohol drinking in mice selectively bred for drinking to intoxication. The HDID lines of mice were selected for high blood alcohol (ethanol) concentration (BEC) after drinking in the dark (DID), and routinely drink to intoxicating blood levels in a limited-access test (Crabbe et al., 2009, Crabbe et al., 2014). These mice have been extensively behaviorally phenotyped to determine correlated responses to selection and possible factors promoting their high drinking (for review, see Barkley-Levenson & Crabbe, 2014). Here, we tested whether drinking during the DID test is sufficient to produce alcohol-induced anxiolysis, and whether differences in anxiolytic response to alcohol or basal anxiety may underlie the high drinking phenotype of HDID mice. We also used existing inbred mouse strain data sets to assess the genetic relationship between anxiety-like behavior and alcohol DID.