Sex differences in neuronal activation during aversion-resistant alcohol consumption

Abstract

Background: One of the DSM-5 criteria for Alcohol Use Disorder is continued alcohol consumption despite negative consequences. This has been modeled in mice using adulteration of alcohol solution with the bitter tastant quinine. Mice that continue to consume alcohol despite this adulteration are considered aversion resistant. The limited number of studies dissecting the underlying neuronal mechanisms of aversion-resistant drinking behaviors used only male subjects. We have previously shown that female mice are more resistant to quinine adulteration of alcohol than males. Our aim here is to identify potential sex differences in neuronal activation that may underlie this behavior.

Methods: Male and female C57BL/6J mice were allowed continuous access to 20% alcohol in a two-bottle choice procedure. To test aversion-resistance, the alcohol was adulterated with increasing concentrations (0.03, 0.1, and 0.2 mM) of quinine hydrochloride. After consumption rates were calculated, brains were extracted to examine neuronal activation using Fos immunohistochemistry.

Results: We found that female mice suppressed their intake to a lesser extent than males when the alcohol solution was adulterated with quinine. Our Fos staining revealed three regions of interest that exhibit a sex difference during quinine-adulterated alcohol drinking: the ventromedial prefrontal cortex (vmPFC), the posterior insular cortex (PIC), and the ventral tegmental area (VTA). Both the vmPFC and the PIC exhibited higher neuronal activation in males during quinine-adulterated alcohol consumption. However, females showed higher Fos activation in the VTA during quinine-adulterated alcohol consumption.

Conclusions: Females more readily exhibit aversion-resistant alcohol intake than their male counterparts and exhibit some differences in neuronal activation patterns. We conclude that there are sex differences in neurocircuitry that may underlie compulsive drinking behaviors.

Keywords: EtOH; Fos activation; aversion; consumption; sex differences.

Figure 1.

Sex differences in baseline and compulsive drinking behaviors. (A) Timeline of compulsive drinking study. (B) Comparison of the average of the last three days of baseline alcohol intake indicates that females drink significantly more than males. (C) Alcohol intake pattern over the ten days of baseline drinking and the average consumption rates of quinine-adulterated alcohol. (D) Effect of sex on quinine-adulterated alcohol intake. Intake expressed as percent change from baseline (last 3 days of 20% alcohol consumption prior to quinine exposure). Bonferroni post hoc analysis revealed a sex difference at 0.1mM quinine concentration. *p<0.05, ****p<0.0001

Figure 2.

Fos positive cell counts per field of males and females consuming either 20% alcohol or 0.1mM quinine-adulterated 20% alcohol (Q-Alc). (A) vmPFC exhibited highest neuronal activation in males that consumed Q-Alc. (B) Representative image of Fos immunohistochemistry from the vmPFC. (C) PIC had highest activation in males that consumed Q-Alc (D) Representative image of Fos immunohistochemistry from the PIC. (E) VTA had highest neuronal activation in females that consumed Q-Alc. (F) Representative image of Fos immunohistochemistry from the VTA. *p<0.05, **p<0.01

Figure 3.

Pearson correlation analysis assessing possible interconnections between brain regions identified and the vmPFC, PIC, and VTA. (A) Male analysis in the vmPFC identified correlations in the LHb, AIC, and dmPFC. No significant correlation in the PIC or VTA. (B) Female analysis in VTA exhibited a negative correlation in the RMTg, no correlation in the vmPFC, and positive correlations in the PIC to the NAcShell, pPVT, and CeA. The color legend at the bottom of the correlogram shows positive correlations represented by blue color and negative correlations with red. The size of the circle in the correlogram is proportional to the correlation coefficient, with larger circles representing a greater correlation between the two brain regions. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

Fos positive cell counts in males and females consuming 0.1mM quinine-adulterated water (A) Quinine preference at concentrations of 0.03mM and 0.1mM quinine-adulterated water did not reveal a sex difference. (B) Quantification and representative images of Fos immunohistochemistry in the vmPFC, which did not exhibit any significant sex differences. (C) Quantification and representative images of Fos immunohistochemistry in the PIC, which did not exhibit any significant sex differences. (D) Quantification and representative images of Fos immunohistochemistry in the VTA, which did not exhibit any significant sex differences.