Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol

Abstract

Background: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling-type transition from moderate to escalated use, leading to dependence. Intermittent 24-hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats.

Methods: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short-term, limited access "binge" drinking procedure, similar to drinking-in-the-dark (DID). B6 mice were also assessed for ethanol dependence with handling-induced convulsion, a reliable measure of withdrawal severity.

Results: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2-hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h).

Discussion: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.

Fig. 1

Ethanol intake (g/kg) over 24 hours for male C57BL/6J mice given continuous access (CA, gray circles, n = 12) or intermittent access (IA, black circles, n = 15) to alcohol (Panel A). Ethanol concentrations were faded from 3%, 6%, 10% ethanol to be maintained on 20% ethanol for the remainder of the experiment. Mice receiving alcohol intermittently had significantly higher alcohol consumption during 3-week maintenance phase on 20% ethanol. Data are mean intake ± SEM. ** p < 0.01 difference between groups. Ethanol intake (g/kg) over 24 hours for male (M, black circles, n = 12) and female (F, gray triangles, n = 12) C57BL/6J mice with intermittent access to alcohol (Panel B). Ethanol concentrations were faded from 3%, 6%, 10% ethanol to be maintained on 20% ethanol for the remainder of the experiment. Female mice had significantly higher alcohol consumption during 3-week maintenance phase on 20% ethanol. Data are mean intake ± SEM. ** p < 0.01 difference between groups.

Fig. 2

Preference ratios were calculated for male C57BL/6J mice by dividing the amount of ethanol solution consumed by the total volume of fluid consumed over 24 hours on alcohol-drinking days. Mice given intermittent access (IA, black circles, n = 15) had a significantly higher preference for 20% ethanol than mice given continuous access (CA, gray circles, n = 12). Data are mean ethanol preference ± SEM. ** p < 0.01 difference between groups.

Fig. 3

Handling-induced convulsion (HIC) ratings of withdrawal severity (Goldstein, 1972) were measured every two hours after ethanol withdrawal for male C57BL/6J mice given intermittent access for 16 weeks (black, n = 9; Panel A), mice given intermittent access for four weeks (dark gray, n = 6; Panel B), and mice given continuous access for four weeks (light gray, n = 6; Panel C). In Panels A, B, and C, median HIC scores are shown with interquartile ranges for each 2-hour measurement after ethanol was removed. ** p < 0.01 difference between groups. Panel D compares the area-under-the-curve (AUC) of HIC scores over time between 16-week intermittent access (IA 16, black), 4-week intermittent access (IA 4, dark gray), and 4-week continuous access (CA 4, light gray) groups.

Fig. 4

Panel A compares 2-hour 20% ethanol intake (g/kg) for male C57BL/6J mice in the limited access “binge” drinking procedure (DID, white, n = 12), continuous access (CA, light gray, n = 11), and intermittent access (IA, black, n = 15) procedures. IA intake (g/kg) was significantly higher than both DID and CA intake. Data are mean ± SEM. * p < 0.05, ** p < 0.01 difference between groups. Panel B shows the correlation between ethanol intake (g/kg/2h) and blood ethanol concentrations (BEC) (mg/dl) after 2-hour access period for a subset of individuals in the IA (black circles, n = 10) and CA (light gray, n = 8) schedules of ethanol presentation.