Long-Evans rats acquire operant self-administration of 20% ethanol without sucrose fading

Abstract

A major obstacle in the development of new medications for the treatment of alcohol use disorders (AUDs) has been the lack of preclinical, oral ethanol consumption paradigms that elicit high consumption. We have previously shown that rats exposed to 20% ethanol intermittently in a two-bottle choice paradigm will consume two times more ethanol than those given continuous access without the use of water deprivation or sucrose fading (5-6 g/kg every 24 h vs 2-3 g/kg every 24 h, respectively). In this study, we have adapted the model to an operant self-administration paradigm. Long-Evans rats were given access to 20% ethanol in overnight sessions on one of two schedules: (1) intermittent (Monday, Wednesday, and Friday) or (2) daily (Monday through Friday). With the progression of the overnight sessions, both groups showed a steady escalation in drinking (3-6 g/kg every 14 h) without the use of a sucrose-fading procedure. Following the acquisition phase, the 20% ethanol groups consumed significantly more ethanol than did animals trained to consume 10% ethanol with a sucrose fade (1.5 vs 0.7 g/kg every 30 min) and reached significantly higher blood ethanol concentrations. In addition, training history (20% ethanol vs 10% ethanol with sucrose fade) had a significant effect on the subsequent self-administration of higher concentrations of ethanol. Administration of the pharmacological stressor yohimbine following extinction caused a significant reinstatement of ethanol-seeking behavior. Both 20% ethanol models show promise and are amenable to the study of maintenance, motivation, and reinstatement. Furthermore, training animals to lever press for ethanol without the use of sucrose fading removes a potential confound from self-administration studies.

Figure 1

Ethanol consumption was significantly higher for animals trained on the intermittent 20% ethanol group than the daily 20% ethanol group during the 12 overnight operant training sessions. The values are expressed as mean ethanol intake g/kg±SEM (two-way ANOVA followed by Newman–Keuls post hoc test). ^*p<0.05, ^**p<0.01, ^***p<0.001, n=15 for the intermittent group and n=13 for the daily group.

Figure 2

Ethanol consumption (g/kg) and blood ethanol concentrations (BECs) were significantly higher for both groups trained with 20% ethanol compared with the group trained to consume 10% ethanol with a sucrose-fading procedure. Both the intermittent 20% ethanol (a) and daily 20% ethanol (b) models yielded significantly higher baseline consumption than did the 10% ethanol group. The values are expressed as mean ethanol intake (g/kg every 30 min)±SEM (two-way ANOVA followed by Newman–Keuls post hoc test). ^*p<0.05, ^**p<0.01, ^***p<0.001 compares ethanol consumption within each day for the 20% ethanol intermittent group and the 10% ethanol group in (a) and the 20% ethanol daily group and the 10% ethanol group in (b). Blood samples were taken immediately following an operant session (one sample per rat collected between sessions 23 and 25) to analyze and calculate blood ethanol concentrations (BECs). The amount of ethanol consumed correlated significantly with the measured BECs (linear regression): 10% ethanol (c): r²=0.65, p< 0.001, n=13; intermittent 20% ethanol (d): r²=0.83, p< 0.0001, n=15; daily 20% ethanol (e): r²=0.52, p<0.01, n=13.

Figure 3

A 20% ethanol challenge in animals trained in the traditional 10% ethanol model with sucrose fading yielded significantly greater ethanol intake (a) and BECs (b). The values are expressed as mean ethanol intake g/kg every 30 min±SEM (repeated measures ANOVA followed by Newman–Keuls post hoc test). ^**p<0.01, ^***p<0.001 compares each of the 20% ethanol days (26–30) with the last 10% ethanol day (25). Blood samples were collected from the lateral tail vein immediately following the final three 30-min FR3 sessions at each concentration (one sample per rat with all samples collected over 3 days, days 23–25 for 10% ethanol and days 28–30 for 20% ethanol) for determination of BECs for 10% ethanol and 20% ethanol, respectively. The BECs following the 20% ethanol challenge were significantly greater than those seen with 10% ethanol (b). The values are expressed as mean blood ethanol concentration, mg% ±SEM (paired t-test), ^*p<0.05. Linear regression analysis revealed that the amount of 20% ethanol consumed correlated significantly with the measured BECs (c): r²=0.70, p<0.001.

Figure 4

Animals trained using the daily-access 20% ethanol model exhibited significantly higher levels of active lever responding (a), ethanol consumption (b), and BECs (c) compared with animals trained to self-administer 10% ethanol with a sucrose-fading procedure when each group was challenged with the same five concentrations of ethanol. The values are expressed as mean active lever presses, ethanol consumption (g/kg), BEC (mg%) ±SEM measured on the third and fourth sessions at each concentration (repeated measures two-way ANOVA followed by Newman–Keuls post hoc test). ^*p<0.05, ^**p<0.01, ^***p<0.001, n=13 for each group.

Figure 5

Following a period of extinction, yohimbine significantly reinstated ethanol seeking in animals from all three groups. Extinction levels are from the last three extinction sessions before the reinstatement test. Rats were pretreated with yohimbine (2 mg/kg, i.p.) or its vehicle 30 min before the operant session. Vehicle tests were performed 1 week preceding the yohimbine tests. The extinction, vehicle, and yohimbine values are expressed as the average number of active lever presses±SEM (two-way ANOVA followed by Newman–Keuls post hoc test). ^**p<0.01, ^***p<0.001 compares the yohimbine challenge for each group with their corresponding vehicle response, n=12 for the 10% ethanol group, n=7 for the intermittent 20% ethanol group, and n=13 for the daily 20% ethanol group.