Role of a genetic polymorphism in the corticotropin-releasing factor receptor 1 gene in alcohol drinking and seeking behaviors of marchigian sardinian alcohol-preferring rats

Abstract

Marchigian Sardinian alcohol-preferring (msP) rats exhibit innate preference for alcohol, are highly sensitive to stress and stress-induced alcohol seeking. Genetic analysis showed that over-expression of the corticotropin-releasing factor (CRF) system of msP rats is correlated with the presence of two single nucleotide polymorphisms (SNPs) occurring in the promoter region (position -1836 and -2097) of the CRF1 receptor (CRF1-R) gene. Here we examined whether these point mutations were associated to the innate alcohol preference, stress-induced drinking, and seeking. We have recently re-derived the msP rats to obtain two distinct lines carrying the wild type (GG) and the point mutations (AA), respectively. The phenotypic characteristics of these two lines were compared with those of unselected Wistar rats. Both AA and GG rats showed similar patterns of voluntary alcohol intake and preference. Similarly, the pharmacological stressor yohimbine (0.0, 0.625, 1.25, and 2.5 mg/kg) elicited increased operant alcohol self-administration under fixed and progressive ratio reinforcement schedules in all three lines. Following extinction, yohimbine (0.0, 0.625, 1.25, and 2.5 mg/kg) significantly reinstated alcohol seeking in the three groups. However, at the highest dose this effect was no longer evident in AA rats. Treatment with the CRF1-R antagonist antalarmin (0, 5, 10, and 20 mg/kg) significantly reduced alcohol-reinforced lever pressing in the AA line (10 and 20 mg/kg) while a weaker or no effect was observed in the Wistar and the GG group, respectively. Finally, antalarmin significantly reduced yohimbine-induced increase in alcohol drinking in all three groups. In conclusion, these specific SNPs in the CRF1-R gene do not seem to play a primary role in the expression of the msP excessive drinking phenotype or stress-induced drinking but may be associated with a decreased threshold for stress-induced alcohol seeking and an increased sensitivity to the effects of pharmacological blockade of CRF1-R on alcohol drinking.

Keywords: CRF; SNP; msP; relapse; self-administration; yohimbine.

Figure 1

Elevated alcohol drinking of the two msP lines GG (n = 8) and AA (n = 8) derived from the original msP line as assessed in the two-bottle free choice drinking paradigm. GG and AA msP rats show minimal changes in (A) drinking patterns and (B) alcohol preference across a period of 15 days. Values are presented as the daily mean g/kg of alcohol intake (±SEM) and percent (%) of alcohol preference (±SEM), respectively. **p < 0.01 and ***p < 0.001, significant difference between the two msP rat lines. GG: gray line; AA: black line. For detailed statistics, see “Results.”

Figure 2

(A) Operant alcohol self-administration in Wistar (n = 10), as well as GG (n = 14), and AA (n = 10) msP rats under a fixed ratio 3 (FR-3) schedule of reinforcement is significantly increased by the systemic (i.p.) administration of the pharmacological stressor yohimbine (0.0, 0.625, 1.25, 2.5 mg/kg) at the dose of 0.625 and 1.25 mg/kg. Values presented are the mean number of rewards earned in 30 min (±SEM). (B) Operant alcohol self-administration in Wistar (n = 10), as well as GG (n = 10), and AA (n = 10) msP rats under a progressive ratio (PR) schedule of reinforcement is significantly increased by the systemic (i.p.) administration of the pharmacological stressor yohimbine at the dose of 0.625 mg/kg. Values presented are the mean of break point measure (last ratio completed by the animal ±SEM). **p < 0.01, ***p < 0.001, significant difference from the collapsed means of vehicle-treated groups (0.0 mg/kg). Wistar: white bars; GG: gray bars; AA: black bars. For detailed statistics, see “Results.”

Figure 3

(A) Dose-response curve of antalarmin (0, 5, 10, 20 mg/kg) when systemically (i.p.) injected in Wistar (n = 7), as well as GG (n = 12), and AA (n = 14) msP rats as assessed on operant alcohol self-administration on a fixed ratio 3 (FR-3) schedule of reinforcement. The AA msP rat line shows increased sensitivity to antalarmin treatment compared to the other rat line examined. Data are the mean (±SEM) number of rewards earned in 30 min. **p < 0.01, difference from the vehicle-treated groups (0 mg/kg). (B) I.p. pre-treatment with antalarmin (10 mg/kg) fully blocks the escalation of alcohol self-administration (FR-3) elicited by systemic (i.p.) treatment with yohimbine at the dose of 0.625 mg/kg in all rat lines examined [Wistar (n = 8), as well as GG (n = 10) and AA (n = 15) msP rats]. Results are the mean (±SEM) number of rewards earned in 30 min. ***p < 0.001, difference from the groups receiving both vehicle-treatments of antalarmin and yohimbine (−/−); ^###p < 0.001, difference from the groups receiving yohimbine 0.625 mg/kg (±). Wistar: white bars; GG: gray bars; AA: black bars. For detailed statistics, see “Results.”

Figure 4

Systemic (i.p.) administration of yohimbine (0.0, 0.625, 1.25, 2.5 mg/kg) elicits reinstatement of alcohol seeking in Wistar (n = 7), as well as GG (n = 8), and AA (n = 9) msP rats following extinction. The AA msP line shows decreased threshold for yohimbine-induced reinstatement due to different sensitivity on responding to the effects of 2.5 mg/kg yohimbine dose. Data are the mean (±SEM) of total number of responses in 30 min. **p < 0.01, ***p < 0.001, difference from the vehicle-treated groups (0.0 mg/kg); ^#p ≤ 0.05, difference from the collapsed means of both the GG msP and the Wistar lines. Wistar: white bars; GG: gray bars; AA: black bars. For detailed statistics, see “Results.”