Mifepristone in the central nucleus of the amygdala reduces yohimbine stress-induced reinstatement of ethanol-seeking

Abstract

Chronic ethanol exposure leads to dysregulation of the hypothalamic-pituitary-adrenal axis, leading to changes in glucocorticoid release and function that have been proposed to maintain pathological alcohol consumption and increase vulnerability to relapse during abstinence. The objective of this study was to determine whether mifepristone, a glucocorticoid receptor antagonist, plays a role in ethanol self-administration and reinstatement. Male, Long-Evans rats were trained to self-administer either ethanol or sucrose in daily 30 min operant self-administration sessions using a fixed ratio 3 schedule of reinforcement. Following establishment of stable baseline responding, we examined the effects of mifepristone on maintained responding and yohimbine-induced increases in responding for ethanol and sucrose. Lever responding was extinguished in separate groups of rats and animals were tested for yohimbine-induced reinstatement and corticosterone release. We also investigated the effects of local mifepristone infusions into the central amygdala (CeA) on yohimbine-induced reinstatement of ethanol- and sucrose-seeking. In addition, we infused mifepristone into the basolateral amygdala (BLA) in ethanol-seeking animals as an anatomical control. We show that both systemic and intra-CeA (but not BLA) mifepristone administration suppressed yohimbine-induced reinstatement of ethanol-seeking, while only systemic injections attenuated sucrose-seeking. In contrast, baseline consumption, yohimbine-induced increases in responding, and circulating CORT levels were unaffected. The data indicate that the CeA plays an important role in the effects of mifepristone on yohimbine-induced reinstatement of ethanol-seeking. Mifepristone may be a valuable pharmacotherapeutic strategy for preventing relapse to alcohol use disorders and, as it is FDA approved, may be a candidate for clinical trials in the near future.

Figure 1

Systemic administration of the glucocorticoid receptor (GR) antagonist mifepristone significantly attenuates yohimbine-induced reinstatement of ethanol- and sucrose-seeking, but does not affect sera corticosterone (CORT) levels. Animals were pretreated with mifepristone (0, 5, or 30 mg/kg) to examine its effects on yohimbine- (2 mg/kg, intraperitoneally (i.p.)) induced reinstatement of ethanol- and sucrose-seeking. (a) Yohimbine significantly reinstates active lever responding in ethanol-trained rats, an effect that is attenuated by mifepristone (^#p<0.05 comparing yohimbine reinstatement following mifepristone vehicle pretreatment to reinstatement following 30 mg/kg mifepristone pretreatment). (b) Yohimbine significantly reinstates active lever responding in sucrose-trained rats, an effect that is attenuated by mifepristone (^#p<0.05 comparing yohimbine reinstatement following mifepristone vehicle and 5 mg/kg pretreatment to reinstatement following 30 mg/kg mifepristone pretreatment). (c) Mifepristone pretreatment (30 mg/kg) has no effect on yohimbine-induced increases in CORT. Data are presented as mean±SEM (n=24 for ethanol reinstatement study, n=9 for sucrose reinstatement study, and n=12 for CORT study). Statistical analysis was performed by analysis of variance (ANOVA) with Newman–Keuls post hoc testing.

Figure 2

Intra-central amygdala (CeA), but not basolateral amygdala (BLA), administration of mifepristone significantly attenuates yohimbine-induced reinstatement of ethanol-seeking. Animals were pretreated with mifepristone (0, 5, or 10 μg) into the CeA and BLA to examine its effects on yohimbine- (2 mg/kg, intraperitoneally (i.p.)) induced reinstatement of ethanol-seeking. (a) Yohimbine significantly reinstates active lever responding (∞, p<0.01 comparing baseline vehicle to baseline yohimbine: ^***p<0.001; ^**p<0.01 compared with yohimbine vehicle treatment for animals pretreated with mifepristone vehicle and 5 μg, respectively), an effect that is dose-dependently attenuated by mifepristone (^###p<0.001 and ^##p<0.01 comparing yohimbine reinstatement following mifepristone vehicle and 5 μg pretreatment, respectively, to reinstatement following 10 μg mifepristone pretreatment). (b) Schematic representations adapted from Paxinos and Watson of the injection cannulae placements in coronal sections of the CeA of rats included in the data analysis. (c) Intra-BLA mifepristone has no effect on yohimbine-induced reinstatement of active lever responding (p<0.01 comparing baseline vehicle to baseline yohimbine; ^*p<0.05 comparing phosphate-buffered saline (PBS) vehicle to PBS yohimbine). (d) Schematic representations adapted from Paxinos and Watson of the injection cannulae placements in coronal sections of the BLA of rats included in the data analysis. Data are presented as mean active lever presses±SEM (n=11 for CeA and n=8 for BLA). Statistical analysis was performed by paired t-test for baseline and PBS data and repeated-measures two-way analysis of variance (ANOVA) with Newman–Keuls post hoc testing for animals pretreated with mifepristone.

Figure 3

Intra-central amygdala (CeA) infusions of dimethylsulfoxide (DMSO) or mifepristone do not cause cell death in the amygdala. (a) Representative slide for amygdala slices with cannula and CeA highlighted. Morphological studies were conducted in CeA sections of rats pretreated with six microinfusions of (b) phosphate-buffered saline (PBS), (c) 100% DMSO, and (d) mifepristone dissolved in 100% DMSO using Hoechst 33342 staining 24 h after the last infusion. (e) Quantification of viable cells was performed by the Imaris Neuroscience software pack. Data are presented as mean±SEM, (n=3). Statistical analysis was performed by one-way analysis of variance (ANOVA).

Figure 4

Intra-central amygdala (CeA) administration of mifepristone has no effect on yohimbine-induced reinstatement of sucrose-seeking. Animals were pretreated with mifepristone (0, 5, or 10 μg) into the CeA to examine its effects on yohimbine- (2 mg/kg, intraperitoneally (i.p.)) induced reinstatement of sucrose-seeking. Mifepristone had no effect on yohimbine-induced reinstatement of active lever responding (∞, p<0.01 comparing baseline vehicle to baseline yohimbine). Data are presented as mean active lever presses±SEM (n=10). Statistical analysis was performed by paired t-test for baseline data and repeated-measures two-way analysis of variance (ANOVA) for animals pretreated with mifepristone.

Figure 5

Mifepristone has no effect on baseline operant responding for ethanol or sucrose. Mifepristone (0, 5, 10, and 30 mg/kg) treatment has no effect on (a) active lever pressing and (b) consumption (g/kg per 30 min) in animals trained to respond for 10% ethanol. Similarly, mifepristone treatment has no effect on (c) active lever pressing and (d) consumption (g/kg per 30 min) in animals trained to respond for 5% sucrose. Data are presented as mean±SEM (n=11 for ethanol-trained rats and n=15 for sucrose-trained rats). Statistical analysis was performed by repeated-measures one-way analysis of variance (ANOVA).

Figure 6

Mifepristone does not attenuate yohimbine-induced increases in ethanol and sucrose responding. Yohimbine (2 mg/kg) significantly increases operant responding for and consumption (g/kg per 30 min) of (a, b) ethanol and (c, d) sucrose (^***p<0.001 compared with baseline responding), while mifepristone pretreatment has no effect on yohimbine-induced increases in ethanol and sucrose responding or consumption. Data are presented as mean±SEM (n=10 for ethanol-trained rats and n=11 for sucrose-trained rats). Statistical analysis was performed by repeated-measures one-way analysis of variance (ANOVA).