cAMP response element-binding protein is required for stress but not cocaine-induced reinstatement

Abstract

Reinstatement of previously extinguished conditioned place preference (CPP) is precipitated by stress or drug exposure. Here, we show that acute exposure to forced swim stress (FS), in a context distinct from conditioning, induces reinstatement of cocaine CPP in wild-type mice. This behavior is accompanied by a pattern of phosphorylated cAMP response element-binding protein (pCREB) activation in discrete brain regions that is distinct from the pattern observed after cocaine-induced reinstatement. For example, previous cocaine conditioning increases pCREB levels in the amygdala, and acute exposure to FS, but not to cocaine, further augments these changes. In contrast, previous cocaine conditioning does not alter levels of pCREB in the nucleus accumbens, but acute exposure to FS increases pCREB levels in this region on reinstatement day. Furthermore, to determine whether these alterations of CREB are necessary in FS or cocaine-induced reinstatement, we examined the effect of these stimuli on reinstatement behavior in mice deficient in alpha and Delta isoforms of CREB. The CREB(alphaDelta) mutant mice show deficits in FS-induced reinstatement of conditioned place preference. In contrast, they show robust cocaine-induced reinstatement. This deficit in stress but not drug-induced reinstatement indicates a specific requirement for CREB in stress-induced behavioral responses to drugs of abuse.

Figure 1.

Experimental paradigm for reinstatement. Preconditioning day (PRE): animals are tested for initial bias. Conditioning phase (CONDITIONING): distinct sides of conditioning boxes are paired with either saline (S) or cocaine (C) (10 mg/kg, i.p.) injections on days 2-9, with one exposure to box per 24 hr. Test is given on day 10 to examine preference. Extinction phase (EXTINCTION): saline injections are given before conditioning on both sides to extinguish preference. Test is given on day 19 to confirm that CPP is extinguished. Arrow represents exposure to either stress or cocaine prime. Stress or consists of a 6 min forced swim test exposure, followed 20 min later by exposure to the conditioning boxes. Cocaine prime is a cocaine injection (10 mg/kg, i.p.) given before testing.

Figure 2.

Acute exposure to the forced swim test (FST) precipitates reinstatement of extinguished place preference for cocaine (10 mg/kg) in wild-type mice. Wild-type mice paired with cocaine (COC/NS, black bar; COC/FST, horizontal stripe) show a significant place preference to the cocaine-paired side on test day. With repeated pairings of saline in both sides, mice previously paired with cocaine do not show place preference on extinction day. When mice are then exposed to a session of acute FST, neither the non-stress groups (SAL/NS, white bars; COC/NS, black bars) nor the saline-paired stressed group (SAL/FST, diagonal stripe) shows significant place preference; however, the mice previously paired with cocaine (COC/FST, horizontal stripe) reveal robust reinstatement of preference after an acute exposure to FST. Data are expressed as mean ± SEM; five to six mice per group. ^*p < 0.05 from corresponding saline group (ANOVA; Bonferroni-Dunn post hoc test).

Figure 3.

Increases in pCREB after FST-induced reinstatement in wild-type mice. Western immunoblots of pCREB change after FST-induced reinstatement. Wild-type mice were paired with saline (SAL) or cocaine (COC). Of those, some were exposed to swim stress on RI day (SAL/FST or COC/FST); the rest were not stressed (SAL/NS or COC/NS). A, Acute exposure to forced swimming significantly increases pCREB expression in the NAc after both saline (SAL/FST) and cocaine (COC/FST) conditioning when compared with the corresponding non-stress groups (SAL/NS or COC/NS). B, Cocaine conditioning increases pCREB expression in the amygdala (COC/NS) when compared with all other groups. Acute exposure to forced swimming (COC/FST) further increases pCREB levels when compared with the NS group. C, There were no changes in pCREB expression after forced swimming or cocaine conditioning in the VTA or the BNST (D). Data are expressed as mean ± SEM; four to six mice per group. ^*p < 0.05 from SAL/NS group; ^**p < 0.05 from COC/NS group (ANOVA; Bonferroni-Dunn post hoc test).

Figure 4.

CREB mutant mice do not show FST-induced reinstatement of place preference to cocaine (10 mg/kg). Both wild-type and mutant mice paired with cocaine (WT/COC/NS, WT/COC/FST, MUT/COC/NS, MUT/COC/FST) show a significant place preference to the cocaine-paired side on test day. With repeated pairings of saline in both sides, mice previously paired with cocaine do not show place preference on extinction day. Although wild-type mice reveal reinstatement of preference after a single exposure to FST (WT/COC/FST), the CREB mutant mice donot show reinstatement (MUT/COC/FST). Data are expressed as mean ± SEM; 10-12 mice per group. ^*p < 0.05 from corresponding saline group (ANOVA; Bonferroni-Dunn post hoc test).

Figure 5.

Both wild-type and CREB mutant mice show cocaine prime-induced reinstatement of extinguished place preference to cocaine (10 mg/kg). Both wild-type and mutant mice paired with cocaine (WT/COC/SAL, WT/COC/COC, MUT/COC/SAL, MUT/COC/COC) show a significant place preference to the cocaine-paired side on test day. With repeated pairings of saline in both sides, all mice previously paired with cocaine do not show place preference on extinction day. Both wild-type and CREB mutant mice show cocaine prime-induced reinstatement of extinguished place preference. Data are expressed as mean ± SEM; five to six mice per group. ^*p < 0.05 from corresponding saline group (ANOVA; Bonferroni-Dunn post hoc test).

Figure 6.

Increases in pCREB after cocaine-induced reinstatement in wild-type mice. Western immunoblots of pCREB changes after cocaine prime-induced reinstatement. Wild-type mice were paired with saline (SAL) or cocaine (COC). One group in each condition was exposed to cocaine prime injection before reinstatement testing (SAL/COC or COC/COC); the other group was injected with saline (SAL/SAL or COC/SAL). A, Priming injection of cocaine prime does not increase pCREB expression in the NAc. B, Cocaine conditioning (COC/COC and COC/SAL) significantly increases pCREB expression when compared with saline conditioning (SAL/COC and SAL/SAL) in the amygdala; however, there is no effect of cocaine prime injection on pCREB levels in the amygdala. C, Injection of the cocaine prime in cocaine-conditioned animals significantly increases pCREB expression in the VTA when compared with corresponding saline group (COC/COC vs SAL/COC). D, There are no significant differences in pCREB expression in the BNST across any of the groups. Data are expressed as mean ± SEM; four to six mice per group. ^*p < 0.05 from corresponding saline-conditioned group (ANOVA; Bonferroni-Dunn post hoc test).