Involvement of the AMPA receptor GluR-C subunit in alcohol-seeking behavior and relapse

Abstract

Craving and relapse are core symptoms of drug addiction and alcoholism. It is suggested that, after chronic drug consumption, long-lasting neuroplastic changes within the glutamatergic system are important determinants of addictive behavior. Here, we show that the AMPA type glutamate receptor plays a crucial role in alcohol craving and relapse. We observed, in two animal models of alcohol craving and relapse, that the AMPA antagonist GYKI 52466 [1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2, 3-benzodiazepine] dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior and the alcohol deprivation effect. The involvement of the AMPA receptor in these phenomena was further studied using mice deficient for the GluR-C AMPA subunit [GluR-C knock-out (KO)]. GluR-C KOs displayed a blunted, cue-induced reinstatement response and alcohol deprivation effect, when compared with wild-type controls; however, no differences between genotypes could be observed regarding ethanol self-administration under operant or home cage drinking conditions. These results imply a role for GluR-C in alcohol relapse, although this phenotype could also be attributable to a reduction in the total number of AMPA receptors in specific brain areas. In conclusion, AMPA receptors seem to be involved in the neuroplastic changes underlying alcohol seeking behavior and relapse. Thus, AMPA receptors represent a novel therapeutic target in preventing relapse.

Figure 1.

Effects of the AMPA receptor antagonist GYKI 54266 on cue-induced reinstatement of ethanol-seeking behavior in Wistar rats (n = 8 per group). Data are shown as mean ± SEM of the reinforced lever presses after the presentation of a composed stimulus, paired previously with ethanol (S⁺/CS⁺; A) or water (S⁻/CS⁻; B) availability (for details, see Materials and Methods). (*p < 0.01 compared with the last extinction session; ^#p < 0.01 compared with GYKI 5466 10 mg/kg).

Figure 2.

Effects of the AMPA antagonist GYKI 54266 on ADE in Wistar rats (n = 8 per group). Data are shown as mean ± SEM ethanol consumption (grams per kilogram per day). Vehicle or GYKI 54266 injections are indicated by arrows (for details, see Materials and Methods) (*p < 0.01 compared with corresponding baseline values; ^#p < 0.01 compared with saline control group). BL1, Baseline 1; BL2, baseline 2; BL3, baseline 3.

Figure 3.

Generation of GluR-C knock-out mice. A, Schematic representation of the X-chromosomally localized WT GluR-C (WT), the targeted GluR-C^3lox (3lox), and the GluR-C^1lox (1lox) allele after Cre-mediated recombination. Boxes represent exon 11 and the pgk-neo selection cassette. LoxP elements are indicated by black triangles. Filled circles mark the 5′ and 3′ borders of the targeting construct. B, Diagnostic BglII restriction sites are indicated. The position of the 3′ outside probe for genomic Southern blot is indicated by a black bar. Primers routinely used for mouse PCR genotyping are given as P1–P4. B, Southern blot analysis of BglII-digested ES cell DNA. Lanes represent, from left to right, WT ES cell DNA, positive GluR-C^3lox clone P59K1, a 1:1 mixture of WT and P59K1 DNA, and GluR-C^1lox ES cell clone #284, obtained after transient transfection of clone P59K1 with Cre recombinase. The probe detected fragments with the expected sizes of 4.95 kb (WT), 2.32 kb (3lox), and 2.24 kb (1lox), as indicated on the left. Note: R1 ES cells are of male origin. Because Gria3 is located on the X-chromosome, there is only one copy of the GluR-C gene present. C, Western blot analysis of WT, GluR-C^3lox, and GluR-C^1lox mice. Total brain lysates of 15-d-old male mice were checked for protein expression by a monoclonal antibody directed against the N-terminal portion of GluR-C. The antibody detected a prominent protein band at the expected size of ∼105 kDa. In GluR-C^1lox animals, no full-length or truncated forms of GluR-C could be detected. Size markers are indicated on the left in kilodaltons.

Figure 4.

A, B, Changes in AMPA receptor subunit expression of the AMPA receptor subunits GluR-A, GluR-B, GluR-C, and GluR-D in hippocampus (A) and cerebellum (B) of wild-type (WT) and GluR-C knock-out (GluR-C −/−) mice from P2 to P90. Total proteins of the hippocampus and the cerebellum were isolated, and for each sample, 10 μg of protein was separated on a 7.5% SDS-PAGE and transferred on nitrocellulose. The glutamate receptor subunits were visualized by selective antibodies and monitored by autoradiography. The α-subunit of the Ca²⁺/calmodulin-dependent protein kinase II (α-CaMKII) and βIII-tubulin (βIII-Tub.) was used as a positive control for developmental controlled gene expression in hippocampus and cerebellum, respectively. Loading was controlled by β-actin. The experiment was repeated with at least three sets of mice. One representative example for each subunit is given.

Figure 5.

Operant ethanol self-administration and cue-induced reinstatement in WT and GluR-C knock-out mice (n = 14 per group). A, Mean ± SEM of ethanol (10% v/v)-reinforced lever presses per session during the last 10 self-administration sessions. B, Mean ± SEM of lever presses in the previously ethanol-reinforced lever for each genotype elicited by a CS⁺ (light) after 15 extinction sessions (for details, see Materials and Methods). Open and filled symbols depict data for WT and GluR-C knock-out mice, respectively (*p < 0.05 between genotypes).

Figure 6.

Two-bottle free-choice ethanol self-administration, preference and alcohol deprivation effect in WT (n = 26) and GluR-C knock-out mice (n = 32). A, Ethanol consumption (grams per kilogram per day) per ethanol concentration. B, Ethanol preference observed during the same conditions depicted in A. C depicts ADE in the same group of animals by comparing ethanol self-administration 3 d before and 3 d after a 3 week deprivation period. All data are expressed as mean ± SEM (for details, see Materials and Methods) (*p < 0.01 between genotypes). B1, Baseline 1; B2, baseline 2; B3, baseline 3.