Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice

Abstract

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Figure 1.

Locomotor activity after acute morphine treatment. A, Basal locomotor activity in WT and βarr2-KO mice during a 1 hr habituation period (for time, F_(11,1022) = 32.89, p < 0.0001; for genotype, F_(1,93) = 8.26, p < 0.005; n = 46-49). B, Effects of an acute dose of morphine. Morphine (10 mg/kg, s.c.) was administered to wild-type (n = 8), heterozygotes (n = 8), and βarr2-KO (n = 8) mice after a 1 hr habituation period. Locomotor activity was assessed as the number of infrared beam breaks in 5 min intervals. The values were averaged across mice, and the means ± SEM are shown here. Morphine-induced locomotor activity in all three groups, yet the genotypes did not differ significantly at this dose (for genotype, F_(23,483) = 27.94, p < 0.0001; for dose, F_(2,21) = 2.62, p = 0.0965). C, Morphine dose-response curve for locomotor activity. The number of beam breaks was summed over the 120 min test period, and the mean ± SEM after morphine or saline (10 μl/g, s.c.) administration is presented. The sum of the beam breaks in B is included in this graph, representing the 10 mg/kg dose. Overall, morphine produced greater increases in locomotor activity in WT mice than in the βarr2-KO mice (for genotype, F_(1,95) = 70.22, p < 0.001; for dose, F_(4,95) = 36.07, p<0.0001; n = 8-16). Bonferroni post hoc analysis revealed significant differences between the genotypes at the doses of 5, 20, and 40 mg/kg subcutaneous morphine (WT vs KO; ^*p < 0.05; ^**p < 0.001).

Figure 2.

Locomotor activity after acute cocaine treatment. A, Cocaine (20 mg/kg, i.p.) was administered to WT (n = 15) and βarr2-KO (n = 12) mice after a 1 hr habituation period. Locomotor activity was assessed as described in Figure 1. Although cocaine produced marked increases in locomotor activity in both genotypes, WT mice displayed slightly higher activity than the βarr2-KO mice after morphine (for time, F_(17,391) = 12.40, p < 0.0001; for genotype, F_(1,23) = 0.96, p = 0.3371). B, Dose-response curve for cocaine-induced locomotor activity. Data represent the sum of the activity after each dose over a 90 min period. There were no significant differences between the genotypes (F_(1,62) = 0.08; p = 0.7723; n = 7-15).

Figure 3.

Locomotor sensitization to morphine. Locomotor activity was initially assessed in WT and βarr2-KO mice during a 30 min habituation period and for 90 min after an acute dose of morphine (10 mg/kg, s.c.). After this observation, mice received one daily dose of morphine (10 mg/kg, s.c.) for 6 additional days in their home cage. After 1 d of rest, the mice were then challenged again with the same dose of morphine (10 mg/kg, s.c.), and locomotor activity was assessed (WT-S, βarr2-KO-S). Locomotor activity was increased significantly after this regimen in both WT and βarr2-KO mice, indicating that sensitization to morphine developed in both genotypes (for sensitization, F_(1,36) = 5.68, p < 0.05; for genotype, F_(1,36) = 3.78, p = 0.06; n = 10 of each genotype.)

Figure 4.

Locomotor sensitization to cocaine. Locomotor activity was initially assessed in WT and βarr2-KO mice during a 30 min habituation period and for 90 min after an acute dose of cocaine (20 mg/kg, i.p.). Mice then received one daily dose of cocaine (20 mg/kg, i.p.) for 4 additional days in their home cage. After 1 d of rest, the mice were then challenged again with the same dose of cocaine (20 mg/kg, i.p.), and locomotor activity was again assessed (WT-S, βarr2-KO-S). Data represent the sum of the number of beam breaks recorded during 60 min after drug administration. Both groups of mice became sensitized to cocaine to a similar extent (for time, F_(1,39) = 7.67, p < 0.01; for genotype, F_(1,39) = 0.00, p = 0.9474; n = 7-14.)

Figure 5.

Invivo microdialysis after acute morphine. The effects of morphine (10 mg/kg, s.c.) and saline (10 μl/g, s.c.) on extracellular DA, DOPAC, and HVA levels in the striatum of freely moving mice as measured using in vivo microdialysis. Data are presented as the percentage of the average level of dopamine or metabolite measured in at least three samples collected before the drug administration. Analysis of area under curve values for 3 hr after administration (data not shown) revealed a significant effect of morphine on extracellular dopamine and metabolites in both genotypes: WT+Sal vs WT+Mor, dopamine, DOPAC, and HVA, p < 0.01; βarr2-KO+Sal vs βarr2-KO+Mor, dopamine, DOPAC, and HVA, p < 0.01; Mann-Whitney U test (two-tailed). Analysis of the 1 hr period after morphine administration (data not shown) revealed that βarr2-KO mice produced a greater accumulation of dopamine and metabolites: WT (n = 9) vs βarr2-KO (n = 6) after morphine, DA, p < 0.05; DOPAC and HVA, p < 0.01; Mann-Whitney U test (two-tailed). Similar analysis of saline effects (1 or 3 hr period) revealed no significant differences between the genotypes (WT, n = 5; βarr2-KO, n = 5; p > 0.05; two-tailed Mann-Whitney U test).

Figure 6.

In vivo microdialysis after acute cocaine. The effects of cocaine (20 mg/kg, i.p.) and saline (10 μl/g, i.p.) on extracellular DA levels in the striatum of freely moving mice as measured using in vivo microdialysis. Data are presented as the percentage of the average level of dopamine measured in at least three samples collected before the drug administration. Analysis of area under curve values for 2 hr after cocaine treatment (data not shown) revealed a significant increase in DA in both genotypes compared with the saline treatment of the respective genotype (p < 0.01; two-tailed Mann-Whitney U test). No significant differences were observed between genotypes after saline (WT, n = 5; βarr2-KO, n = 5) or cocaine (WT, n = 5; KO, n = 4; p > 0.05; two-tailed Mann-Whitney U test).

Figure 7.

Morphine-induced conditioned place preference. Mice were assessed for the time spent in either the black or white compartment on the preconditioning day. Drug was alternatively paired with either side for both groups of mice as described in Materials and Methods. After the 6 d conditioning regimen (drug on days 1, 3, and 5; saline on days 2, 4, and 6), the time spent in each compartment was assessed in the absence of drug or saline (postconditioning). A, Data are shown as the difference in the time spent in the drug-paired (morphine, 3 mg/kg, s.c.) compartment on the postconditioning day and the preconditioning day when the data are analyzed at 10, 15, or 20 min preconditioning and postconditioning times (mean ± SEM). The βarr2-KO mice spent more time in the drug-paired side than the WT mice when compared over all times (for genotype, F_(1,11) = 5.53, p < 0.05; for time, F_(2,22) = 15.20, p < 0.0001). B, Dose-response curve at 20 min preconditioning and postconditioning times. Morphine produced a dose-dependent increase in preference for the morphine-paired compartment in both genotypes; however, when comparing genotypes, the effect was significantly greater in the βarr2-KO mice compared with their WT littermates (for genotype, F_(1,41) = 6.31, p < 0.05; for dose, F_(2,41) = 7.05, p < 0.01; n = 9 per group).

Figure 8.

Cocaine-induced conditioned place preference. Conditioning was performed as described in Figure 7A with cocaine (10 mg/kg, i.p.). Data are shown as the difference in the time spent in the drug-paired compartment on the postconditioning day and the preconditioning day when the preconditioning and postconditioning times were set at 10, 15, or 20 min (mean ± SEM). Cocaine (10 mg/kg, i.p.) produced preference for the cocaine-paired compartment in both genotypes that did not differ under any of the conditions (for genotype, F_(1,16) = 0.05, p = 0.8179; for time, F_(2,32) = 5.95, p < 0.01; n = 9-10 per group).