Role of cannabinoid type 1 receptors in locomotor activity and striatal signaling in response to psychostimulants

Abstract

A single administration of cocaine or D-amphetamine produces acute hyperlocomotion and long-lasting increased sensitivity to subsequent injections. This locomotor sensitization reveals the powerful ability of psychostimulants to induce brain plasticity and may participate in the alterations that underlie addiction. We investigated the role of cannabinoid receptor type 1 (CB1-R) in the effects of a single injection of psychostimulants. The acute locomotor response to cocaine was normal in mice pretreated with the CB1-R inverse agonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), whereas no sensitization was observed in response to a second administration a week later. Locomotor responses to cocaine and D-amphetamine were decreased in CB1-R-deficient mice, and sensitization was impaired. To determine how CB1-R controls long-lasting effects of psychostimulants, we studied cocaine-activated signaling pathways. Cocaine-induced cAMP-dependent phosphorylation of glutamate receptor 1 was altered in the striatum of CB1-R mutant mice but not of AM251-treated mice. In contrast, cocaine-induced phosphorylation of extracellular signal-regulated kinase (ERK) was blocked in both CB1-R mutant and antagonist-pretreated mice. Conditional deletion of CB1-R in forebrain principal neurons or GABAergic neurons prevented cocaine-induced ERK activation in dorsal striatum and nucleus accumbens. Our results provide strong evidence for the role of the endocannabinoid system in regulating neuronal circuits critical for long-lasting effects of cocaine, presumably by acting on CB1-R located on terminals of striatal medium spiny neurons.

Figure 1.

AM251, a CB1-R inverse agonist, does not alter acute locomotor effects but prevents locomotor sensitization induced by a single injection of cocaine. Mice received a cocaine injection [10 (A) or 20 (B, C) mg/kg, i.p.] on days 1 and 7. Fifteen minutes before cocaine administration, mice were pretreated with vehicle or with AM251 [3 (A, B) or 10 (C) mg/kg, i.p.] on day 1 (middle) or day 7 (right). Data for each mouse are shown individually. Arrows indicate the mean locomotor activity for each group (n = 8–11 mice per group). Statistical analysis was done with two-way repeated-measures ANOVA for matching data. Induction of behavioral sensitization: A, interaction between days and AM251 pretreatment (F_(1,18) = 26.38; p < 0.01); effect of days (F_(1,18) = 52.78; p < 0.01); effect of AM251 (F_(1,18) = 8.50; p < 0.01); B, interaction between days and AM251 pretreatment (F_(1,14) = 7.80; p < 0.05); effect of days (F_(1,14) = 36.18; p < 0.01); effect of AM251 (F_(1,14) = 6.64; p < 0.05); C, interaction between days and AM251 pretreatment (F_(1,18) = 10.36; p < 0.01); effect of days (F_(1,18) = 15.54; p < 0.01); effect of AM251 (F_(1,18) = 20.70; p < 0.01). Expression of behavioral sensitization: A, interaction between days and AM251 pretreatment [F_(1,14) = 0.03; not significant (NS)]; effect of days (F_(1,14) = 87.11; p < 0.01); effect of AM251 (F_(1,14) = 2.59; NS); B, interaction between days and AM251 pretreatment (F_(1,14) = 0.2; NS); effect of days (F_(1,14) = 48.07; p < 0.01); effect of AM251 (F_(1,14) = 0.87; NS); C, interaction between days and AM251 pretreatment (F_(1,18) = 0.01; NS); effect of days (F_(1,18) = 46.38; p < 0.01); effect of AM251 (F_(1,18) = 0.00; NS). Post hoc comparisons were done with Bonferroni's posttest; p values for comparisons between the first and second injection are indicated on the figure for each group.

Figure 2.

Acute and sensitized locomotor responses induced by psychostimulant drugs are altered in CB1-R mutant mice. A, B, Locomotor responses to an injection of cocaine (10, 20, and 30 mg/kg, i.p.; A) or d-amph (2 and 4 mg/kg, i.p.; B) in wild-type (WT) and CB1-R-null (KO) mice. Data (means ± SEM; n = 10–11 mice per group) were compared using two-way ANOVA analysis: interaction between genotype and dose of cocaine (F_(3,64) = 5.49; p < 0.01); effect of dose (F_(3,64) = 44.57; p < 0.01); effect of genotype (F_(1,64) = 23.02; p < 0.01); interaction between genotype and dose of d-amphetamine [F_(2,50) = 2.58; not significant (ns)]; effect of dose (F_(2,50) = 67.27; p < 0.01); effect of genotype (F_(1,50) = 9.74; p < 0.01). Bonferroni's posttest: °p < 0.05, °°p < 0.01 compared with wild type. C, Comparison of the locomotor effects of a first injection of cocaine (20 mg/kg) and of a second injection 7 d later. Data for each mouse are shown individually. The position of the mean is indicated by an arrow for each group. Statistical analysis was done with two-way repeated-measures ANOVA: interaction between genotype and days (F_(1,18) = 1.97; NS); effect of days (F_(1,18) = 27.91; p < 0.01); effect of genotype (F_(1,18) = 29.55; p < 0.01). Post hoc comparisons were done with Bonferroni's posttest. Comparison between genotypes: day 1 WT versus KO (p < 0.01); day 7 WT versus KO (p < 0.01). D, Ratios: cocaine-induced locomotor activity at day 7/locomotor activity at day 1. Variances were significantly different (Bartlett test, p < 0.03). E, Same as in C except that d-amphetamine (2 mg/kg) was used instead of cocaine. Two-way repeated-measures ANOVA: interaction between genotype and days (F_(1,18) = 4.15; NS); effect of days (F_(1,18) = 23.20; p < 0.01); effect of genotype (F_(1,18) = 8.16; p < 0.05). Post hoc comparisons were done with Bonferroni's posttest. Comparison between genotypes: day 1 WT versus KO (p < 0.05); day 7 WT versus KO (p < 0.01). F, Ratios as in D. The variances were different (Bartlett test, p < 0.001).

Figure 3.

Effects of cocaine on protein phosphorylation in the dorsal striatum of CB1-R-deficient mice. Wild-type (WT) and CB1-R-null (KO) mice were injected with 20 mg/kg saline (−) or cocaine (+) and killed 10 min later. Their heads were rapidly frozen, and the striata were dissected without thawing. A, Immunoblot analysis of phosphorylation of GluR1 at Ser-845 (P-GluR1; top) and total GluR1 (bottom). Immunoreactive bands were quantified by laser scanning, and results were expressed as ratios of phosphorylated/total protein and normalized to the mean of saline-treated wild-type mice (percentage of control). Data (means ± SEM; n = 7–9 mice per group) were analyzed using two-way ANOVA: interaction between genotype and treatment (F_(1,32) = 20.28; p < 0.01); effect of treatment (F_(1,32) = 12.27; p < 0.01); effect of genotype [F_(1,32) = 2.25; not significant (NS)]. B, Same as in A except that the proteins analyzed were phospho-Thr-34-DARPP-32 (P-Thr34; top) and total DARPP-32 (bottom). Data (means ± SEM; n = 6 mice per group) were analyzed using two-way ANOVA: interaction between genotype and treatment (F_(1,18) = 4.51; p < 0.05); effect of treatment (F_(1,18) = 6.23; p < 0.05); effect of genotype (F_(1,18) = 0.66; NS). C, Same as in A except that the proteins analyzed were P-ERK1/2 (top) and total ERK1 and ERK2 (bottom). Note that P-ERK1 was barely detectable in most experiments. Data (means ± SEM; n = 7–9 mice per group) were analyzed using two-way ANOVA: interaction between genotype and treatment (F_(1,28) = 9.78; p < 0.01); effect of treatment (F_(1,28) = 14.95; p < 0.01); effect of genotype (F_(1,28) = 0.93; NS). Bonferroni's posttest, *p < 0.05, **p < 0.01 compared with saline; °p < 0.05, °°p < 0.01 compared with WT.

Figure 4.

Cocaine-induced ERK phosphorylation and c-Fos induction in the dorsal striatum and nucleus accumbens in CB1-R-null mice. A, P-ERK immunostaining in the shell of the NAcc in wild-type (WT) and CB1-R-null (KO) mice 10 min after the injection of saline or cocaine (20 mg/kg), as indicated. Scale bars, 50 μm. B, Quantification of P-ERK-positive cells in sections of the dorsal striatum and nucleus accumbens shell (Acc. shell) and core (Acc. core). Data (means ± SEM; n = 8–10 per group) were analyzed using two-way ANOVA: dorsal striatum, interaction between treatment and genotype (F_(1,30) = 6.67; p < 0.05); effect of treatment (F_(1,30) = 13.53; p < 0.01); effect of genotype (F_(1,30) = 6.85; p < 0.05). Acc. shell, interaction between treatment and genotype (F_(1,30) = 10.72; p < 0.01); effect of treatment (F_(1,30) = 17.24; p < 0.01); effect of genotype (F_(1,30) = 15.94; p < 0.01). Acc. core, interaction between treatment and genotype (F_(1,30) = 6.30; p < 0.05); effect of treatment (F_(1,30) = 16.45; p < 0.01); effect of genotype (F_(1,30) = 12.53; p < 0.01). C, c-Fos immunostaining in the shell of the NAcc in WT and KO mice 90 min after the injection of saline or cocaine (20 mg/kg), as indicated. Scale bars, 50 μm. D, Quantification of c-Fos-positive cells in sections of the dorsal striatum, Acc. shell, and Acc. core. Data (means ± SEM; n = 4 per group) were analyzed using two-way ANOVA: dorsal striatum, interaction between treatment and genotype (F_(1,12) = 15.39; p < 0.01); effect of treatment (F_(1,12) = 13.04; p < 0.01); effect of genotype (F_(1,12) = 12.74; p < 0.01). Acc. shell, interaction between treatment and genotype (F_(1,12) = 4.4; p = 0.057); effect of treatment [F_(1,12) = 3.92; not significant (NS)]; effect of genotype (F_(1,12) = 7.45; p < 0.05). Acc. core, interaction between treatment and genotype (F_(1,12) = 2.52; NS); effect of treatment (F_(1,12) = 3.5; NS); effect of genotype (F_(1,12) = 7.21; p < 0.05). Bonferroni's posttest, **p < 0.01 compared with saline; °p < 0.05, °°p < 0.01 compared with WT.

Figure 5.

Effects of the CB1-R inverse agonist AM251 on cocaine-induced phosphorylation of ERK and GluR1 in the mouse striatum. Mice were pretreated with vehicle (Veh.) or AM251 (10 mg/kg, i.p.) 15 min before the injection of 20 mg/kg saline (−) or cocaine (+) and killed 10 min later. Their heads were rapidly frozen, and the striata were dissected without thawing. A, Immunoblot analysis of phosphorylation of GluR1 at Ser-845 (P-GluR1; top) and total GluR1 (bottom). Immunoreactive bands were quantified by laser scanning, and results were expressed as ratios of phosphorylated protein to total protein and normalized to the mean of saline-treated wild-type mice (percentage of control). Data (means ± SEM; n = 7–9 mice per group) were analyzed using two-way ANOVA: interaction between cocaine and AM251 treatment [F_(1,33) = 0.62; not significant (NS)]; effect of cocaine (F_(1,33) = 64.02; p < 0.01); effect of AM251 treatment (F_(1,33) = 1.66; NS). B, Same as in A except that the proteins analyzed were P-ERK1/2 (top) and total ERK1 and ERK2 (bottom). Note that P-ERK1 was barely detectable in most experiments. Data (means ± SEM; n = 8–9 mice per group) were analyzed using two-way ANOVA: interaction between cocaine and AM251 treatment (F_(1,35) = 4.13; p < 0.05); effect of cocaine (F_(1,35) = 17.37; p < 0.01); effect of AM251 treatment (F_(1,35) = 3.16; NS). Bonferroni's posttest, **p < 0.01 compared with saline; °p < 0.05 compared with vehicle.

Figure 6.

Effects of CB1-R inverse agonists on cocaine-induced ERK phosphorylation in the dorsal striatum and nucleus accumbens. A, Quantification of P-ERK-positive cells in sections of the dorsal striatum and nucleus accumbens shell (Acc. shell) and core (Acc. core) from mice pretreated with vehicle (Veh.) or AM251 (10 mg/kg, i.p.; 15 min), injected with saline or cocaine and perfused 10 min later. Data (means ± SEM; n = 4 per group) were analyzed using two-way ANOVA: dorsal striatum, interaction between cocaine and AM251 pretreatment (F_(1,12) = 5.74; p < 0.05); effect of cocaine (F_(1,12) = 11.17; p < 0.01); effect of AM251 (F_(1,12) = 5.53; p < 0.05). Acc. shell, interaction between cocaine and AM251 pretreatment (F_(1,12) = 7.04; p < 0.05); effect of cocaine (F_(1,12) = 5.96; p < 0.01); effect of AM251 [F_(1,12) = 3.96; not significant (NS)]. Acc. core, interaction between cocaine and AM251 pretreatment (F_(1,12) = 9.6; p < 0.01); effect of cocaine (F_(1,12) = 15.32; p < 0.01); effect of AM251 (F_(1,12) = 9.6; p < 0.01). B, Same as in A except that rimonabant (3 mg/kg, i.p.) was used instead of AM251. Data (means ± SEM; n = 5–7 per group) were analyzed using two-way ANOVA: dorsal striatum, interaction between cocaine and rimonabant pretreatment (F_(1,22) = 44.25; p < 0.01); effect of cocaine (F_(1,22) = 37.12; p < 0.01); effect of rimonabant (F_(1,22) = 55.01; p < 0.01). Acc. shell, interaction between cocaine and rimonabant pretreatment (F_(1,22) = 8.34; p < 0.01); effect of cocaine (F_(1,22) = 5.82; p < 0.01); effect of rimonabant (F_(1,22) = 0.16; NS). Acc. core, interaction between cocaine and rimonabant pretreatment (F_(1,22) = 8.56; p < 0.01); effect of cocaine (F_(1,22) = 8.56; p < 0.01); effect of rimonabant (F_(1,22) = 1.04; NS). Bonferroni's posttest, *p < 0.05, **p < 0.01 compared with saline; °p < 0.05, °°p < 0.01 compared with vehicle.

Figure 7.

Effects of conditional deletion of CB1-R in different neuronal subpopulations on cocaine-induced ERK phosphorylation in the striatum. A, Micrographs showing CB1-R mRNA expression in the dorsal striatum and cortex of CB1^f/f, CB1^{f/f;CaMKIIα-Cre}, and CB1^{f/f;Dlx5/6-Cre}. Note the absence of CB1-R expression in the dorsal striatum of CB1^{f/f;CaMKIIα-Cre} and CB1^{f/f;Dlx5/6-Cre} mice. B, Micrographs showing double in situ hybridization of CB1-R (red staining) together with GAD65 mRNA (silver grains in the cortex of CB1^f/f, CB1^{f/f;CaMKIIα-Cre}, and CB1^{f/f;Dlx5/6-Cre}. In CB1^f/f, CB1-R is present in both GABAergic interneurons and principal neurons. In CB1^{f/f;CaMKIIα-Cre}, CB1-R is only expressed by some GABAergic interneurons, whereas in CB1^{f/f;Dlx5/6-Cre}, CB1-R is only present in glutamatergic neurons. Blue staining corresponds to toluidine blue nuclear staining. Solid arrows, GABAergic interneurons (GAD65 positive) expressing CB1-R mRNA. Open arrows, GABAergic interneurons lacking CB1-R expression. C, P-ERK immunostaining in the dorsal striatum in wild-type mice (CB1^f/f) and in CB1-R conditional mutant mice (CB1^{f/f;CaMKIIα-Cre} and CB1^{f/f;Dlx5/6-Cre}) 10 min after acute administration of saline or cocaine (20 mg/kg). Scale bar, 100 μm. D, Quantification of P-ERK-positive cells in sections of the dorsal striatum and nucleus accumbens shell (Acc. shell) and core (Acc. core) from wild-type (CB1^f/f) and CB1-R conditional mutant (CB1^{f/f;CaMKIIα-Cre} and CB1^{f/f;Dlx5/6-Cre}) mice 10 min after injection of cocaine (20 mg/kg). Data (means ± SEM; n = 4–8 per group) were analyzed using two-way ANOVA: dorsal striatum, interaction between genotypes and treatment (F_(2,29) = 7.73; p < 0.01); effect of treatment (F_(1,29) = 20.83; p < 0.01); effect of genotypes (F_(2,29) = 7.53; p < 0.01); Acc. shell, interaction between genotypes and treatment [F_(2,29) = 1.13; not significant (NS)]; effect of treatment (F_(1,29) = 5.26; p < 0.05); effect of genotypes (F_(2,29) = 1.56; NS); Acc. core, interaction between genotypes and treatment (F_(2,29) = 3.24; NS); effect of treatment (F_(1,29) = 10.29; p < 0.01); effect of genotype (F_(2,29) = 3.36; p < 0.05). Bonferroni's posttest, *p < 0.05, **p < 0.01 compared with saline; °p < 0.05, °°p < 0.01 compared with wild type.