Cannabinoid action depends on phosphorylation of dopamine- and cAMP-regulated phosphoprotein of 32 kDa at the protein kinase A site in striatal projection neurons

Abstract

Herbal cannabis, smoked in the form of marihuana or hashish, is the most common illicit drug consumed in the Western world. In the brain, cannabinoids interact with neuronal CB1 receptors, thereby producing a marked reduction of motor activity. Here, we report that the motor depressant effect produced by the cannabinoid receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) is attenuated by genetic inactivation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), which is abundantly expressed in the medium spiny neurons of the striatum. Point mutation of Thr34, the protein kinase A (PKA) phosphorylation site of DARPP-32, produces a similar reduction in the effect of the CB1 agonist. In contrast, point mutation of Thr75, a site on DARPP-32 specifically phosphorylated by cyclin-dependent kinase 5, does not affect the behavioral response to CP55,940. Activation of CB1 receptors, either by an agonist or by inhibition of reuptake of endogenous cannabinoids, stimulates phosphorylation at Thr34, thereby converting DARPP-32 into an inhibitor of protein phosphatase-1. Genetic inactivation either of dopamine D2 receptors or of adenosine A2A receptors reduces the phosphorylation of DARPP-32 at Thr34 and the motor depression produced by CP55,940. Our data indicate that a considerable proportion of the psychomotor effect of cannabinoids can be accounted for by a signaling cascade in striatal projection neurons involving PKA-dependent phosphorylation of DARPP-32, achieved via modulation of dopamine D2 and adenosine A2A transmission.

Figure 1.

Activation of CB1 receptors or blockade of endogenous cannabinoid reuptake stimulates DARPP-32 phosphorylation. a, d, Mice were treated intraperitoneally with CP55,940 (a) or AM404 (d) and decapitated 15 min (a) or 30 min(d) after injection. b, e, Mice were treated intraperitoneally with 0.5 mg/kg CP55,940 (b) or with 10 mg/kg AM404 (e) and decapitated at various times (15–120 min) after injection. c, f, Mice were treated intraperitoneally with 5.0 mg/kg SR141716A (specific CB1 inhibitor) 15 min before administration of 0.5 mg/kg CP55,940 (c) or 10 mg/kg AM404 (f) and decapitated 45 min later. The striatal levels of phosphoThr34-DARPP-32 were determined as described in Materials and Methods. The top panels show representative gels; the bottom panels show summaries of each group of experiments. The amount of phosphorylated DARPP-32 is expressed as a percentage of that determined after vehicle administration. Data represent means ± SEM (n = 7–12). *p < 0.01 and **p < 0.001 versus vehicle-treated group; one-way ANOVA followed by Dunnett's test. Treatment with SR141716A significantly reduced the effects of CP55,940 (F_(1,31) = 10.39; p < 0.01) and AM404 (F_(1,33) = 6.62; p < 0.05); two-way ANOVA.

Figure 2.

Thr34 of DARPP-32 is required for the motor depressant effect of CP55,940. Catalepsy was determined 15, 30, 60, 120, and 180 min after administration of CP55,940 (0.5 mg/kg) to wild-type (WT) mice (a–c), DARPP-32 knock-out mice (a), Thr34 mutant (Thr34 → Ala) mice (b), or Thr75 mutant (Thr75 → Ala) mice (c). Data represent means ± SEM (n = 6–10). The effect of CP55,940 was significantly reduced in DARPP-32 knock-out mice (F_(1,29) = 4.40; p < 0.05) and Thr34 → Ala mice (F_(1,24) = 4.42; p < 0.05; repeated-measures ANOVA).

Figure 3.

Dopamine D₂ receptors are required for the biochemical and behavioral effects of CP55,940. a, Wild-type (WT) or dopamine D₂ receptor knock-out (D₂ KO) mice were treated intraperitoneally with vehicle (filled bars) or CP55,940 (0.5 mg/kg, i.p.; open bars) and killed 60 min later. The striatal levels of phosphoThr34-DARPP-32 were determined as described in Materials and Methods. The top panel shows representative gels; the bottom panel shows summaries of experiments. The amount of phosphorylated DARPP-32 is expressed as a percentage of that determined in wild-type mice after vehicle administration. Data represent means ± SEM(n=6–8). The effect of CP55,940 was significantly reduced in D₂ receptor knock-out mice (F_(1,24) = 5.76; p < 0.05); two-way ANOVA. b, Catalepsy was determined 15, 30, 60, 120, and 180 min after administration of CP55,940 (0.5 mg/kg) to wild-type or D₂ knock-out mice. Data represent means ± SEM (n = 6). The effect of CP55,940 was significantly reduced in D₂ knock-out mice (F_(1,20) = 4.44; p < 0.05; repeated-measures ANOVA).

Figure 4.

Adenosine A_2A receptor activation is required for the biochemical and behavioral effects of CP55,940. a, b, Wild-type (WT; CD1 background) or adenosine A_2A receptor knock-out (A_2A KO) mice were treated intraperitoneally with vehicle (filled bars) or CP55,940 (0.5 mg/kg, i.p.; open bars). c, d, C57BL/6 mice were treated intraperitoneally with vehicle, CP55,940, KW6002, or a combination of the two drugs (injected at the same time). Mice were killed 60 min after vehicle or drug administration, and the striatal levels of phosphoThr34-DARPP-32 were determined as described in Materials and Methods. The top panels show representative gels; the bottom panels show summaries of experiments. The amount of phosphorylated DARPP-32 is expressed as a percentage of that determined in wild-type mice after vehicle administration. Data represent means ± SEM (n = 8–11). The effect of CP55,940 was significantly reduced in A_2A receptor knock-out mice (F_(1,36) = 51.45; p < 0.01), and treatment with KW6002 significantly reduced the effect of CP55,940 (F_(1,33) = 5.006; p < 0.05; two-way ANOVA). b, d, Catalepsy was determined 15, 30, 60, 120, and 180 min after administration of CP55,940 (0.5 mg/kg) to wild-type or A_2A knock-out mice (b) or administration of CP55,940, KW6002, or a combination of the two drugs to C57BL/6 mice (d). Data represent means ± SEM (n = 6–15). The effect of CP55,940 was significantly reduced in A_2A knock-out mice (F_(1,26) = 4.60; p < 0.05); treatment with KW6002 significantly reduced the effect of CP55,940 (F_(1,96) = 9.015; p < 0.05; repeated-measures ANOVA).