Dissecting the role of CB1 and CB2 receptors in cannabinoid reward versus aversion using transgenic CB1- and CB2-knockout mice

Abstract

Cannabinoids produce both rewarding and aversive effects in humans and experimental animals. However, the mechanisms underlying these conflicting findings are unclear. Here we examined the potential involvement of CB₁ and CB₂ receptors in cannabinoid action using transgenic CB₁-knockout (CB₁-KO) and CB₂-knockout (CB₂-KO) mice. We found that Δ⁹-tetrahydrocannabinol (Δ⁹-THC) induced conditioned place preference at a low dose (1 mg/kg) in WT mice that was attenuated by deletion of the CB₁ receptor. At 5 mg/kg, no subjective effects of Δ⁹-THC were detected in WT mice, but CB₁-KO mice exhibited a trend towards place aversion and CB₂-KO mice developed significant place preferences. This data suggests that activation of the CB₁ receptor is rewarding, while CB₂R activation is aversive. We then examined the nucleus accumbens (NAc) dopamine (DA) response to Δ⁹-THC using in vivo microdialysis. Unexpectedly, Δ⁹-THC produced a dose-dependent decrease in extracellular DA in WT mice, that was potentiated in CB₁-KO mice. However, in CB₂-KO mice Δ⁹-THC produced a dose-dependent increase in extracellular DA, suggesting that activation of the CB₂R inhibits DA release in the NAc. In contrast, Δ⁹-THC, when administered systemically or locally into the NAc, failed to alter extracellular DA in rats. Lastly, we examined the locomotor response to Δ⁹-THC. Both CB₁ and CB₂ receptor mechanisms were shown to underlie Δ⁹-THC-induced hypolocomotion. These findings indicate that Δ⁹-THC's variable subjective effects reflect differential activation of cannabinoid receptors. Specifically, the opposing actions of CB₁ and CB₂ receptors regulate cannabis reward and aversion, with CB₂-mediated effects predominant in mice.

Keywords: Aversion; CB(1) receptor; CB(2) receptor; Dopamine; Reward; Δ(9)-Tetrahydrocannabinol (Δ(9)-THC).

Figure 1.

Δ⁹-THC place conditioning in wild type, CB₁^−/− and CB₂^−/−. (A) Experimental timeline of events for each day of CPP conditioning and testing. (B) WT mice displayed a significant increase in CPP score at 1 mg/kg (p<0.05). but not at 5 mg/kg (p>0.05). (C) CB₁^−/− mutants displayed a dose-dependent trend toward CPA after Δ⁹-THC administration. (D) Δ⁹-THC produced a robust dose-dependent place preference in CB₂^−/− mice. *p<0.05, ***p< 0.001, relative to preconditioning. #p< 0.05 compared to WT mice.

Figure 2:

Effects of Δ⁹-THC on extracellular DA in the NAc. Δ⁹-THC produced a dose-dependent decrease in extracellular DA in WT (A) and CB₁^−/− mice (B), while CB₂^−/− mice demonstrated a dose-dependent increase in extracellular DA relative to baseline (C). (D) Δ⁹-THC, at 3 mg/kg, reduced extracellular DA in CB₁^−/− mice and elevated DA in CB₂^−/− mice compared to WT controls. (E, F) A representative image and histological verification showing that microdialysis probes are located in the shell and core of the NAc. *p<0.05, **p<0.01, ***p<0.001.

Figure 3:

Effects of Δ⁹-THC and SR141716A on extracellular DA in the NAc in rats. (A) Systemic administration of Δ⁹-THC or vehicle produced a small decrease in extracellular DA, but no difference was found between the vehicle and THC-treatment groups. (B) Systemic administration of SR141716A failed to alter extracellular DA in the NAc. (C) Local perfusion of Δ⁹-THC failed to alter extracellular DA, while SR141716A elevated extracellular DA in a concentration-dependent manner. *p<0.05, ***p<0.001, compared to baseline.

Figure 4:

Genetic deletion of CB₁ and CB₂ receptors differentially alters basal levels of open-field locomotor activity. (A) Time course of locomotion before and after vehicle injection in WT, CB₁^−/− and CB₂^−/− mice. (B) Area under curve (AUC) data during the initial 1-h habituation (before vehicle injection) in open-field locomotion chambers. (C) AUC data during 2-h maintenance (after vehicle) in locomotion chambers in three different genotypes of mice. *p<0.05, ***p<0.001, relative to WT mice.

Figure 5:

Open field locomotion following Δ⁹-THC administration in mice. (A, B, C) Original locomotor activity (A), normalized data (% baseline) (B), and ΔAUC (C) in WT mice, illustrating that Δ⁹-THC dose-dependently decreased locomotor activity (n = 8). (D, E, F) Original locomotor activity (D), normalized data over baseline (% baseline) (E), and ΔAUC (F) in CB₁-KO mice, illustrating that CB₁-KO mice displayed significantly lower basal level of locomotion and lower locomotor response (amplitude) to Δ⁹-THC (D) (n = 8). Unexpectedly, the normalized data (E, F) also revealed dose-dependent locomotor depression after Δ⁹-THC administration in CB₁-KO mice. (G, H, I) Original locomotor activity (G), normalized data over baseline (% baseline) (H), and ΔAUC (I) in CB₂-KO mice, illustrating that Δ⁹-THC had no effect locomotor activity (n = 7). *p<0.05, **p<0.01 relative to vehicle.

Figure 6:

Graphic diagram illustrating how Δ⁹-THC modulates the mesolimbic DA system and cannabinoid action via CB₁ and CB₂ receptors. Δ⁹-THC may produce rewarding effects by binding to CB₁ receptors on GABAergic neurons or afferents in the VTA, thereby reducing GABA-mediated inhibition of VTA DA neurons and increasing DA release in the NAc. Conversely, Δ⁹-THC may produce aversive effects by activating CB₁ receptors on glutamatergic neurons or afferents in the VTA as well as CB₂ receptors on DA neurons, thereby inhibiting VTA DA release to the NAc. The subjective effects of Δ⁹-THC may thus depend on the balance of opposing effects produced by activation of CB₁ and CB₂ receptors in different phenotypes of neurons. VTA: Ventral tegmental area. NAc: Nucleus accumbens, DA: Dopamine.