Role of CB2 cannabinoid receptors in the rewarding, reinforcing, and physical effects of nicotine

Abstract

This study was aimed to evaluate the involvement of CB2 cannabinoid receptors (CB2r) in the rewarding, reinforcing and motivational effects of nicotine. Conditioned place preference (CPP) and intravenous self-administration experiments were carried out in knockout mice lacking CB2r (CB2KO) and wild-type (WT) littermates treated with the CB2r antagonist AM630 (1 and 3 mg/kg). Gene expression analyses of tyrosine hydroxylase (TH) and α3- and α4-nicotinic acetylcholine receptor subunits (nAChRs) in the ventral tegmental area (VTA) and immunohistochemical studies to elucidate whether CB2r colocalized with α3- and α4-nAChRs in the nucleus accumbens and VTA were performed. Mecamylamine-precipitated withdrawal syndrome after chronic nicotine exposure was evaluated in CB2KO mice and WT mice treated with AM630 (1 and 3 mg/kg). CB2KO mice did not show nicotine-induced place conditioning and self-administered significantly less nicotine. In addition, AM630 was able to block (3 mg/kg) nicotine-induced CPP and reduce (1 and 3 mg/kg) nicotine self-administration. Under baseline conditions, TH, α3-nAChR, and α4-nAChR mRNA levels in the VTA of CB2KO mice were significantly lower compared with WT mice. Confocal microscopy images revealed that CB2r colocalized with α3- and α4-nAChRs. Somatic signs of nicotine withdrawal (rearings, groomings, scratches, teeth chattering, and body tremors) increased significantly in WT but were absent in CB2KO mice. Interestingly, the administration of AM630 blocked the nicotine withdrawal syndrome and failed to alter basal behavior in saline-treated WT mice. These results suggest that CB2r play a relevant role in the rewarding, reinforcing, and motivational effects of nicotine. Pharmacological manipulation of this receptor deserves further consideration as a potential new valuable target for the treatment of nicotine dependence.

Figure 1

Nicotine-induced CPP in CB2KO and WT mice and effects of AM630. (a and b) The bars represent the time spent in seconds in the drug-paired compartment during the preconditioning test and the postconditioning test. *P<0.01, **P>0.01, significant difference in the time spent in the drug-paired compartment in preconditioning vs postconditioning tests.

Figure 2

Acquisition of nicotine self-administration and motivation for nicotine in CB2KO and WT mice and effects of AM630. (a) Mean number of active and inactive nose pokes during FR1 schedule of reinforcement to obtain nicotine at the dose of 0.03 mg/kg/infusion i.v. in 1-h daily sessions during 10 days; (b) mean breaking point in a session of progressive ratio that was conducted once and lasted 3 h for WT and CB2KO mice; (c) mean number of active and inactive nose pokes in WT mice during FR1 schedule of reinforcement to obtain nicotine at the dose of 0.03 mg/kg/infusion i.v. in 1-h daily sessions during 7 days; (d) after acquisition, the administration of AM630 (1 and 3 mg/kg, i.p.) decreased the number of responses in the active hole when tested on day 8 and day 12, respectively; and (e) mean breaking point in a session of progressive ratio that was conducted on day 15 and lasted 3 h for mice administered with vehicle or AM630 (3 mg/kg, i.p.). The number of mice was 12 for the WT group and 18 for the KO group in panels a and b. The total number of subjects was 21 in panel c. The number of mice was 7 for the vehicle group and 14 for the AM630 group in panels d and e. Data are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 comparison between groups (the Student–Newman–Keuls test or one-way ANOVA).

Figure 3

Real-time PCR studies of TH and α3-nAChRs/α4-nAChRs in the VTA of WT mice. Evaluation of TH and α3- and α4-nAChR gene expression in the VTA under baseline conditions. Columns represent the means and vertical lines ±SEM of 2^−(ΔΔCt) of relative TH (a), α3-nAChR (b), and α4-nAChR (c) gene expression in WT and CB2KO mice. *Represents values from CB2KO mice that are significantly different (Student's t-test, P<0.05) from WT mice.

Figure 4

Immunolabeling for CB2r and α3-nAChRs/α4-nAChRs in the NAcc and VTA of WT mice. Confocal photomicrographs showing immunolabeling for CB2r (green cells in panels aA, bA, cA, and dA) and either for α3-nAChRs (red cells in panels aB and bB) or for α4-nAChRs (red cells in panels cB and dB) in NAcc and VTA of WT mice. In NAcc, double labeling (yellow cells in panels aC and cC) indicates that α3- and α4-nAChRs colocalize with CB2r immunoreactive cells. In VTA, scarce α3- and α4-nAChR immunoreactive cells are not labeled for CB2r (arrow in panel bB and arrowhead in panel dB). Arrows in panel dA point two CB2r-labeled cells that are immunonegative for α4-nAChRs.

Figure 5

Somatic signs of mecamylamine-precipitated nicotine withdrawal in WT and CB2KO or WT mice treated with AM630. Columns represent the means and vertical lines ±SEM of number of rearings (a, f), groomings (b, g), scratches (c, h), teeth chattering (d, i), and body tremors (e, h). *Values from WT nicotine-treated mice that are significantly different from WT saline-treated mice (P<0.05).^&Values from CB2KO nicotine-treated mice that are significantly different from WT nicotine-treated mice. ^#Values from WT nicotine- and vehicle-treated mice that are significantly different from WT saline- and vehicle-treated mice (P<0.05).^ΦValues from WT nicotine- and AM630-treated mice that are significantly different from WT nicotine- and vehicle-treated mice (P<0.05).