Endocannabinoid brain mechanisms and addiction

点击翻译按钮获取中文摘要

During the present reporting period, significant progress was made on this research project. Cannabinoid CB1 receptors (CB1Rs) have been shown to be a promising target in medication development for the treatment of addiction. However, clinical trials with SR141716A (rimonabant, a selective CB1R antagonist/inverse agonist) for the treatment of obesity and smoking cessation failed due to unwanted side effects. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models with fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. We therefore studied whether systemic administration of AM4113 (3 and 10 mg/kg) alters heroin, cocaine, or methamphetamine effects. We found that AM4113 dose-dependently inhibited i.v. self-administration of heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10 mg/kg) dose-dependently inhibited the self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A (3 and 10 mg/kg) dose-dependently increased the BSR stimulation threshold (i.e., decreased brain reward), but AM4113 had no effect on BSR at the same doses, suggesting that SR141716A may produce aversive effects while AM4113 may not. Together, these findings show that neutral CB1R antagonists such as AM4113 deserve further research as a new class of CB1R-based medications for the treatment of opioid addiction without SR141716A-like aversive effects. Growing evidence indicates the presence of functional cannabinoid CB2 receptors (CB2Rs) in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at 40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs. Further, we re-examined the roles of both CB1 and CB2 receptors in the effects of cannabis. We used electrical brain-stimulation reward (BSR) to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). Two mixed CB1/CB2 receptor agonists, delta-9-tetrahydrocannabinol (THC) and WIN55,212-2, produced biphasic effects - mild enhancement of BSR at low doses but inhibition at higher doses. The CB1R antagonist AM251 attenuated the low dose-enhanced BSR, while the CB2R receptor AM630 attenuated high dose-inhibited BSR. Rats were also treated with selective CB1R and CB2R agonists, which produced BSR enhancement and inhibition, respectively. We conclude that the subjective effects of cannabis depend on the balance of these opposing effects, and explain why cannabis can be either rewarding or aversive in humans, as expression of CB1Rs and CB2Rs may differ in the brains of different subjects. Further, we studied whether combining CB1R antagonism with CB2R agonism produces additive anti-addiction effects. Delta-8-tetrahydrocannabivarin (delta-8-THCV) is a delta-9-THCV analogue with combined CB1 antagonist/CB2 agonist properties. We tested delta-8-THCV in seven different rodent models relevant to nicotine dependence - nicotine self-administration, cue-triggered nicotine-seeking behavior following forced abstinence, nicotine-triggered reinstatement of nicotine-seeking behavior, acquisition of nicotine-induced conditioned place preference, anxiety-like behavior induced by nicotine withdrawal, somatic withdrawal signs induced by nicotine withdrawal, and hyperalgesia induced by nicotine withdrawal. Delta-8-THCV significantly attenuated intravenous nicotine self-administration and both cue-induced and nicotine-induced relapse to nicotine-seeking behavior in rats. Delta-8-THCV also significantly attenuated nicotine-induced conditioned place preference and nicotine withdrawal in mice. We conclude that delta-8-THCV may have therapeutic potential for the treatment of nicotine dependence. We also suggest that tetrahydrocannabivarins be tested for anti-addiction efficacy in a broader range of preclinical animal models, against other addictive drugs, and eventually in humans. We also studied cannabinoid-induced tetrad effects - analgesia, hypothermia, catalepsy, and suppressed locomotion, which were previously believed to be mediated by the activation of cannabinoid CB1Rs. Given recent findings of CB2 and GPR55 receptors in the brain, we examined whether these receptors are also involved in cannabinoid action. We found that administration of delta-9-THC, WIN55212-2, or XLR11 produced dose-dependent tetrad effects in wild-type (WT) mice. Genetic deletion or pharmacological blockade of CB1Rs abolished tetrad effects produced by all three cannabinoids. Unexpectedly, genetic deletion of CB2Rs abolished analgesia and catalepsy produced by delta-9-THC or WIN55212-2, but not by XLR11. Microinjections of delta-9-THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1-KO mice. CB2-KO mice displayed a reduction in intraventricular delta-9-THC-induced analgesia and catalepsy. In contrast, genetic deletion of GPR55 receptors caused enhanced responses to delta-9-THC or WIN55212-2. Antagonism of CB1Rs, CB2Rs, or GPR55Rs produced alterations similar to those observed in each genotype mouse line. These findings suggest that in addition to CB1Rs, both CB2Rs and GPR55Rs are also involved in pharmacological effects produced by cannabinoids. CB1Rs/CB2Rs, in contrast to GPR55Rs, appear to play opposite roles in cannabinoid actions. Finally, we found that beta-Caryophyllene, a dietary terpenoid, inhibits nicotine taking and nicotine seeking behaviors in rodents.