Endocannabinoid brain mechanisms and addiction

在本报告所述期间，这项研究项目取得了重大进展。大麻素CB1受体（Cannabinoid CB1 receptor, CB1Rs）已被证明是药物开发中治疗成瘾的一个有希望的靶点。然而，SR141716A（利莫那班，一种选择性CB1R拮抗剂/逆激动剂）用于治疗肥胖和戒烟的临床试验由于不良副作用而失败。最近的临床前研究表明，中性CB1R拮抗剂AM4113可能在尼古丁自我给药模型中保留SR141716A的治疗性抗成瘾作用，并且副作用更少。然而，AM4113是否对其他滥用药物，如阿片类药物和精神兴奋剂也有效，以及它是否在实验动物中产生类似SR141716A的抑郁副作用，目前尚不清楚。因此，我们研究了全身给药AM4113（3和10mg /kg）是否会改变海洛因、可卡因或甲基苯丙胺的作用。我们发现，AM4113对海洛因静脉注射自我给药具有剂量依赖性，但对可卡因或甲基苯丙胺没有抑制作用，而SR141716A（3和10 mg/kg）对海洛因和甲基苯丙胺的自我给药具有剂量依赖性，但对可卡因没有抑制作用。在脑电刺激奖励（BSR）范式中，SR141716A（3和10 mg/kg）剂量依赖性地增加了BSR刺激阈值（即减少脑奖励），而AM4113在相同剂量下对BSR没有影响，表明SR141716A可能产生厌恶效应，而AM4113可能不会产生厌恶效应。综上所述，这些发现表明，中性CB1R拮抗剂如AM4113作为一种新的基于CB1R的药物，值得进一步研究，以治疗阿片类药物成瘾，没有sr141716a样的不良反应。越来越多的证据表明，大脑中存在功能性大麻素CB2受体（CB2Rs）。然而，由于CB2R抗体信号的特异性，这一发现存在争议。我们使用目前可用的两株cb2部分敲除（CB2-KO）小鼠作为对照，四种抗大鼠或抗小鼠CB2R抗体，并通过mRNA定量进一步解决这一问题。使用这四种抗体进行Western blot检测，在脑和脾脏均检测到40 kD的cb2r样带。值得注意的是，在大脑中检测到的条带多于在脾脏中检测到的条带，特异性免疫肽阻断了条带检测。免疫组化法检测小鼠中脑多巴胺神经元的cb2样免疫染色。CB2-KO小鼠的CB2R缺失可能降低或保持CB2R样免疫反应性不变，这取决于抗体表位。受体缺失区表位抗体检测到n端缺失Deltagen和c端缺失Zimmer菌株CB2-KO小鼠的CB2R带密度和免疫染色显著降低。在野生型和CB2-KO小鼠中，在预测受体未缺失区域具有表位的其他抗体检测到相似的带密度和免疫染色。在Deltagen或Zimmer CB2- ko小鼠中，定量RT-PCR检测CB2 mRNA表达，使用靶向上游或下游基因序列的探针，而不是靶向基因缺失序列的探针。这些发现表明，所测试的四种多克隆抗体均不具有小鼠cb2r的高度特异性。非特异性结合可能与部分CB2- ko小鼠中突变或截断的cb2r样蛋白的表达以及抗大鼠CB2抗体的使用有关，因为大鼠和小鼠CB2Rs的表位不同。此外，我们重新检查了CB1和CB2受体在大麻作用中的作用。采用脑电刺激奖励（BSR）方法，研究了不同大麻素药物对实验大鼠脑奖励的影响，以及CB1和CB2受体激活在脑奖励功能中的作用。两种混合CB1/CB2受体激动剂，δ -9-四氢大麻酚（THC）和WIN55,212-2，产生双相效应-低剂量时轻度增强BSR，高剂量时抑制。CB1R拮抗剂AM251能减弱低剂量增强的BSR，而CB2R受体AM630能减弱高剂量抑制的BSR。给大鼠注射选择性CB1R和CB2R激动剂，分别增强和抑制BSR。我们得出结论，大麻的主观影响取决于这些相反影响的平衡，并解释了为什么大麻在人类中可能是有益的或厌恶的，因为不同受试者的大脑中CB1Rs和CB2Rs的表达可能不同。进一步，我们研究了CB1R拮抗剂与CB2R拮抗剂联合使用是否能产生加性抗成瘾作用。δ -8-四氢大麻素（δ -8- thcv）是δ -9- thcv类似物，具有CB1拮抗剂/CB2激动剂的联合特性。我们在七种与尼古丁依赖相关的啮齿动物模型中测试了δ -8- thcv，这些模型包括尼古丁自我给药、强迫戒断后的线索引发的尼古丁寻求行为、尼古丁引发的尼古丁寻求行为恢复、尼古丁引发的条件位置偏好获得、尼古丁戒断诱导的焦虑样行为、尼古丁戒断诱导的躯体戒断体征和尼古丁戒断诱导的痛觉过敏。δ -8- thcv显著减弱大鼠静脉内尼古丁自我给药以及线索诱导和尼古丁诱导的尼古丁寻求行为复发。δ -8- thcv还能显著减弱小鼠尼古丁诱导的条件位置偏好和尼古丁戒断。我们得出结论，δ -8- thcv可能具有治疗尼古丁依赖的治疗潜力。我们还建议在更广泛的临床前动物模型中测试四氢大麻素的抗成瘾功效，对抗其他成瘾药物，最终在人类身上进行测试。我们还研究了大麻素诱导的四联体效应——镇痛、低温、猝睡和运动抑制，这些效应之前被认为是由大麻素CB1Rs的激活介导的。鉴于最近在大脑中发现的CB2和GPR55受体，我们研究了这些受体是否也参与大麻素的作用。我们发现δ -9- thc、WIN55212-2或XLR11在野生型（WT）小鼠中产生剂量依赖的四体效应。基因缺失或药理阻断CB1Rs可消除所有三种大麻素产生的四体效应。出乎意料的是，CB2Rs的基因缺失消除了delta-9-THC或WIN55212-2产生的镇痛和麻痹，而XLR11则没有。向侧脑室微量注射δ -9-四氢大麻酚也能在WT小鼠中产生四分体效应，但在CB1-KO小鼠中没有。CB2-KO小鼠显示脑室内δ -9-四氢大麻酚诱导的镇痛和麻痹减少。相比之下，GPR55受体的基因缺失导致对delta-9-THC或WIN55212-2的反应增强。CB1Rs、CB2Rs或GPR55Rs的拮抗作用产生的变化与在每种基因型小鼠系中观察到的变化相似。这些发现表明，除了CB1Rs， CB2Rs和GPR55Rs也参与大麻素产生的药理作用。与GPR55Rs相比，CB1Rs/CB2Rs似乎在大麻素作用中发挥相反的作用。最后，我们发现-石竹烯，一种膳食萜类化合物，可以抑制啮齿动物的尼古丁摄入和尼古丁寻求行为。

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.