Endocannabinoid brain mechanisms and addiction

在本报告所述期间，这一研究项目取得了非常重大的进展。到目前为止，大麻素CB2受体在大脑中的存在一直存在争议。到目前为止，大多数证据表明，在大脑和中枢神经系统中只发现了CB1大麻素受体，而大麻素CB2受体仅限于身体的外周--主要是免疫系统。然而，最近关于CB2受体存在于中枢神经系统的说法以及最近关于CB2受体调节突触活动的说法挑战了这一观点。因此，我们使用高选择性的CB2激动剂和拮抗剂，结合使用CB1和CB2受体基因缺失的小鼠，研究CB2参与可卡因的行为和神经化学效应。我们发现CB2受体选择性激动剂JWH133减弱了野生型和CB1基因缺失小鼠静脉注射可卡因的自我给药，但对CB2基因缺失小鼠没有影响。这种作用被CB2受体选择性拮抗剂AM630所消除。为了证实我们的发现，我们还使用了CB2选择性激动剂GW405833，并在野生型小鼠中发现了类似的静脉注射可卡因自我给药的抑制作用。在累进比率强化条件下，我们发现JWH133抑制了自我给药的激励动机，这一点从累进比率突变点的显著降低可见一斑。JWH133鼻腔给药(通过筛板直接进入大脑)或直接脑内伏隔核微量注射JWH133时，也有类似的效果。同样，这种效应在野生型小鼠中也能看到，但在CB2受体基因缺失的小鼠中却没有。通过静脉给药和条件性位置偏爱/厌恶实验，发现JWH133本身没有增强或厌恶作用。此外，JWH133抑制了野生型和CB1基因缺失小鼠的可卡因增强的运动，但不抑制CB2基因缺失小鼠的运动。在野生型和CB1基因缺失的小鼠中，JWH133本身对轨迹运动有抑制作用，而在CB2基因缺失的小鼠中则没有。CB2选择性拮抗剂AM630对野生型和CB1基因缺失小鼠的运动有刺激作用，但对CB2基因缺失小鼠无此作用。实时脑微透析法测定，JWH133本身对伏隔核外核多巴胺有抑制作用。JWH133还抑制基础和可卡因增强的伏隔核细胞外核多巴胺，通过实时活体脑微透析测量。这种作用可被CB2选择性拮抗剂AM630阻断。AM630本身被微量注射到伏隔核内，注入伏隔基底核细胞外的多巴胺。我们得出结论，CB2大麻素受体存在于大脑中，CB2受体功能调节中脑伏隔核-多巴胺系统，CB2受体功能调节多巴胺介导的行为。此外，我们使用脑电刺激奖赏临床前动物模型来研究几种大麻素的奖赏和/或厌恶效应，以及这些作用背后的受体机制。我们发现CB1/CB2类大麻激动剂Delta9-四氢大麻酚(THC)和WIN55212-2具有双相脑奖赏效应--低剂量增强脑奖赏机制，高剂量抑制脑奖赏机制。另一方面，选择性CB1受体激动剂ACEA只产生大脑奖励增强，而选择性CB2受体激动剂只产生大脑奖励抑制。此外，选择性大麻素CB1受体拮抗剂AM251选择性地阻断低剂量THC或WIN55212-2产生的增强的脑奖赏，而选择性大麻样CB2受体拮抗剂AM630选择性地阻断高剂量THC或WIN55212-2产生的脑奖赏抑制。TRPV1拮抗剂卡萨西平(一些研究人员认为它通过非CB1、非CB2大麻受体起作用)不能改变THC或WIN55212-2诱导的大脑奖赏的变化。此外，CB1受体选择性拮抗剂AM251，而不是CB2受体选择性拮抗剂AM630，可以阻断ACEA增强的大脑奖励。CB2受体选择性拮抗剂AM630，而不是CB1受体选择性拮抗剂AM251，可阻断JWH133诱导的脑奖赏抑制。JWH133鼻腔给药抑制脑奖赏，CB2受体选择性拮抗剂AM630鼻腔联合给药可阻断这种作用。我们的结论是，大麻素激动剂对脑奖赏具有双相效应，低剂量增强，高剂量抑制脑奖赏机制。我们进一步得出结论，大麻素诱导的奖赏增强是通过激活脑CB1受体介导的，而大麻素诱导的脑奖赏机制的抑制是通过激活脑中的CB2受体介导的。这些研究结果表明，大脑CB1和CB2受体连接的神经系统可能以一种相互对立的方式在功能上相互对抗。这种机制知识可以帮助寻找新的有效的药物治疗化合物来治疗药物成瘾和依赖。我们还将这项工作扩展到实验室大鼠，看看大脑CB2受体在不同的哺乳动物物种中是否发挥不同的作用。我们还开始了JWH133对多巴胺能神经元放电的电生理学研究-在单个分离的腹侧被盖区多巴胺神经元，在中脑切片制备的腹侧被盖区多巴胺神经元，以及在活体麻醉动物腹侧被盖区多巴胺神经元。

During the present reporting period, very significant progress was made on this research project. The existence of cannabinoid CB2 receptors in the brain has been heretofore controversial. Most evidence has heretofore suggested that only CB1 cannabinoid receptors are found in brain and central nervous system while cannabinoid CB2 receptors are restricted to the body's periphery - primarily in the immune system. However, this view has been challenged by recent claims that CB2 receptors are present in the central nervous system and by recent claims that CB2 receptors modulate synaptic activity. Therefore, we used highly selective CB2 agonists and antagonists, combined with the use of CB1 and CB2 receptor gene-deleted mice, to study CB2 involvement in cocaine's behavioral and neurochemical effects. We found that the CB2 receptor-selective agonist JWH133 attenuates intravenous cocaine self-administration in wild-type and CB1 gene-deleted mice, but not in CB2 gene-deleted mice. This effect was abolished by the CB2 receptor-selective antagonist AM630. To confirm our findings, we also used the CB2-selective agonist GW405833 and found a similar inhibition of intravenous cocaine self-administration in wild-type mice. Under progressive-ratio reinforcement conditions, we found that JWH133 inhibits incentive motivation to self-administer cocaine, as evidenced by strong reductions in the progressive-ratio break-point. Similar effects were found when JWH133 was administered intra-nasally (for direct passage into the brain via the cribiform plate) or administered by direct intracerebral microinjections of JWH133 into the nucleus accumbens. Again, the effect was seen in wild-type but not in CB2 receptor gene-deleted mice. JWH133 by itself was found to have no reinforcing or aversive effects, as assessed by intravenous self-administration and by conditioned place preference/aversion experiments. Further, JWH133 inhibited cocaine-enhanced locomotion in wild-type and CB1 gene-deleted mice, but not in CB2 gene-deleted mice. JWH133 by itself had an inhibitory effect on locmotion, both with systemic administration and with intracerebral microinjection into the nucleus accumbens in wild-type and CB1 gene-deleted mice, but not in CB2 gene-deleted mice. The CB2 selective antagonist AM630 had a stimulatory effect on locomotion, both with systemic administration and with intracerebral microinjection into the nucleus accumbens in wild-type and CB1 gene-deleted mice, but not in CB2 gene-deleted mice. JWH133 by itself inhibited extracellular nucleus accumbens dopamine as measured by real-time in vivo brain microdialysis. JWH133 also inbited basal and cocaine-enhanced extracellular nucleus accumbens dopamine as measured by real-time in vivo brain microdialysis. This effect was blocked by the CB2-selective antagonist AM630. By itself, AM630 - microinjected intracerebrally into the nucleus accumbens - aumented basal extracellular nucleus accumbens dopamine. We conclude that CB2 cannabinoid receptors exist in the brain, that CB2 receptors functionally modulate the meso-accumbens dopamine system, and that CB2 receptors functionally modulate dopamine-mediated behaviors. In addition, we used the electrical brain-stimulation reward preclinical animal model to study the rewarding and/or aversive effects of several cannabinoids and the receptor mechanisms underlying these actions in laboratory rats. We found that the mixed CB1/CB2 cannabinoid agonists delta-9-tetrahydrocannabinol (THC) and WIN55212-2 produce biphasic effects on brain reward - low doses enhancing brain reward mechanism and high doses inhibiting them. On the other hand, the selective CB1 cannabinoid receptor agonist ACEA produces only brain-reward enhancement, while the selective CB2 receptor agonist produces only brain-reward inhibition. Further, the selective cannabinoid CB1 receptor antagonist AM251 selectively blocks the enhanced brain reward produced by low dose THC or WIN55212-2, while the selective cannabinoid CB2 receptor antagonist AM630 selectively blocks the brain reward inhibition produced by high dose THC or WIN55212-2. The TRPV1 antagonist capsazepine (posited by some researchers to act via a non-CB1, non-CB2 cannabinoid receptor) fails to alter THC- or WIN55212-2-induced changes in brain reward. In addition, the CB1 receptor selective antagonist AM251, but not the CB2 receptor selective antagonist AM630, blocks ACEA-enhanced brain reward. The CB2 receptor selective antagonist AM630, but not the CB1 receptor selective antagonist AM251, blocks JWH133-induced inhibition of brain reward. Intranasal JWH133 inhibits brain reward, an effect that is blocked by intranasal co-administration of the CB2 receptor selective antagonist AM630. We conclude that cannabinoid agonists produce biphasic effects on brain reward, with low doses enhancing and high doses inhibiting brain reward mechanisms. We further conclude that cannabinoid-induced reward enhancement is mediated by activation of brain CB1 receptors, while cannabinoid-induced inhibition of brain reward mechanisms is mediated by activation of CB2 receptors in the brain. These research findings suggest that brain CB1 and CB2 receptor-linked neural systems may functionally antagonize each other in a reciprocal mutually antatagonistic manner. Such mechanistic knowledge can aid in the search for new and effective pharmacotherapeutic compounds for the treatment of drug addiction and dependence. We have also extended this work to laboratory rats, to see if brain CB2 receptors act differently in a different mammalian species. We have also started electrophysiological studies of the effects of JWH133 on dopaminergic neuronal cell firing - in single dissociated ventral tegmental area dopamine neurons, in ventral tegmental area dopamine neurons in midbrain slice preparations, and in ventral tegmental area dopamine neurons in live anesthetized animals.