Basic brain mechanisms underlying drug addiction, craving, and relapse

在本报告所述期间，由于可用研究资源严重减少，我们在这一领域的研究受到限制。尽管如此，我们在三个不同的领域开展了研究：第一，继续研究抗尼古丁戒烟药物varenicline的基本脑作用机制；第二，研究大脑中大麻素CB2受体的存在；第三，在我们的实验室建立了奖励延迟折扣行为模型。在本报告期间，我们对伐尼克兰的研究是对我们之前对伐尼克兰的广泛研究的后续研究，我们证明了伐尼克兰的α 4 β 2烟碱部分激动剂特性对其戒烟治疗效果至关重要，而其α 7烟碱激动剂特性与其治疗作用无关。因此，未来的抗尼古丁成瘾抗吸烟药物疗法可以设计为仅具有α 4 β 2烟碱部分激动剂特性，从而提高治疗效果并减少产生不良副作用的可能性。在大麻素和内源性大麻素的研究领域，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，只有CB1大麻素受体存在于大脑和中枢神经系统，而大麻素CB2受体仅限于身体的外周-主要是在免疫系统中。然而，这一观点受到了最近CB2受体存在于中枢神经系统和CB2受体调节突触活动的说法的挑战。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作用。我们发现CB2受体选择性激动剂JWH133减少了野生型和CB1基因缺失小鼠的静脉可卡因自我给药，但在CB2基因缺失小鼠中没有。这种作用被CB2受体选择性拮抗剂AM630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抑制作用。在递进比强化条件下，我们发现JWH133抑制了自我服用可卡因的激励动机，这可以通过递进比断点的显著降低来证明。当JWH133通过鼻内（通过筛板直接进入大脑）或直接将JWH133脑内显微注射到伏隔核时，也发现了类似的效果。同样，这种效果在野生型小鼠中出现，而在CB2受体基因缺失的小鼠中没有出现。通过静脉注射自我给药和条件位置偏好/厌恶实验，发现JWH133本身没有强化或厌恶作用。此外，JWH133在野生型和CB1基因缺失小鼠中抑制可卡因增强的运动，但在CB2基因缺失小鼠中没有。在野生型和CB1基因缺失的小鼠中，JWH133本身具有全身给药和脑内微注射到伏隔核的抑制运动作用，但在CB2基因缺失的小鼠中没有。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，无论是全身给药还是脑内微注射到伏隔核，但对CB2基因缺失小鼠没有刺激作用。通过实时活体脑微透析检测，JWH133本身抑制伏隔核胞外多巴胺。通过实时体内脑微透析测量，JWH133还抑制基础和可卡因增强的细胞外伏隔核多巴胺。这种作用被cb2选择性拮抗剂AM630阻断。AM630在脑内注入伏隔核，增加伏隔核基底细胞外多巴胺。我们得出的结论是，CB2大麻素受体存在于大脑中，CB2受体功能调节中伏多巴胺系统，CB2受体功能调节多巴胺介导的行为，并且大脑CB1和CB2受体相关的神经系统可能以相互拮抗的方式相互作用。这种机制知识可以帮助寻找治疗药物成瘾和依赖的新的有效药物治疗化合物。此外，在本报告期间，我们将一种新的动物模型引入我们的成瘾临床前动物模型。在人类的层面上，无法延迟满足是药物成瘾的一种病态症状。因此，我们在实验室中引入了奖励延迟贴现任务。在这项任务中，实验鼠的实验室内有两个壁挂式杠杆。按下一个杠杆会立即提供食物奖励。在长达60秒的延迟期后，按压另一个杠杆将获得更大的奖励。动物必须选择是想要一个小的即时奖励还是一个大的延迟奖励。通过这种方式，冲动性选择可以被测量和量化。我们的目的是利用这种新的动物模型来衡量长期服用成瘾性药物对冲动选择的影响，并确定我们假设的抗成瘾药物疗法是否能改变成瘾性药物改变的冲动选择，使其朝着假设的治疗方向发展。我们相信，这个来源于行为经济学的新模型的加入，为我们在临床前动物模型水平上评估潜在的治疗性抗成瘾、抗渴望、抗复发药物提供了一个全新的临床相关视角。

During the present reporting period, our research in this area was limited due to severe reductions in available research resources. Nevertheless, we carried out research in three distinct domains - first, continued research into the basic brain mechanisms of action of the anti-nicotine smoking-cessation medication varenicline, second, research into the existence of cannabinoid CB2 receptors in the brain, and third, setting up a reward delay discounting behavioral model in our labortory. Our verenicline work during the present reporting period was a follow-up to our extensive previous research on varenicline in which we proved that varenicline's alpha4beta2 nicotinic partial agonist properties are essential to its therapeutic effect on smoking cessation while its alpha7 nicotinic agonist properties are irrelevant to its therapeutic action. Thus, future anti-nicotine-addiction anti-smoking pharmacotherapies can be designed with only alpha4beta2 nicotinic partial agonist properties - thus enhancing therapeutic efficacy and diminishing the liklihood of producing unwanted side effects. In the cannabinoid and endocannabinoid realm of research, 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, that CB2 receptors functionally modulate dopamine-mediated behaviors, and that the 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. In additiom, during this reporting period, we introduced a new animal model into our battery of preclinical animal models of addiction. At the human level, inability to delay gratification is a pathognomonic symptom of drug addiction. Therefore, we have introduced a reward delay discounting task into our laboratory. In this task, laboratory rats are presented with two wall-mounted levers in their test chambers. Depression of one lever delivers a food reward immediately. Depression of the other lever delivers a larger reward after a delay period ranging up to 60 seconds. The animal must choose whether it desires a small immediate reward or a larger delayed reward. In this manner, impulsive choice can be measured and quantified. It is our intention to use this new animal model to measure the effect of chronic administration of addictive drugs on impulsive choice, and also to determine whether any of our putative anti-addiction pharmacotherapies can change addictive-drug-altered impulsice choice in a putatively therapeutic direction. We believe that the addition of this new model - derived from behavioral economics - gives us an entirely new clinically-relevant perspective to evaluate potentially therapeutic anti-addiction anti-craving anti-relapse medications at the preclinical animal model level.