Basic brain mechanisms underlying drug addiction, craving, and relapse

在本报告所述期间，由于现有研究资源严重减少，我们在这一领域的研究受到限制。然而，我们在三个不同的领域进行了研究-第一，继续研究抗尼古丁戒烟药物varenicline的基本大脑作用机制，第二，研究大脑中是否存在大麻素CB2受体，第三，在我们的实验室中建立奖励延迟折扣行为模型。在本报告所述期间，我们的verenicline工作是我们先前对varenicline进行的广泛研究的后续工作，在该研究中，我们证明varenicline的α4beta2烟碱部分激动剂特性对于其戒烟疗效至关重要，而其alpha7尼古丁激动剂特性与其治疗作用无关。因此，未来的抗尼古丁成瘾反吸烟药物疗法可以设计成只具有α4beta2尼古丁部分激动剂特性-从而提高治疗效果并减少产生不想要的副作用的可能性。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，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受体存在于大脑中，CB2受体功能上调节中伏核-多巴胺系统，CB2受体功能上调节多巴胺介导的行为，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拮抗。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。此外，在本报告期间，我们在临床前成瘾动物模型组中引入了一种新的动物模型。在人类层面上，无法延迟满足是吸毒成瘾的病征。因此，我们在实验室中引入了奖励延迟折扣任务。在这项任务中，实验大鼠在它们的测试室中被展示了两个安装在墙上的杠杆。一个杠杆的压抑会立即带来食物奖励。在长达60秒的延迟期后，按下另一个杠杆会带来更大的奖励。动物必须选择它是想要一个小的即时奖励，还是一个更大的延迟奖励。通过这种方式，冲动的选择可以被测量和量化。我们的目的是利用这一新的动物模型来衡量长期服用成瘾药物对冲动选择的影响，并确定我们推定的任何抗成瘾药物疗法是否可以改变成瘾药物改变的冲动选择，朝着推测的治疗方向发展。我们相信，这个源自行为经济学的新模型的加入，给了我们一个全新的临床相关视角，在临床前动物模型水平上评估潜在的治疗、抗成瘾、抗渴求、抗复发药物。

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.