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。迄今为止，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，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，我们采用高选择性的CB2激动剂和拮抗剂，结合使用CB1和CB2受体基因缺失小鼠，以及结合电生理学的分子神经生物学技术，研究了CB2受体在大脑中的存在和功能。首先，我们研究了大鼠腹侧被盖区（VTA）多巴胺神经元上功能性大麻素CB2受体的表达。这项工作的基本原理是，我们之前已经报道了小鼠中脑多巴胺神经元中功能性大麻素CB2受体的表达。然而，关于CB2受体在大鼠脑中是否有类似的表达，人们知之甚少。采用原位杂交和免疫组化检测方法，在VTA多巴胺神经元中检测CB2基因和受体。可卡因自我给药后，VTA多巴胺神经元上的CB2受体表达上调。电生理实验表明，CB2选择性受体激动剂JWH133激活CB2受体可抑制单个游离神经元的VTA多巴胺神经元放电。伏隔核局部微注射JWH133抑制可卡因增强的细胞外多巴胺和静脉可卡因自我给药。这种作用被AM630阻断，AM630是一种选择性CB2受体拮抗剂。这些数据表明，CB2受体在VTA多巴胺神经元上表达，并在功能上调节大鼠多巴胺能神经元的活性和可卡因自我给药行为。然后，我们研究了大麻素CB2受体的物种差异以及大鼠与小鼠对可卡因自我给药的受体反应。我们发现，小鼠和大鼠在CB2受体mRNA剪接和表达、蛋白质序列以及受体对CB2特异性配体的反应方面存在显著的物种差异。JWH133是一种高度选择性的CB2受体激动剂，在小鼠中，在固定比例强化计划下，全身给予JWH133显著且剂量依赖性地抑制静脉内可卡因自我给药，但在大鼠中没有。然而，在递进比强化下，JWH133显著提高了可卡因自我给药大鼠的递进比断点，从而降低了可卡因的激励动机特性。然后我们检查了脑内CB2受体基因表达和受体结构。除了CB2a和CB2b mRNA亚型外，我们还发现了新的大鼠特异性CB2c和CB2d mRNA亚型。使用原位杂交RNAscope检测，我们发现小鼠与大鼠相比，不同脑区和细胞类型的CB2受体mRNA水平更高。通过比较CB2受体编码区域，我们在小鼠CB2受体基因中发现了一个过早停止密码子，该密码子截断了13个氨基酸残基，包括细胞内c端功能性自磷酸化位点。这些发现表明，CB2受体基因和受体结构的剪接和表达的物种差异可能部分解释了CB2受体选择性配体对小鼠和大鼠可卡因自我给药的不同影响。此外，我们还研究了CB2受体介导的对海马神经元可塑性的影响。内源性大麻素系统的功能主要归因于充分记录的突触前大麻素CB1受体的逆行激活。然而，我们发现动作电位驱动的内源性大麻素释放导致海马主细胞中独立于CB1受体的长期膜电位超极化。这种超极化是海马CA2和CA3锥体细胞特异性的，依赖于CB2受体的激活，如药物遗传学和免疫组织化学联合方法所示。激活后，它们调节碳酸氢钠共转运体的活性，导致神经元的超极化。CB2受体的激活以自我调节的方式发生，有力地改变了CA3海马锥体细胞的输入/输出功能，并调节了体内的伽马振荡。因此，我们首次在海马中发现了一种细胞类型特异性的可塑性机制，为CB2受体的神经元表达提供了强有力的证据，并强调了它们在基本神经元传递中的重要性。最后，我们探讨了新型大麻素化合物δ -8-四氢大麻素（THCV）对尼古丁对啮齿动物的影响。我们发现THCV抑制酒精偏好(P)大鼠的尼古丁自我给药，抑制渴望动物模型中P大鼠的线索诱导的尼古丁寻求行为，抑制复发动物模型中P大鼠的尼古丁诱导的尼古丁寻求行为复发，阻止尼古丁诱导的条件位置偏好小鼠的获得。显着减弱尼古丁戒断小鼠的焦虑样行为（如在正迷宫中测量），显着减弱尼古丁戒断小鼠的躯体戒断迹象，显着减弱尼古丁戒断小鼠的痛觉过敏（如使用热板试验测量）。由于THCV是CB1拮抗剂和CB2激动剂的联合作用，这些发现与我们之前关于大麻素CB1拮抗剂和CB2激动剂显著抗成瘾作用的报道完全一致。此外，我们认为四氢大麻素是开发抗成瘾、抗渴望和抗复发药物的一个令人兴奋的新目标。

During the present reporting period, 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, and molecular neurobiology techniques combined with electrophysiology, to study the existence and function of CB2 receptors in the brain. Firstly, we studied the expression of functional cannabinoid CB2 receptors on dopamine neurons within the ventral tegmental area (VTA) in rats. The rationale for this work was that we had previously reported the expression of functional cannabinoid CB2 receptors in midbrain dopamine neurons in mice. However, little was known as to whether CB2 receptors are similarly expressed in rat brain. We used in situ hybridization and immunohistochemical assays, and detected CB2 gene and receptors in dopamine neurons of the VTA. The CB2 receptors on VTA dopamine neurons were up-regulated by cocaine self-administration. Electrophysiological experiments showed that activation of CB2 receptors by the CB2-selective receptor agonist JWH133 inhibited VTA dopamine neuronal firing in single dissociated neurons. Local administration of JWH133 by micro-injection into the nucleus accumbens inhibited cocaine-enhanced extracellular dopamine and intravenous cocaine self-administration. This effect was blocked by AM630, a selective CB2 receptor antagonist. These data suggest that CB2 receptors are expressed on VTA dopamine neurons and functionally modulate dopaminergic neuronal activity and cocaine self-administration behavior in rats. We then studied species differences in cannabinoid CB2 receptors and receptor responses to cocaine self-administration in rats versus mice. We found that there are significant species differences in CB2 receptor mRNA splicing and expression, protein sequences, and receptor responses to CB2-specific ligands in mice versus rats. Systemic administration of JWH133, a highly selective CB2 receptor agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed ratio schedule of reinforcement in mice, but not in rats. However, under progressive-ratio reinforcement, JWH133 significantly increased the progressive-ratio break-point in cocaine self-administering rats - thus decreasing cocaine's incentive motivational properties. We then examined CB2 receptor gene expression and receptor structure in the brain. We found novel rat-specific CB2c and CB2d mRNA isoforms in addition to CB2a and CB2b mRNA isoforms. Using in situ hybridization RNAscope assays, we found higher levels of CB2 receptor mRNA in different brain regions and cell types in mice versus rats. By comparing CB2 receptor-encoding regions, we found a premature stop codon in the mouse CB2 receptor gene that truncated 13 amino acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2 receptor genes and receptor structures may in part explain the different effects of CB2 receptor-selective ligands on cocaine self-administration in mice versus rats. In addition, we studied CB2 receptor-mediated effects on neuronal plasticity in the hippocampus. The functionality of the endocannabinoid system is primarily ascribed to the well-documented retrograde activation of presynaptic cannabinoid CB1 receptors. However, we found that action potential-driven endocannabinoid release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1 receptors. This hyperpolarization, which is specific to hippocampal CA2 and CA3 pyramidal cells, depends on the activation of CB2 receptors, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to hyperpolarization of the neuron. CB2 receptor activation occurred in a self-regulatory manner, robustly altered the input/output function of CA3 hippocampal pyramidal cells, and modulated gamma oscillations in vivo. Thus, we found - for the first time - a cell-type specific plasticity mechanism in the hippocampus that provides robust evidence for the neuronal expression of CB2 receptors and emphasizes their importance in basic neuronal transmission. Finally, we explored the effects of the novel cannabinoid compound delta-8-tetrahydrocannabivarin (THCV) on nicotine's effects in rodents. We found that THCV inhibits nicotine self-administration in alcohol-preferring (P) rats, inhibits cue-induced nicotine-seeking behavior in P rats tested in the incubation of craving animal model, inhibits nicotine-induced relapse to nicotine-seeking behavior in P rats tested in the reinstatement animal model of relapse, prevents acquisition of nicotine-induced conditioned place preference in mice, significantly attenuates anxiety-like behavior (as measured in the plus maze) in mice placed into nicotine-withdrawal, significantly attenuates somatic signs of withdrawal in mice placed into nicotine-withdrawal, and significantly attenuates the hyperalgesia (as measured using the hot-plate test) in mice placed into nicotine-withdrawal. As THCV is a combined CB1 antagonist and CB2 agonist, such findings are fully congruent with our previous reports of significant anti-addiction actions of cannabinoid CB1 antagonists and CB2 agonists. Further, we propose that the tetrahydrocannabivarins constitute an exciting new target for the development of anti-addiction, anti-craving, and anti-relapse medications.