Endocannabinoid brain mechanisms and addiction

内源性大麻素脑机制和成瘾

• 批准号: 9555591
• 负责人: Eliot Gardner
• 金额: $ 41.89万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9555591
• 关键词: AM630; Action Potentials; Addictive Behavior; Agonist; Alpha Cell; Amino Acid Sequence; Amino Acids; Animal Model; Attenuated; Behavior; Biochemical; Biological Assay; Brain; Brain region; CB2 receptor antagonist; CNR1 gene; CNR2 gene; Cannabinoids; Cells; Chemicals; Cocaine; Cues; Data; Development; Dopamine; Dose; Electrical Stimulation of the Brain; Electrophysiology (science); Endocannabinoids; Enzymes; Exposure to; Gene Expression; Genes; Goals; High Pressure Liquid Chromatography; Hippocampus (Brain); Hyperalgesia; Immune system; In Situ Hybridization; Incentives; Injection of therapeutic agent; Intravenous; Laboratory Animals; Ligands; Measures; Mediating; Membrane Potentials; Messenger RNA; Microdialysis; Midbrain structure; Molecular Neurobiology; Motivation; Mus; Neuraxis; Neuronal Plasticity; Neurons; Neurotransmitters; Nicotine; Nicotine Withdrawal; Nonsense Codon; Nucleus Accumbens; Output; Pharmaceutical Preparations; Pharmacogenetics; Polymerase Chain Reaction; Property; Protein Isoforms; Proteins; Psychological reinforcement; Pyramidal Cells; RNA; RNA Splicing; Rattus; Receptor Activation; Receptor Gene; Reinforcement Schedule; Relapse; Reporting; Research Project Grants; Reverse Transcription; Rewards; Rodent; Role; Sampling; Self Administration; Self-Administered; Site; Sodium Bicarbonate; Stress; Structure; Synapses; System; Techniques; Testing; Time; Ventral Tegmental Area; Western Blotting; Withdrawal; Work; addiction; alcohol preferring rats; anxiety-like behavior; brain cell; cell type; craving; dopaminergic neuron; endogenous cannabinoid system; experimental study; extracellular; in vivo; neurotransmission; novel; overexpression; pre-clinical; preference; presynaptic; prevent; receptor; response; species difference; symporter

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.

本报告期内，该研究项目取得重大进展。 迄今为止，大脑中大麻素 CB2 受体的存在一直存在争议。迄今为止，大多数证据表明，仅在大脑和中枢神经系统中发现了 CB1 大麻素受体，而大麻素 CB2 受体仅限于身体的外周 - 主要是在免疫系统中。然而，这种观点受到最近关于 CB2 受体存在于中枢神经系统中的说法以及最近关于 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受体基因缺失的小鼠，以及分子神经生物学技术结合电生理学，研究大脑中CB2受体的存在和功能。 首先，我们研究了大鼠腹侧被盖区（VTA）内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，对于 CB2 受体在大鼠大脑中是否也有类似表达，人们知之甚少。 我们采用原位杂交和免疫组化方法，检测了腹侧被盖区多巴胺神经元的CB2基因和受体。 自我给予可卡因可上调 VTA 多巴胺神经元上的 CB2 受体。 电生理学实验表明，CB2 选择性受体激动剂 JWH133 激活 CB2 受体可抑制单个分离神经元中的 VTA 多巴胺神经元放电。 通过伏隔核微量注射 JWH133 局部给药可抑制可卡因增强的细胞外多巴胺和静脉注射可卡因自我给药。 这种作用被 AM630（一种选择性 CB2 受体拮抗剂）阻断。 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的多巴胺能神经元活动和可卡因自我给药行为。 然后，我们研究了大鼠和小鼠大麻素 CB2 受体的物种差异以及受体对可卡因自我给药的反应。 我们发现小鼠与大鼠的 CB2 受体 mRNA 剪接和表达、蛋白质序列以及受体对 CB2 特异性配体的反应存在显着的物种差异。 JWH133（一种高度选择性的 CB2 受体激动剂）的全身给药，在固定比例的强化方案下，可显着且剂量依赖性地抑制小鼠静脉内可卡因的自我给药，但在大鼠中则不然。 然而，在渐进比率强化下，JWH133 显着增加了可卡因自我给药大鼠的渐进比率断点，从而降低了可卡因的激励动机特性。 然后我们检查了大脑中 CB2 受体基因表达和受体结构。 除了 CB2a 和 CB2b mRNA 亚型之外，我们还发现了新型大鼠特异性 CB2c 和 CB2d mRNA 亚型。 使用原位杂交 RNAscope 检测，我们发现小鼠不同大脑区域和细胞类型的 CB2 受体 mRNA 水平高于大鼠。 通过比较 CB2 受体编码区，我们在小鼠 CB2 受体基因中发现了一个过早终止密码子，该密码子截短了 13 个氨基酸残基，包括细胞内 C 末端的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与大鼠可卡因自我给药的不同影响。 此外，我们研究了 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会导致海马主细胞出现持久的膜电位超极化，这种超极化与 CB1 受体无关。 这种超极化是海马 CA2 和 CA3 锥体细胞特有的，取决于 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，它们调节碳酸氢钠协同转运蛋白的活性，导致神经元超极化。 CB2受体激活以自我调节的方式发生，强烈改变CA3海马锥体细胞的输入/输出功能，并调节体内伽马振荡。 因此，我们首次发现海马体中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 delta-8-四氢大麻二酚 (THCV) 对啮齿类动物尼古丁作用的影响。 我们发现 THCV 抑制酒精偏好 (P) 大鼠的尼古丁自我给药，抑制在渴望动物模型孵化中测试的 P 大鼠中线索诱导的尼古丁寻求行为，抑制在复吸动物模型中测试的尼古丁诱导的尼古丁寻求行为复发，防止尼古丁诱导的条件性位置的获得 显着减弱尼古丁戒断小鼠的焦虑样行为（如在十字迷宫中测量），显着减弱尼古丁戒断小鼠的躯体戒断症状，并显着减弱尼古丁戒断小鼠的痛觉过敏（如使用热板测试测量）。 由于 THCV 是 CB1 拮抗剂和 CB2 激动剂的组合，因此这些发现与我们之前关于大麻素 CB1 拮抗剂和 CB2 激动剂的显着抗成瘾作用的报道完全一致。 此外，我们建议四氢大麻素构成抗成瘾、抗渴求和抗复发药物开发的令人兴奋的新靶标。