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.

在本报告期间，该研究项目取得了重大进展。 迄今为止，大脑中大麻素CB2受体的存在引起了争议。迄今为止，大多数证据都表明，在大脑和中枢神经系统中仅发现CB1大麻素受体，而大麻素CB2受体仅限于人体的周围 - 主要在免疫系统中。但是，最近的说法挑战了这种观点，即中枢神经系统中存在CB2受体，并且最近声称CB2受体调节突触活动。因此，我们使用了高度选择性的CB2激动剂和拮抗剂，并结合使用CB1和CB2受体基因删除小鼠，以及结合电生理学的分子神经生物学技术来研究大脑中CB2受体的存在和功能。 首先，我们研究了大鼠腹侧段区域（VTA）内多巴胺神经元上功能性大麻素CB2受体的表达。 这项工作的理由是，我们以前曾报道过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受体是否在大鼠脑中类似表达的知之甚少。 我们在VTA的多巴胺神经元中使用了原位杂交和免疫组织化学测定，并检测到CB2基因和受体。 可卡因自我给药上上调了VTA多巴胺神经元上的CB2受体。 电生理实验表明，CB2选择性受体激动剂JWH133激活CB2受体，抑制了单个解离神经元中VTA多巴胺神经元触发。 通过微注射对伏隔核的局部给药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。 这种效果被选择性CB2受体拮抗剂AM630阻塞。 这些数据表明，CB2受体是在VTA多巴胺神经元上表达的，并在功能上调节了大鼠多巴胺能神经元活性和可卡因自给行为。 然后，我们研究了大麻素CB2受体中的物种差异以及对可卡因自我给药的受体反应在大鼠与小鼠中的自我给药。 我们发现，在小鼠与大鼠中，CB2受体mRNA剪接和表达，蛋白质序列以及对CB2特异性配体的受体反应存在显着的物种差异。 系统性地给药JWH133是一种高度选择性的CB2受体激动剂，显着和剂量依赖性地抑制了在小鼠的固定增强比例下，但在大鼠中却不在固定的增强比例下，而不是大鼠。 然而，在渐进率增强下，JWH133显着增加了可卡因自我管理大鼠的渐进率突破点 - 从而降低了可卡因的激励动机性能。 然后，我们检查了大脑中的CB2受体基因表达和受体结构。 除了CB2A和CB2B mRNA同工型外，我们还发现了新型大鼠特异性CB2C和CB2D mRNA同工型。 使用原位杂交RNASCOPE分析，我们发现不同脑区域的CB2受体mRNA和小鼠与大鼠的细胞类型中的CB2受体mRNA较高。 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的终止密码子，该密码子截断了13个氨基酸残基，包括细胞内C末端中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有据可查的突触前大麻素CB1受体的逆行激活。 但是，我们发现动作电位驱动的内源性大麻素释放导致海马主要细胞中持久的膜潜在超极化，而与CB1受体无关。 如海马Ca2和CA3锥体细胞，这种超极化取决于CB2受体的激活，如一种结合的药物遗传学和免疫组织化学方法所示。 激活后，它们调节双碳酸氢钠共转产蛋白的活性，从而导致神经元的超极化。 CB2受体激活以自我调节方式发生，可牢固地改变Ca3海马金字塔细胞的输入/输出功能，并在体内调节γ振荡。 因此，我们首次发现了海马中的细胞类型特异性可塑性机制，为CB2受体的神经元表达提供了可靠的证据，并强调了它们在基本神经元传播中的重要性。最后，我们探索了新型大麻素化合物Delta-8-四氢糖蛋白（THCV）对啮齿动物中尼古丁效应的影响。 我们发现，THCV抑制了饮酒（P）大鼠抑制尼古丁的自我给药，抑制在渴望动物模型孵化的P大鼠中，在P大鼠中抑制提示诱导的尼古丁寻求行为，抑制尼古丁诱导的尼古丁诱导的复发，从而在PARTIDED的相对模型中，在PARTIONS中，在PARTIDENCTATED ANDIDED SECATION in NIC模型中脱离了烟碱的行为。偏爱小鼠的偏爱会大大减轻焦虑的行为（如在Plus迷宫中的测量），在尼古丁 - 垂直绘制的小鼠中，显着减弱了将小鼠戒断的体细胞迹象，并在尼古丁 - 尼古拉德拉德（Nicotine-WithDrawal）中戒断，并显着减弱杀伤力（使用热板测试中的小鼠中的小鼠中的小鼠中的小鼠）将其置于尼古丁中。 由于THCV是CB1拮抗剂和CB2激动剂的组合，因此此类发现与我们先前关于大麻素CB1拮抗剂和CB2激动剂的重大抗吸气作用的报道完全一致。 此外，我们建议四氢大麻素蛋白构成了抗瘾，抗捕获和抗释放药物的激动人心的新目标。