Brain Cannabinoid Signaling: Selectivity and Adaptation

大脑大麻素信号传导：选择性和适应

• 批准号: 6472884
• 负责人: Laura J Sim-Selley
• 金额: $ 22.5万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6472884
• 关键词: G protein; activation analysis; autoradiography; brain; brain mapping; cannabinoid receptor; cannabinoids; confocal scanning microscopy; drug administration rate /duration; drug tolerance; guanosine triphosphate; immunocytochemistry; immunoprecipitation; laboratory mouse; laboratory rat; neurochemistry; protein localization; radiotracer; receptor coupling; receptor expression; receptor sensitivity

DESCRIPTION (provided by applicant): Cannabinoid (CB1) receptors in brain mediate the effects of delta 9-tetrahydrocannabinol (THC) and endocannabinoids, primarily by activation of inhibitory G-proteins of the Gi/Go family. However, it is our premise that CB1 receptor- G-protein coupling is not uniform throughout the brain, and CB1 receptors may couple to multiple G-proteins to account for the multiplicity of cannabinoid effects. Indeed, our progress to date demonstrates regional differences in CB1 receptor-mediated G-protein activity in both normal brain and in adaptive responses to chronic THC administration. Moreover, the pattern of tolerance development is not identical for all THC effects, a finding we hypothesize is related to these regional differences in CB1 receptor-G-protein coupling. The proposed studies will investigate the relationship between cannabinoid tolerance and CB1 receptor desensitization and downregulation, and examine whether selective coupling of CB1 receptors to specific G-protein subtypes is correlated with regional differences in CB1 receptor adaptation. We propose examining in a systematic manner the role of CB1 receptor- G-protein coupling in neuroadaptation not only as a means of understanding cannabinoid tolerance but as a way of characterizing the endocannabinoid system. In order to address the role of receptor occupancy in adaptation, the magnitude of THC tolerance will be varied by administering different doses of THC. We will then assess 1) tolerance to cannabinoid-mediated hypoactivity, antinociception, hypothermia and memory impairment in behavioral assays and 2) CB1 receptor downregulation and desensitization using radiolabeled ligand and agonist- stimulated [35S]GTPgammaS autoradiography. We also hypothesize that differences in CB1 receptor-G-protein coupling throughout the brain account for differences in recovery of tolerance to separate THC-mediated behavioral effects. Therefore, the temporal relationship between recovery of tolerance and CB1 receptor function will be evaluated by treating mice with THC, then evaluating tolerance and downregulation/desensitization at different times after cessation of treatment. We will also conduct experiments to determine whether CB1 receptor coupling to different G-protein subtypes is responsible for variations in cannabinoid actions in different brain regions. We will examine co-localization of CB1 receptors and specific G-beta and G-gamma subtypes using immunocytochemistry to determine whether there is selective co-localization of CB1 receptors and specific subunits in different regions. We will then examine whether chronic THC administration selectively alters CB1 receptor coupling to specific G-alpha subtypes using agonist- stimulated [35S]GTPgammaS binding with subsequent immunprecipitation of activated G-alpha subtypes. These studies will contribute to elucidation of the mechanisms of action of CB1 receptors in brain, as well as determine the effects of chronic cannabinoid administration on cellular function during tolerance.

描述（由申请人提供）：大脑中的大麻素 (CB1) 受体 介导 Delta 9-四氢大麻酚 (THC) 和内源性大麻素的作用， 主要是通过激活 Gi/Go 家族的抑制性 G 蛋白。然而， 我们的前提是 CB1 受体 - G 蛋白偶联不均匀 CB1 受体可能与多种 G 蛋白偶联， 解释大麻素效应的多重性。事实上，我们的进步 数据显示 CB1 受体介导的 G 蛋白的区域差异 正常大脑和对慢性 THC 的适应性反应的活动 行政。此外，耐受性发展的模式也不尽相同。 对于所有 THC 效应，我们假设的发现与这些区域有关 CB1受体-G-蛋白偶联的差异。拟议的研究将 研究大麻素耐受性与CB1受体之间的关系 脱敏和下调，并检查是否选择性偶联 特定 G 蛋白亚型的 CB1 受体与区域性相关 CB1受体适应的差异。我们建议系统地进行审查 CB1受体-G蛋白偶联在神经适应中的作用不仅 作为理解大麻素耐受性的一种手段，但作为一种方式 表征内源性大麻素系统。为了解决这个角色 适应过程中受体占据情况不同，THC 耐受程度也会不同 通过施用不同剂量的 THC。然后我们将评估 1) 的耐受性 大麻素介导的活动减退、镇痛、体温过低和记忆 行为测定受损以及 2) CB1 受体下调和 使用放射性标记配体和激动剂刺激的脱敏 [35S]GTPgammaS放射自显影。我们还假设 CB1 的差异 整个大脑中的受体-G-蛋白耦合导致了 恢复对单独 THC 介导的行为效应的耐受性。所以， 耐受恢复与CB1受体之间的时间关系 通过用 THC 治疗小鼠来评估功能，然后评估耐受性 以及停止后不同时间的下调/脱敏 治疗。我们还将进行实验来确定CB1受体是否 与不同 G 蛋白亚型的耦合导致了 大麻素在不同大脑区域的作用。我们将检查共本地化 CB1受体和特定的G-beta和G-gamma亚型使用 免疫细胞化学以确定是否存在选择性共定位 不同区域的CB1受体和特定亚基。然后我们将检查 长期使用 THC 是否会选择性地改变 CB1 受体偶联 使用激动剂刺激的 [35S]GTPgammaS 与特定 G-α 亚型结合 随后对活化的 G-α 亚型进行免疫沉淀。这些研究将 有助于阐明 CB1 受体的作用机制 大脑，以及确定长期服用大麻素的影响 耐受期间细胞功能的影响。