Brain Cannabinoid Signaling: Selectivity and Adaptation

描述(申请人提供)：大脑中的大麻素(CB1)受体 介导β-四氢大麻酚(THC)和内源性大麻素的作用， 主要通过激活GI/GO家族的抑制性G蛋白。然而， CB1受体-G蛋白偶联不均匀是我们的前提 整个大脑，CB1受体可能与多个G蛋白偶联以 解释了大麻素效应的多样性。事实上，我们在这方面的进展 日期显示CB1受体介导的G蛋白的地区差异 正常脑和慢性THC适应性反应中的活动 行政管理。此外，宽容发展的模式也不尽相同 对于所有的影响，我们假设的一个发现与这些地区性的 CB1受体-G蛋白偶联的差异。拟议的研究将 大麻素耐受与CB1受体关系的研究 脱敏和下调，并检查选择性偶联是否 特异性G蛋白亚型的CB1受体与地区性 CB1受体适应性的差异。我们建议系统地研究 CB1受体-G蛋白偶联在神经适应中的作用 作为了解大麻素耐受性的一种手段，但作为一种 内源性大麻素系统的特征。为了解决以下问题： 受体在适应中的占有率，耐受性的大小会有所不同 通过给予不同剂量的THC。然后我们将评估对以下各项的容忍度 大麻素介导的低活动、抗伤害性、体温过低与记忆 行为分析的损害和2)CB1受体下调和 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中的差异 整个大脑的受体-G-蛋白偶联解释了 恢复耐受性以分离THC介导的行为效应。因此， CB1受体与耐受性恢复的时间关系 通过用THC治疗小鼠，然后评估耐受性来评估功能 以及停药后不同时间的下调/脱敏 治疗。我们还将进行实验，以确定CB1受体 与不同G蛋白亚型的偶联是导致 大麻素在不同脑区的作用。我们将研究共同本地化 CB1受体和特定的G-β和G-伽马亚型 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受体和特异性亚基。然后我们将检查 慢性THC给药是否选择性改变CB1受体偶联 用激动剂刺激的[35S]GTP-GammaS结合特异性G-α亚型 随后免疫沉淀活化的G-α亚型。这些研究将 有助于阐明CB1受体在脑出血中的作用机制 大脑，以及确定长期服用大麻素的影响 耐受性过程中的细胞功能。

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.