Brain Cannabinoid Signaling: Selectivity and Adaptation

描述（由申请人提供）：脑中的大麻素（CB 1）受体 介导δ 9-四氢大麻酚（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，在这方面， 我们假设CB 1受体-G蛋白偶联是不均匀的， CB 1受体可能与多种G蛋白偶联， 解释了大麻素作用的多样性。事实上，我们的进展， 数据显示CB 1受体介导的G蛋白的区域差异 正常大脑和对慢性THC的适应性反应中的活性 局此外，耐受性发展的模式是不相同的 对于所有THC的影响，我们假设的一个发现与这些区域有关。 CB 1受体-G蛋白偶联的差异。拟议的研究将 探讨大麻素耐受与CB 1受体的关系 脱敏和下调，并检查是否选择性偶联 特异性G蛋白亚型的CB 1受体与区域性 CB 1受体适应的差异。我们建议在一个系统的 CB 1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， 表征内源性大麻素系统。为了发挥 在适应过程中，THC耐受性的大小将有所不同 注射不同剂量的四氢大麻酚然后我们将评估1）耐受性， 大麻素介导的活动减退、抗伤害感受、体温降低和记忆 行为测定的损害和2）CB 1受体下调， 使用放射性标记的配体和激动剂刺激的脱敏 [35 S] GTP γ S放射自显影。我们还假设CB 1的差异 受体-G蛋白偶联在整个大脑中的差异， 恢复对单独THC介导的行为效应的耐受性。因此，我们认为， 耐受性恢复与CB 1受体的时间关系 通过用THC治疗小鼠，然后评估耐受性， 和在停止后的不同时间下调/脱敏。 治疗我们还将进行实验，以确定CB 1受体是否 与不同G蛋白亚型的偶联导致了 大麻素在大脑不同区域的作用。我们将研究共同本地化 CB 1受体和特定的G-β和G-γ亚型， 免疫细胞化学，以确定是否有选择性共定位的 CB 1受体和不同区域的特异性亚基。然后我们将检查 慢性THC给药是否选择性地改变CB 1受体偶联， 使用激动剂刺激的[35 S] GTP γ S结合的特异性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CB 1受体的作用机制 大脑，以及确定慢性大麻素管理的影响 对细胞功能的影响

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.