LOCALIZATION OF RECEPTOR ACITIVITY FOR DRUGS OF ABUSE

描述：(申请人摘要) 利用[35S]GTPgS放射自显影技术开发了一种新技术 定位受体激活的G蛋白。这项技术是基于 以前使用激动剂刺激的[35S]GTPgS结合的研究，在 存在过剩的GDP，以测量受体激活的G蛋白 膜。[35S]GTPgS放射自显影的优越性 技术是定位具有功能活性的G蛋白的能力。这个 这项技术已经被用来证明由几个 受体：阿片、大麻素和GABA-B。[35S]GTPgS放射自显影 也被用来鉴定ORL1肽刺激的[35S]GTPg结合。这 演示了该技术的另一个优势；因为[35S]GTPgS是 使用放射性配基，任何未标记的激动剂都可以用于这项技术。 [35S]GTPgS放射自显影也被用来检测G蛋白的活性 在接受慢性药物治疗后。在这些研究中，慢性吗啡 治疗降低MU阿片类药物刺激的特异性[35S]GTPgS结合 脑干核团：中缝背核、斑核、蓝斑、臂旁核 孤束核和连合核。建议进行的研究 将从开发[35S]GTPgS放射自显影的实验开始 附加的受体系统。然后将进行研究，以检查 长期服用大麻素或可卡因对受体的影响 G蛋白的激活。最初，研究将检查大麻素和 多巴胺刺激的[35S]GTPgS结合，然而，后续研究将 也要检查其他受体系统。第三系列实验 将使用一种新的解剖学方法，其中特定区域被损害， 然后进行[35S]GTPgS放射自显影以1)识别来源 核内的传入神经和2)决定这些神经元上的受体是否 纤维是突触前或突触后的。这些研究将集中在ORL1和 大麻素系统，因为几乎没有解剖学信息 关于这些系统。这个项目将提供一个极好的机会 西姆博士利用她的神经解剖学训练，同时发展 在信号转导和药理学领域的培训。西姆博士将 在奇尔德斯博士的指导下进行这项研究，奇尔德斯博士是 药物信号转导机制研究人员 虐待。此外，还与以下组织成员建立了合作关系 药物滥用神经科学研究中心，教职员工专门从事 行为学、药理学、生理学和分子生物学。这个项目将 允许辛博士发展成为受过培训的独立调查员 解剖学、药理学和信号转导。

DESCRIPTION: (Applicant's Abstract) A novel technique has been developed using [35S]GTPgS autoradiography to localize receptor-activated G-proteins. This technique is based upon previous studies using agonist-stimulated [35S]GTPgS binding, in the presence of excess GDP, to measure receptor-activated G-proteins in membranes. The advantage of [35S]GTPgS autoradiography over other techniques is the ability to localize functionally active G-proteins. The technique has been used to demonstrate G-proteins activated by several receptors: opioid, cannabinoid and GABA-B. [35S]GTPgS autoradiography has also been used to identify ORL1 peptide-stimulated [35S]GTPg binding. This demonstrates another advantage of the technique; since [35S]GTPgS is the radioligand used, any unlabeled agonist can be used in this technique. [35S]GTPgS autoradiography has also been used to examine G-protein activity following chronic drug treatment. In these studies, chronic morphine treatment decreased mu opioid-stimulated [35S]GTPgS binding in specific brainstem nuclei: the dorsal raphe nucleus, locus, coeruleus, parabrachial nucleus and commissural nucleus tractus solitarius. The proposed studies will begin with experiments to develop [35S]GTPgS autoradiography for additional receptor systems. Studies will then be performed to examine the effects of chronic cannabinoid or cocaine administration on receptor activation of G-proteins. Initially, studies will examine cannabinoid and dopamine-stimulated [35S]GTPgS binding, however, subsequent studies will examine other receptor systems as well. The third series of experiments will use a novel anatomical approach in which specific regions are lesioned, then [35S]GTPgS autoradiography is performed to 1) identify the origin of afferents to the nucleus and 2) determine whether the receptors on those fibers are pre- or post-synaptic. These studies will focus on the ORL1 and cannabinoid systems, since little anatomical information is available regarding these systems. This project will provide an excellent opportunity for Dr. Sim to utilize her neuroanatomical training, while developing training in the areas of signal transduction and pharmacology. Dr. Sim will conduct this research under the mentorship of Dr. Childers, an established investigator in the field of signal transduction mechanisms of drug of abuse. In addition collaborations have been established with members of the Neuroscience of Drug Abuse Research Center, with faculty specializing in behavior, pharmacology, physiology and molecular biology. This project will allow Dr. Sim to develop into an independent investigator trained in anatomy, pharmacology and signal transduction.