LOCALIZATION OF RECEPTOR ACITIVITY FOR DRUGS OF ABUSE

描述：（申请人摘要） 利用[~（35）S]GTPgS放射自显影技术， 定位受体激活的G蛋白。 这项技术是基于 以前的研究使用激动剂刺激的[35 S]GTPgS结合，在 过量GDP的存在，以测量受体激活的G蛋白， 膜。 [35 S]GTPgS放射自显影术优于其他放射自显影术 技术是定位功能活性G蛋白的能力。 的 技术已被用来证明G蛋白激活的几个 受体：阿片类、大麻素和GABA-B。 [35 S]GTPgS放射自显影具有 还用于鉴定ORL 1肽刺激的[35 S]GTPg结合。 这 证明了该技术的另一个优点;由于[35 S]GTPgS是 尽管使用了放射性配体，但是任何未标记的激动剂都可以用于该技术。 [35 S]GTPgS放射自显影也被用于检查G蛋白活性 慢性药物治疗后。 在这些研究中，慢性吗啡 治疗降低μ阿片刺激的[35 S]GTPgS结合， 脑干核团：中缝背核、斑、蓝斑、臂旁核 孤束核和连合核。 拟议的研究 将开始实验，开发[35 S]GTPgS放射自显影， 额外的受体系统。 然后将进行研究，以检查 慢性给予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[35 S]GTPgS结合，然而，随后的研究将 也检查其他受体系统。 第三组实验 将使用一种新的解剖学方法，在这种方法中，特定的区域被损伤， 然后进行[35 S]GTPgS放射自显影，以1）鉴定 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ORL 1和 大麻素系统，因为几乎没有解剖学信息可用 对于这些系统。 这个项目将提供一个极好的机会 沈博士利用她的神经解剖学训练， 信号传导和药理学领域的培训。 沈医生会 在Childers博士的指导下进行这项研究， 药物信号转导机制领域的研究者， 虐待 此外，还与联合国开发计划署成员建立了合作关系， 药物滥用神经科学研究中心，教师专门从事 行为学、药理学、生理学和分子生物学。 该项目将 允许沈博士发展成为一名独立的调查员， 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.