PSYCHOSTIMULANT ACTIONS /BIOGENIC AMINE RECEPTORS /TRANS

精神兴奋剂，包括可卡因以及本中心开发的新型可卡因类似物，通过阻断转运体介导的生物胺（多巴胺，5-羟色胺和去甲肾上腺素）的再摄取而起作用。本项目利用新型的莨菪碱和哌甲酯类似物，以及其他中心项目开发的特定动物模型，研究转运体结合位点，并检查慢性药物治疗对g蛋白偶联受体的影响。该项目的第一个目标是利用该中心开发的动物模型来研究精神兴奋剂（可卡因和新型可卡因类似物）的慢性治疗如何影响大脑中生物胺受体与g蛋白的偶联。这些研究将利用[35S]GTPgammaS放射自显影技术，提供g蛋白受体激活的神经解剖学定位。由于该技术允许在同一动物的大脑切片中同时检测许多不同的受体，因此它是有效分析来自中心来源的动物脑组织的理想方法。本项目将继续先前的研究，表明慢性阿片治疗在特定脑干核中选择性地衰减mu阿片激活的g蛋白。将采用三种不同的药物治疗模式：一种是使用强力的莨菪碱类似物WF-23的慢性治疗，一种是Roberts博士在子项目0011中开发的可卡因暴饮模型中的慢性自我给药，另一种是由Dr. Roberts博士开发的快速球（海洛因/可卡因组合）自我给药模型。史密斯和马丁在子项目0005。第二个目标将表征新型不可逆可卡因类似物（包括tropanes和methylphenidates）作为多巴胺转运体结构和功能探针的特性。由于这些化合物将用于其他中心项目的行为研究，我们的实验室将首先在体外和体内表征这些类似物的基本不可逆结合特性，使用膜中的转运体放射配体结合和放射自显影。最有效的类似物将被标记为[1251]，通过SDS-PAGE和从转染了多巴胺转运蛋白的细胞中分离的[1251]标记的色氨酸片段的纯化来检测tropanes和methylphenidates之间结合的差异。

Psychostimulants, including cocaine as well as novel cocaine analogs developed by this Center, act by blocking transporter-mediated reuptake of biogenic amines (dopamine, 5-HT and norepinephrine). This project utilizes novel tropane and methylphenidate analogs, along with specific animals models developed by other Center projects, to study transporter binding sites, and to examine the consequences of chronic drug treatment on G-protein-coupled receptors. The first aim of this project utilizes animal models developed by the Center to examine how chronic treatment with psychostimulants (both cocaine and novel cocaine analogs) affect coupling of biogenic amine receptors to G-proteins in brain. These studies will utilize [35S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for an number of different receptors to be assayed simultaneously in brain sections from the same animal, it is an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies showing that chronic opioid treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. Three different drug treatment paradigms will be employed: a chronic treatment with the highly potent tropane analog WF-23, chronic self-administration in a binge model of cocaine developed by Dr. Roberts in subproject 0011, and a speedball (heroin/cocaine combination) self-administration model developed by Drs. Smith and Martin in subproject 0005. The second aim will characterize the properties of novel irreversible cocaine analogs (both tropanes and methylphenidates) as probes of dopamine transporter structure and function. Since these compounds will be used in behavioral studies by other Center projects, our laboratory will first characterize the basic irreversible binding properties of these analogs both in vitro and in vivo, using transporter radioligand binding in membranes and autoradiography. The most potent analogs will be labeled with [1251] and differences in binding between tropanes and methylphenidates will be examined by SDS-PAGE and purification of [1251]-labeled tryptic peptide fragments isolated from cells transfected with dopamine transporters.