THE ROLE OF GLIAL MONOAMINE TRANSPORTERS IN COCAINE-INDUCED SENSITIZATION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 在美国，有150万人被归类为依赖或滥用可卡因。可卡因通过与高亲和力的单胺转运体结合来抑制多巴胺的再摄取，从而损害从突触中移除多巴胺。这些增加的多巴胺水平与可卡因滥用者报告的奖励效应有关。在大脑中，神经胶质细胞围绕着神经元突起和突触，具有多种类型的单胺转运体，包括不被可卡因阻断的有机阳离子转运体(OCT)。本研究的一般假设是，反复给予可卡因激活星形胶质细胞可增强其OCT介导的单胺摄取，从而降低多巴胺水平以应对药物挑战，并导致对可卡因的耐受性的形成。这一假设将在以下两个具体目标中得到检验： 具体目的1：确定星形胶质细胞在行为敏感化和对可卡因耐受性发展中的作用。我们的初步数据表明，选择性胶质毒素氟柠檬酸盐对胶质代谢的抑制降低了对可卡因的耐受性。我们将确定这种效应是否是由于星形胶质细胞中产生电的转运体抑制了单胺的摄取。 特定目的2：研究重复给予可卡因和单胺对培养的星形胶质细胞和大鼠脑内OCT转运体电流和蛋白水平的影响。我们将确定单胺水平的升高，特别是多巴胺，是否会上调星形胶质细胞上OCT转运蛋白的表达，从而导致对可卡因的明显行为耐受。在目前的电生理和蛋白质印迹实验中，我们将评估单胺类物质对培养的星形胶质细胞(即直接作用)和大鼠对可卡因敏感或耐受后脑片OCT产生电流和蛋白质水平的影响。 具体目的3：确定重复给予可卡因对星形胶质细胞内荧光单胺类似物4-(4-(dimethylamino)-styryl)-N-methylpyridinium；(天冬氨酸+)积聚的影响。利用荧光成像研究DAT、NET和OCT的荧光底物ASP+在急性分离的星形胶质细胞中的积累，我们将测量对照和可卡因处理的急性分离的星形胶质细胞中ASP+的积累。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In the United States, there are 1.5 million people classified as dependent on or abusing cocaine. Cocaine inhibits dopamine reuptake by binding to high-affinity monoamine transporters thereby impairing removal of dopamine from the synapse. These increased dopamine levels have been associated with the rewarding effect reported by cocaine abusers. In the brain, glial cells surround neuronal processes and synapses and have multiple types of monoamine transporters, including the organic cation transporters (OCT) that are not blocked by cocaine. The general hypothesis of the present proposal is that activation of astrocytes by repeated administration of cocaine enhances their OCT- mediated monoamine uptake thus decreasing dopamine levels in response to a drug challenge and leading to the development of tolerance to cocaine. This hypothesis will be tested in the following two specific aims: Specific Aim 1: To determine the role of astrocytes in the development of behavioral sensitization and tolerance to cocaine. Our preliminary data suggest that inhibition of glial metabolism by the selective glial toxin fluorocitrate reduces tolerance to cocaine. We will determine if this effect is due to inhibition of monoamine uptake by the electrogenic transporters in astrocytes. Specific Aim 2: To determine the effect of repeated cocaine and monoamine administration on transporter currents and the protein levels of OCT transporters in cultured astrocytes and rat brain. We will determine if elevated levels of monoamines, particularly dopamine, up-regulate expression of the OCT transporters on astrocytes resulting in apparent behavioral tolerance to cocaine. In the present electrophysiological and western blot experiments, we will assess the effects of monoamines on OCT electrogenic currents and protein levels in both cultured astrocytes (i.e., a direct effect) and in brain slices after rats have become sensitized or tolerant to cocaine. Specific Aim 3: To determine the effects of repeated administration of cocaine on the accumulation in astrocytes of the fluorescent monoamine analog 4-(4-(dimethylamino)-styryl)-N-methylpyridinium; (ASP+). Using fluorescent imaging to study the accumulation of ASP+, a fluorescent substrate for DAT, NET and OCT in acutely separated astrocytes, we will measure the accumulation of ASP+ in acutely isolated astrocytes from control and cocaine treated animals.