THE ROLE OF GLIAL MONOAMINE TRANSPORTERS IN COCAINE-INDUCED SENSITIZATION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 在美国，有150万人被列为依赖或滥用可卡因。 辅酶A通过与高亲和力单胺转运蛋白结合抑制多巴胺再摄取，从而损害多巴胺从突触的清除。 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，神经胶质细胞围绕神经元突起和突触，并具有多种类型的单胺转运蛋白，包括不被可卡因阻断的有机阳离子转运蛋白（OCT）。 本建议的一般假设是，通过重复施用可卡因激活星形胶质细胞增强了它们的OCT介导的单胺摄取，从而降低了响应于药物攻击的多巴胺水平，并导致对可卡因的耐受性的发展。 将在以下两个具体目标中检验这一假设： 具体目标1：确定星形胶质细胞在可卡因行为敏化和耐受发展中的作用。我们的初步数据表明，选择性神经胶质毒素氟柠檬酸盐抑制神经胶质代谢，降低耐受可卡因。我们将确定这种效应是否是由于星形胶质细胞中的生电转运蛋白抑制单胺摄取所致。 具体目标二：确定重复给予可卡因和单胺对培养的星形胶质细胞和大鼠脑中OCT转运体的转运体电流和蛋白水平的影响。 我们将确定是否单胺水平升高，特别是多巴胺，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。 在本电生理学和蛋白质印迹实验中，我们将评估单胺对两种培养的星形胶质细胞（即，一种直接的影响）和在老鼠对可卡因变得敏感或耐受后的脑切片中。 具体目标3：确定可卡因重复给药对荧光单胺类似物4-（4-（二甲氨基）-苯乙烯基）-N-甲基吡啶鎓（ASP+）在星形胶质细胞中蓄积的影响。使用荧光成像研究ASP+（DAT、NET和OCT的荧光底物）在急性分离的星形胶质细胞中的蓄积，我们将测量ASP+在对照和可卡因处理动物的急性分离的星形胶质细胞中的蓄积。

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.