THE ROLE OF GLIAL MONOAMINE TRANSPORTERS IN COCAINE-INDUCED SENSITIZATION

神经胶质单胺转运蛋白在可卡因致敏和耐受性中的作用在美国，有150万人被归类为依赖或滥用可卡因。可卡因通过结合高亲和力的单胺转运体抑制多巴胺的再摄取，从而损害多巴胺从突触的移除。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，神经胶质细胞包围着神经元过程和突触，并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，通过反复给药可卡因激活星形胶质细胞，增强了OCT介导的单胺摄取，从而降低了多巴胺水平，从而导致对可卡因产生耐受性。这一假设将在以下两个具体目标中得到验证：具体目标1：确定星形胶质细胞在可卡因行为致敏和耐受性发展中的作用。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可降低对可卡因的耐受性。我们将确定这种效应是否由于星形胶质细胞中电致转运体对单胺摄取的抑制。目的2：研究可卡因和单胺反复给药对培养的星形胶质细胞和大鼠脑OCT转运体电流和蛋白水平的影响。我们将确定是否升高的单胺，特别是多巴胺，上调星形胶质细胞上OCT转运蛋白的表达，导致对可卡因的明显行为耐受。在当前的电生理和western blot实验中，我们将评估单胺对培养星形胶质细胞（即直接影响）和大鼠对可卡因敏感或耐受后脑切片中OCT电致电流和蛋白质水平的影响。特异性目的3：确定反复给药可卡因对荧光单胺类似物4-（4-（二甲胺）-苯乙烯）- nmethylpyridinium在星形胶质细胞中积累的影响；(ASP +)。利用荧光成像技术研究急性分离星形胶质细胞中ASP+（一种用于DAT、NET和OCT的荧光底物）的积累，我们将测量对照和可卡因治疗动物急性分离星形胶质细胞中ASP+的积累。

The Role of Glial Monoamine Transporters in Cocaine-Induced Sensitization and Tolerance 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-(dimethylaminp)-styryl)-Nmethylpyridinium; (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.