THE ROLE OF GLIAL MONOAMINE TRANSPORTERS IN COCAINE-INDUCED SENSITIZATION

胶质单胺转运蛋白在可卡因引起的致敏中的作用

• 批准号: 8357106
• 负责人: MIKHAIL INYUSHIN
• 金额: $ 5.53万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8357106
• 关键词: Affinity; Animals; Astrocytes; Binding; Biomedical Research; Brain; Cocaine; Cocaine Abuse; Data; Development; Dopamine; Excision; Funding; Grant; Image; Measures; Mediating; Metabolism; National Center for Research Resources; Neuroglia; Neurons; Organic Cation Transporter; Pharmaceutical Preparations; Principal Investigator; Process; Proteins; Rattus; Reporting; Research; Research Infrastructure; Resources; Rewards; Role; Slice; Source; Synapses; Testing; Toxin; United States; United States National Institutes of Health; Western Blotting; analog; behavioral sensitization; behavioral tolerance; cost; fluorocitrate; monoamine; research study; response; reuptake; uptake

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 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单胺转运蛋白结合而抑制多巴胺的再摄取，从而损害了从突触中去除多巴胺。 这些增加的多巴胺水平与可卡因滥用者报告的奖励作用有关。在大脑中，神经胶质细胞围绕神经元过程和突触，并具有多种类型的单胺转运蛋白，包括未被可卡因阻止的有机阳离子转运蛋白（OCT）。 本提案的一般假设是，通过重复给药可卡因通过可卡因的重复激活星形胶质细胞可增强其OCT介导的单胺摄取，从而降低了多巴胺水平，以应对药物挑战并导致对可卡因的耐受性的发展。 该假设将在以下两个具体目标中进行检验： 具体目的1：确定星形胶质细胞在行为敏化和对可卡因耐受性的发展中的作用。我们的初步数据表明，选择性神经胶质毒素氟氯酸酯对神经胶质代谢的抑制可降低对可卡因的耐受性。我们将确定这种作用是否是由于星形胶质细胞中电源性转运蛋白对单胺摄取的抑制。 具体目的2：确定重复可卡因和单胺给药对培养的星形胶质细胞和大鼠脑中OCT转运蛋白的蛋白质水平的影响。 我们将确定单胺的水平升高，尤其是多巴胺的水平是否上调了OCT转运蛋白在星形胶质细胞上的表达，从而导致对可卡因的行为耐受性明显。 在目前的电生理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（即直接效应）对OCT电流和蛋白质水平的影响，在大鼠切片中大脑切片变得敏感或耐受可卡因后。 具体目的3：确定可卡因反复施用对荧光单胺类似物4-（4-（二甲基氨基）-Styryl） - n-甲基吡啶的影响； （ASP+）。使用荧光成像研究ASP+的积累，ASP+的荧光底物在急性分离的星形胶质细胞中，净和OCT的荧光底物，我们将测量来自对照和可卡因治疗的动物的急性分离的星形胶质细胞中ASP+的积累。