Dendritic DAT activity monitored with fluorescent biosensors

使用荧光生物传感器监测树突状 DAT 活性

• 批准号: 7795252
• 负责人: Susan L Ingram
• 金额: $ 31.09万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7795252
• 关键词: Affinity; Amphetamines; Axon; Back; Baculoviruses; Binding; Biosensor; Cells; Chloride Channels; Chloride Ion; Chlorides; Cocaine; Coupled; Data; Dendrites; Drug Addiction; Electrophysiology (science); Glutamates; Image; Intake; Ion Channel; Ions; Measures; Mediating; Midbrain structure; Monitor; Movement; Nerve; Neurons; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Psychostimulant dependence; Regulation; Relative (related person); Rewards; Role; Signal Transduction; Slice; Spinal Cord; Synapses; Testing; Time; Transfection; Work; addiction; brain pathway; craving; dopamine transporter; dopaminergic neuron; drug seeking behavior; extracellular; fluorescence imaging; gamma-Aminobutyric Acid; in vivo; inhibitor/antagonist; neuronal cell body; novel; patch clamp; psychostimulant; research study; response; voltage clamp

DESCRIPTION (provided by applicant): The continued use of psychostimulants, such as amphetamine and cocaine results in addiction that is associated with strong cravings and persistent drug-seeking behaviors. Psychostimulants induce addiction in part by increasing activity of DA neurons in reward pathways of the brain and their abuse potential is closely correlated with their affinity for the dopamine transporter (DAT). Although the DAT has a confirmed role in transporting extracellular DA back into nerve terminals, new data demonstrates that DAT also gates an intrinsic chloride (Cl-) channel that increases excitability of DA neurons. To date the physiological role of this DAT-mediated Cl- channel is not known, but the current could potentially modulate psychostimulant drug addiction. The studies in this proposal are the first to characterize this channel in dendrites of midbrain DA neurons using electrophysiology and imaging of a fluorescent biosensor (YFPQS) that is sensitive to changes in intracellular Cl- concentrations ([Cl-int]). We will test the hypothesis that activation of the DAT increases [Cl-int] in DA neurons and that the change in [Cl-int] increases the firing rate of DA neurons. We will also determine the relationship between activation of the Cl- channel and Cl- flux associated with the transport of DA by DAT. Finally, we will test the hypothesis that the DAT-mediated Cl- current modulates the amplitudes of GABA and glutamate-mediated synaptic currents on DA neurons. These studies will provide important information on the specific role(s) of this intrinsic DAT Cl- channel in modulating the activity of DA neurons in reward pathways. Transporter-mediated conductances represent novel and unexplored avenues for cellular mechanisms of psychostimulant action and these studies may also serve to identify a novel target for the treatment of drug addiction. Psychostimulant addiction is associated with an increase in DA signaling in reward pathways of the brain via interactions at the dopamine transporter (DAT). Recent evidence shows that DAT activates an intrinsic chloride channel in addition to transporting DA. Experiments in this proposal will use simultaneous electrophysiology and fluorescence imaging to determine the physiological role of this chloride channel in modulating activity of DA neurons in midbrain reward pathways.

描述（由申请人提供）：继续使用心理刺激剂，例如苯丙胺和可卡因会导致成瘾，这与强烈的渴望和持续的寻求毒品行为有关。精神刺激因素部分通过增加大脑奖励途径的DA神经元的活性而引起成瘾，其滥用潜力与它们对多巴胺转运蛋白（DAT）的亲和力密切相关。尽管DAT在将细胞外DA转移到神经终端中具有确认的作用，但新数据表明，DAT还闸门固有的氯化物（Cl-）通道，可提高DA神经元的兴奋性。迄今为止，该DAT介导的CL-通道的生理作用尚不清楚，但是电流可能会调节精神刺激药物成瘾。该提案中的研究是第一个使用电生理学和荧光生物传感器（YFPQS）成像的中脑DA神经元树突中表征该通道的研究，该图对细胞内CL-浓度的变化敏感（[CL-INT]）。我们将测试以下假设：DA神经元中DAT的激活增加[Cl-Int]，并且[Cl-Int]的变化会增加DA神经元的发射速率。我们还将确定与DAT的传输相关的Cl-通道的激活与Cl-Flux的激活之间的关系。最后，我们将检验以下假设：DAT介导的CL-电流调节DA神经元上GABA和谷氨酸介导的突触电流的幅度。这些研究将提供有关该内在DAT通道在调节奖励途径中DA神经元活动中的特定作用的重要信息。转运蛋白介导的电导代表了精神刺激作用的细胞机制的新颖和未开发的途径，这些研究也可能有助于确定治疗药物成瘾的新目标。精神刺激性成瘾与通过多巴胺转运蛋白（DAT）的相互作用（DAT）的相互作用在大脑奖励途径中的DA信号增加有关。最近的证据表明，除了运输DA之外，DAT还激活了固有的氯化物通道。该建议中的实验将使用同时的电生理学和荧光成像来确定该氯化物通道在调节中脑奖励途径中DA神经元活性中的生理作用。