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）：持续使用安非他明和可卡因等精神兴奋剂会导致成瘾，并伴有强烈的渴望和持续的寻求药物行为。精神兴奋剂部分通过增加大脑奖赏通路中 DA 神经元的活性来诱发成瘾，其滥用潜力与其对多巴胺转运蛋白 (DAT) 的亲和力密切相关。尽管 DAT 在将细胞外 DA 转运回神经末梢方面具有明确的作用，但新数据表明，DAT 还门控内在氯 (Cl-) 通道，从而增加 DA 神经元的兴奋性。迄今为止，这种 DAT 介导的 Cl 通道的生理作用尚不清楚，但电流可能会调节精神兴奋剂药物成瘾。本提案中的研究首次利用电生理学和对细胞内 Cl- 浓度 ([Cl-int]) 变化敏感的荧光生物传感器 (YFPQS) 成像来表征中脑 DA 神经元树突中的该通道。我们将测试以下假设：DAT 的激活会增加 DA 神经元中的 [Cl-int]，并且 [Cl-int] 的变化会增加 DA 神经元的放电率。我们还将确定 Cl- 通道的激活和与 DAT 运输 DA 相关的 Cl- 通量之间的关系。最后，我们将检验 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的假设。这些研究将提供有关这种内在 DAT Cl 通道在奖赏通路中调节 DA 神经元活动中的具体作用的重要信息。转运蛋白介导的电导代表了精神兴奋剂作用细胞机制的新颖且未经探索的途径，这些研究也可能有助于确定治疗药物成瘾的新靶点。精神兴奋剂成瘾与大脑奖赏通路中通过多巴胺转运蛋白 (DAT) 相互作用而增加的 DA 信号有关。最近的证据表明，DAT 除了运输 DA 之外，还激活内在的氯离子通道。该提案中的实验将使用同时电生理学和荧光成像来确定该氯离子通道在调节中脑奖励通路中 DA 神经元活动中的生理作用。