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: 8302583
• 负责人: Susan L Ingram
• 金额: $ 24.54万
• 依托单位: OREGON HEALTH & SCIENCE 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/8302583
• 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(申请人提供)：持续使用精神刺激剂，如苯丙胺和可卡因，会导致上瘾，并与强烈的渴望和持续的药物寻找行为有关。精神刺激剂诱导成瘾的部分机制是通过增加大脑奖赏通路中DA神经元的活性，其滥用潜能与其与多巴胺转运体(DAT)的亲和力密切相关。虽然DAT在将细胞外DA转运回神经末梢中起到了被证实的作用，但新的数据表明，DAT还能门控一种增加DA神经元兴奋性的内在氯(Cl-)通道。到目前为止，这种DAT介导的氯离子通道的生理作用尚不清楚，但该电流可能潜在地调节精神刺激性药物成瘾。这项研究首次使用电生理学和荧光生物传感器(YFPQS)成像来表征中脑DA神经元树突中的这一通道，该传感器对细胞内氯浓度([Cl-INT])的变化很敏感。我们将验证这样一种假设，即激活DAT会增加DA神经元的[Cl-int]，并且[Cl-int]的变化会增加DA神经元的放电频率。我们还将确定与DAT转运DA相关的氯通道激活和氯离子通量之间的关系。最后，我们将验证DAT介导的氯电流调制GABA和谷氨酸介导的DA神经元突触电流幅度的假设。这些研究将为研究这种固有的DATCl-通道在调节多巴胺能神经元奖赏通路活动中的特殊作用(S)提供重要的信息。转运蛋白介导的电导代表了心理刺激作用的细胞机制的新的和未被探索的途径，这些研究也可能有助于确定治疗药物成瘾的新靶点。通过多巴胺转运体(DAT)的相互作用，精神刺激剂成瘾与大脑奖赏通路中DA信号的增加有关。最近的证据表明，DAT除了运输DA外，还激活一种内在的氯离子通道。在这项提议中的实验将使用同时的电生理学和荧光成像来确定这种氯通道在调节中脑奖励通路中DA神经元的活动中的生理作用。