Dendritic DAT activity monitored with fluorescent biosensors

描述（由申请人提供）：持续使用精神兴奋剂，如安非他明和可卡因，会导致成瘾，这种成瘾与强烈的渴望和持续的药物寻求行为有关。精神兴奋剂诱导成瘾部分是通过增加大脑奖赏通路中多巴胺神经元的活性，其滥用潜力与它们对多巴胺转运体（DAT）的亲和力密切相关。虽然DAT在将细胞外的DA转运回神经末梢中起着确定的作用，但新的数据表明，DAT也开启了一个内在的氯离子（Cl-）通道，增加了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，但电流可能潜在地调节精神兴奋剂药物成瘾。本研究首次利用对细胞内Cl-浓度（[Cl-int]）变化敏感的荧光生物传感器（YFPQS）的电生理学和成像来表征中脑DA神经元树突中的这一通道。我们将验证DAT的激活会增加DA神经元中的[Cl-int]，而[Cl-int]的变化会增加DA神经元的放电速率。我们还将确定Cl-通道的激活与通过DAT运输DA相关的Cl-通量之间的关系。最后，我们将验证dat介导的Cl-电流调节GABA和谷氨酸介导的DA神经元突触电流振幅的假设。这些研究将提供重要的信息，说明这种内在的DAT Cl-通道在调节DA神经元在奖赏通路中的活动中的具体作用。转运体介导的传导为精神兴奋剂作用的细胞机制提供了新的和未被探索的途径，这些研究也可能有助于确定药物成瘾治疗的新靶点。通过多巴胺转运体（DAT）的相互作用，精神兴奋剂成瘾与大脑奖赏通路中多巴胺信号的增加有关。最近的证据表明，除了运输DA外，DAT还激活了一个固有的氯离子通道。本实验将同时使用电生理学和荧光成像来确定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。

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.