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，并伴有强烈的渴望和持续的寻药行为。精神兴奋剂通过增加大脑奖赏通路中DA神经元的活性部分地诱导成瘾，并且它们的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DA转运回神经末梢中具有确认的作用，但新的数据表明DAT还门控增加DA神经元兴奋性的内在氯（Cl-）通道。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。该提案中的研究首次使用对细胞内Cl-浓度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神经元树突中的该通道（[Cl-int]）。我们将检验这样的假设：DAT的激活增加了DA神经元中的[Cl-int]，并且[Cl-int]的变化增加了DA神经元的放电率。我们还将确定激活的Cl-通道和Cl-流量与DA的运输DAT之间的关系。最后，我们将测试的假设，DAT介导的Cl-电流调制的GABA和谷氨酸介导的DA神经元的突触电流的幅度。这些研究将提供关于这种内在DAT Cl-通道在调节DA神经元在奖赏通路中的活性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兴奋剂作用的细胞机制的新的和未探索的途径，这些研究也可以用于确定药物成瘾治疗的新靶点。精神兴奋剂成瘾与通过多巴胺转运蛋白（DAT）相互作用的大脑奖赏通路中DA信号的增加有关。最近的证据表明，DAT激活一个内在的氯离子通道，除了运输DA。在这个建议中的实验将使用同时电生理和荧光成像，以确定这个氯离子通道的生理作用，在调节活动的DA神经元中脑奖励途径。

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.