Dendritic DAT Conductances in Psychostimulant Addiction

描述（由申请人提供）：精神兴奋剂，如安非他明和可卡因，会产生成瘾，与强烈的渴望和持续的药物寻求行为有关。精神兴奋剂的滥用潜力与它们对多巴胺转运体（DAT）的亲和力密切相关，但对成瘾的细胞机制的理解一直难以捉摸。人们普遍认为，DAT通过将释放的多巴胺转运回神经末梢来调节细胞外多巴胺（DA）。我们最近的数据表明，DAT的激活也开启了一个相关的氯离子（CI-）通道，可以调节DA神经元的兴奋性。安非他明和可卡因调节这种Cl-传导的活性，这表明精神兴奋剂的作用机制尚未被探索。我们建议通过结合一种新型Cl敏感荧光生物传感器的成像和同时全细胞膜片钳记录来研究dat介导的电流的生理相关性。这种创新的方法将使我们能够直接测量DA神经元树突中的DAT活动，并确定与DAT相关的Cl-通道的生理相关性。首先，我们提出验证Cl-调控发生在DA神经元树突内严格控制的局部环境中的假设。接下来，我们将确定dat介导的Cl-电导是否调节细胞内Cl-浓度。最后，我们将验证data介导的Cl-电导在调节兴奋性和黑质DA神经元树突突触活动整合中发挥重要作用的假设。这些研究首次探讨了dat介导的Cl-传导的生理作用，并可能显著改变我们对单胺转运体功能及其被精神兴奋剂调节的理解。转运体介导的传导为精神兴奋剂作用的细胞机制和药物成瘾新治疗的潜在靶点提供了新的和未探索的途径。

DESCRIPTION (provided by applicant): Psychostimulants, such as amphetamine and cocaine, produce addiction that is associated with strong cravings and persistent drug-seeking behaviors. The abuse potential of psychostimulants is closely correlated with their affinity for the dopamine transporter (DAT) but an understanding of the cellular mechanisms underlying addiction has been elusive. It is commonly accepted that the DAT regulates extracellular dopamine (DA) by transporting released DA back into nerve terminals. Our recent data indicates activation of DAT also gates an associated chloride (CI-) channel that can modulate the excitability of DA neurons. Amphetamine and cocaine regulate the activity of this Cl- conductance suggesting that psychostimulants have unexplored mechanisms of action. We propose to study the physiological relevance of the DAT-mediated current by combining imaging of a novel Cl- sensitive fluorescent biosensor with simultaneous whole-cell patch-clamp recordings. This innovative approach will allow us to directly measure DAT activity in dendrites of DA neurons and determine the physiological relevance of the Cl- channel associated with the DAT. First, we propose to test the hypothesis that Cl- regulation occurs in tightly controlled local environments within dendrites of DA neurons. Next, we will determine if the DAT-mediated Cl- conductance regulates intracellular Cl- concentrations. Finally, we will test the hypothesis that the DATmediated Cl- conductance plays an important role in the regulation of excitability and in the integration of synaptic activity in dendrites of substantia nigra DA neurons. These studies are the first to address the physiological role of the DAT-mediated Cl- conductance and might significantly alter our understanding of monoamine transporter function and their modulation by psychostimulants. Transporter-mediated conductances represent novel and unexplored avenues for cellular mechanisms of psychostimulant action and potential targets for novel treatments for drug addiction.