Multiphoton detection of dopamine and drug release

描述(申请人提供)：多巴胺对帕金森氏症、滥用药物(如安非他明)和临床精神药物(如抗精神病药物)的作用很重要。由于在光镜下不能直接在活神经元中看到多巴胺，因此很难定位和定量钙依赖的囊泡性和苯丙胺诱导的活体脑组织胞体、树突和终末的非囊泡性多巴胺释放。同样，药物在单胺囊泡中积累和释放的假说也没有得到检验。这种抗精神病药物与其靶向递质的共同释放将导致在需要药物作用的时间和地点(即在活跃的多巴胺和5-羟色胺突触)集中给药，从而产生更高的疗效和特异性。为了能够可视化活神经元中的多巴胺和药物动力学，我们一直在开发基于啮齿动物脑片多光子显微镜的新实验方法。首先，我们发现临床上使用的抗焦虑抗精神病药物胞嘧啶在多光子激发下产生可见的荧光。中脑脑片的多光子成像显示，胞嘧啶受酸性捕获和钙依赖性释放的影响。第二，多光子显微镜检测到黑质多巴胺神经元的自发荧光。苯丙胺诱导多巴胺转运体介导的这种信号的耗竭。同样，去极化导致钙依赖的耗竭。这些结果支持本征多光子自发荧光来源于多巴胺的假说。这项建议建立在这些初步结果的基础上，首先确定大脑切片中的多光子显微镜是否可以成像揭示内容和释放的多巴胺衍生信号。则假设存在抗精神病药物与其目标单胺传递体的共局地释放(即， 多巴胺和5-羟色胺)进行测试。这些实验将探索研究活体脑组织中的多巴胺和药物的新的光学方法。此外，确定是否存在抗焦虑抗精神病药物与5-羟色胺和多巴胺的共同释放对于建立精神药物作用的新范例将是重要的。

DESCRIPTION (provided by applicant): Dopamine is important in Parkinson's disease and the action of abused drugs (e.g. amphetamines) and clinical psychiatric drugs (e.g. antipsychotics). Because dopamine cannot be seen directly in living neurons with the light microscope, it is difficult to localize and quantify Ca2+-dependent vesicular and amphetamine- induced nonvesicular dopamine release from the soma, dendrites and terminals in living brain tissue. Likewise, the hypothesis that drugs accumulate in and are released from monoamine vesicles has not been tested. Such corelease of antipsychotic drugs with their target transmitters would result in concentrated drug delivery when and where drug action is needed (i.e., at active dopamine and serotonin synapses) resulting in greater efficacy and specificity. To be able to visualize dopamine and drug dynamics in living neurons, we have been developing new experimental approaches based on multiphoton microscopy in the rodent brain slice. First, we found that the clinically used anxiolytic antipsychotic drug cyamemazine produces visible fluorescence upon multiphoton excitation. Multiphoton imaging in midbrain slices showed that cyamemazine is subject to acidic trapping and Ca2+-dependent release. Second, multiphoton microscopy detected autofluorescence in substantia nigra dopamine neurons. Amphetamine induced dopamine transporter- mediated depletion of this signal. Likewise, depolarization induced Ca2+-dependent depletion. These results support the hypothesis that intrinsic multiphoton autofluorescence is derived from dopamine. This proposal builds on these preliminary results to first determine whether multiphoton microscopy in the brain slice can image a dopamine-derived signal that reveals content and release. Then the hypothesis that there is colocalized release of an antipsychotic drug with its target monoamine transmitters (i.e., dopamine and serotonin) is tested. These experiments will explore new optical approaches for studying dopamine and drugs in living brain tissue. Furthermore, determining whether there is corelease of an anxiolytic antipsychotic drug with serotonin and dopamine would be important for establishing a new paradigm for psychiatric drug action.