Multiphoton detection of dopamine and drug release

描述（由申请人提供）：多巴胺在帕金森病以及滥用药物（如安非他明）和临床精神病药物（如抗精神病药）的作用中很重要。因为多巴胺不能直接在活的神经元中用光学显微镜观察到，所以很难定位和定量从活的脑组织中的索马、树突和终末的Ca 2+依赖性囊泡和苯丙胺诱导的非囊泡多巴胺释放。同样，药物在单胺囊泡中积累并从单胺囊泡中释放的假设也没有得到验证。抗精神病药物与其靶向递质的这种共释放将导致在需要药物作用的时间和位置的集中药物递送（即，在活跃的多巴胺和5-羟色胺突触）导致更大的功效和特异性。为了能够在活体神经元中可视化多巴胺和药物动力学，我们一直在开发基于啮齿动物脑切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氰氨苯哒嗪在多光子激发下产生可见荧光。中脑切片的多光子成像显示，氰美津受到酸性捕获和Ca 2+依赖性释放。第二，多光子显微镜检测黑质多巴胺神经元的自发荧光。安非他明诱导多巴胺转运蛋白介导的这种信号的耗竭。同样，去极化诱导Ca 2+依赖性耗尽。这些结果支持了内源性多光子自发荧光来源于多巴胺的假设。 该提案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多巴胺衍生的信号，以揭示内容和释放。然后假设存在抗精神病药物与其靶向单胺递质的共定位释放（即， 多巴胺和血清素）进行测试。这些实验将探索新的光学方法来研究活体脑组织中的多巴胺和药物。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释放对于建立精神药物作用的新范式将是重要的。

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.