Multiphoton detection of dopamine and drug release

描述（由申请人提供）：多巴胺在帕金森病和滥用药物（如安非他明）和临床精神药物（如抗精神病药）的作用中很重要。由于在光镜下不能直接在活的神经元中观察到多巴胺，因此很难定位和量化活脑组织中Ca2+依赖性的囊泡性和安非他命诱导的非囊泡性多巴胺释放。同样，药物在单胺类囊泡中积聚和从单胺类囊泡中释放的假设也没有得到验证。这种抗精神病药物与其目标递质的共同释放将导致药物在需要药物作用的时间和地点（即活跃的多巴胺和血清素突触）集中递送，从而产生更大的功效和特异性。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大脑切片的多光子显微镜的新实验方法。首先，我们发现临床使用的抗焦虑抗精神病药物氰胺嗪在多光子激发下产生可见荧光。中脑切片多光子成像显示，氰胺嗪受酸性捕获和Ca2+依赖性释放的影响。其次，多光子显微镜检测黑质多巴胺神经元的自身荧光。安非他明诱导多巴胺转运体介导的该信号耗竭。同样，去极化诱导Ca2+依赖性耗竭。这些结果支持了内在多光子自身荧光来源于多巴胺的假设。该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多巴胺含量和释放的多巴胺衍生信号。然后假设抗精神病药物与目标单胺递质(即，

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.