Multiphoton detection of dopamine and drug release

多巴胺和药物释放的多光子检测

• 批准号: 8780111
• 负责人: EDWIN S LEVITAN
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780111
• 关键词: Affect; Amphetamines; Animal Model; Animals; Anti-Anxiety Agents; Antipsychotic Agents; Brain; Clinical; Cognition; Complex; Corpus striatum structure; DRD2 gene; Dendrites; Detection; Development; Dopamine; Dopamine Antagonists; Dopaminergic Agents; Dose; Drug Delivery Systems; Drug effect disorder; Exocytosis; Feasibility Studies; Fluorescence; Image; Life; Light Microscope; Mediating; Microscopy; Midbrain structure; Moods; Mus; Neurons; Optics; Parkinson Disease; Pharmaceutical Preparations; Pharmacology; Photochemistry; Property; Protocols documentation; Reporting; Rodent; Serotonin; Signal Transduction; Site; Slice; Specificity; Substantia nigra structure; Synapses; Testing; Tryptamines; Vesicle; addiction; atypical antipsychotic; base; brain tissue; clinical efficacy; clinically relevant; dopamine transporter; dopaminergic neuron; dorsal raphe nucleus; drug of abuse; follow-up; insight; monoamine; neuronal cell body; novel; novel strategies; pars compacta; patch clamp; public health relevance; receptor; research study; response; tool

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.

描述（由申请人提供）：多巴胺在帕金森病以及滥用药物（例如安非他明）和临床精神药物（例如抗精神病药物）的作用中很重要。由于光显微镜无法直接在活体神经元中看到多巴胺，因此很难定位和量化活体脑组织中胞体、树突和末梢中 Ca2+ 依赖性囊泡和安非他明诱导的非囊泡多巴胺释放。同样，药物在单胺囊泡中积累和释放的假设尚未得到检验。抗精神病药物与其目标递质的这种共释放将导致在需要药物作用的时间和地点（即，在活跃的多巴胺和血清素突触）集中药物输送，从而产生更大的功效和特异性。为了能够可视化活体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切片中的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胺嗪在多光子激发下产生可见荧光。中脑切片的多光子成像表明氰尿嗪会受到酸性捕获和 Ca2+ 依赖性释放。其次，多光子显微镜检测到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该信号的消耗。同样，去极化诱导 Ca2+ 依赖性消耗。这些结果支持了固有多光子自发荧光源自多巴胺的假设。 该提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对揭示内容和释放的多巴胺衍生信号进行成像。然后假设抗精神病药物与其目标单胺递质（即， 多巴胺和血清素）进行了测试。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，确定抗焦虑抗精神病药物是否与血清素和多巴胺共同释放对于建立精神科药物作用的新范例非常重要。