Architecture and function of striatal dopamine release machinery

纹状体多巴胺释放机制的结构和功能

• 批准号: 9402528
• 负责人: Pascal Simon Kaeser
• 金额: $ 51.47万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9402528
• 关键词: Acute; Address; Anatomy; Appearance; Architecture; Axon; Brain; Brain Diseases; Cell Fractionation; Cognition; Corpus striatum structure; Data; Defect; Dependence; Diffuse; Disease; Docking; Dopamine; Dopamine Receptor; Drug Addiction; Electron Microscopy; Electrophysiology (science); Emotions; Excision; Exocytosis; Extracellular Space; Functional disorder; G-Protein-Coupled Receptors; Gene Targeting; Genetic; Glutamates; Goals; Grant; Impairment; Individual; Knock-out; Knockout Mice; Maps; Mediating; Membrane; Microscopy; Midbrain structure; Molecular; Molecular Target; Mood Disorders; Movement; Mus; Nerve Degeneration; Neuromodulator; Neurotransmitters; Parkinson Disease; Pathology; Pathway interactions; Protein Family; Proteins; Regulation; Role; SNAP receptor; Scaffolding Protein; Schizophrenia; Signal Transduction; Site; Slice; Speed; Structure; Substance abuse problem; Surface; Synapses; Synaptic Vesicles; Testing; Varicosity; Vesicle; cholinergic; density; dopaminergic neuron; experimental study; gamma-Aminobutyric Acid; nerve supply; neuroregulation; optogenetics; postsynaptic; presynaptic; protein structure; receptor; release factor; scaffold; secretory protein; sensor; transmission process

Summary Dopamine is an important neuromodulator and pathologies in dopamine signaling are a hallmark of brain diseases such as neurodegeneration, substance abuse, and schizophrenia. Despite these important roles for dopamine, remarkably little is known about the molecular mechanisms of its release. Because dopamine acts as a volume transmitter, it is not clear whether dopamine release involves molecular machinery that warrants spatial and temporal precision for release. Alternatively, dopamine release could be spread over the surface of an axon, which is consistent with volume transmission. The release of classical transmitters relies on an active zone, a highly organized protein structure that contains scaffolding proteins such as RIM and ELKS and determines the precise localization, speed and accuracy of synaptic vesicle exocytosis. The active zone also provides mechanisms for regulation of release during plasticity. Our preliminary experiments reveal that the presynaptic scaffolding protein RIM is absolutely required for dopamine release in the mouse striatum, but that ELKS is dispensable for dopamine release. This is different from classical fast synapses, where knockout of either protein family leads to a reduction of 50-80% of release. We thus hypothesize that dopamine release necessitates mechanistically specialized release sites. This hypothesis is bolstered by superresolution microscopy in striatal brain slices, which shows that several release site scaffolding proteins are clustered inside dopamine axons. We pursue a two-pronged approach to address this central hypothesis. In aim one, we use rigorous conditional mouse genetics and electrophysiology in acute brain slices of the mouse striatum to systematically address the necessity of scaffolding proteins, priming proteins and Ca2+ channel tethers in dopamine release and in co-release of GABA and glutamate from dopamine neurons. This is the first study on the requirements of molecular scaffolds for dopamine secretion and it will lead to a comprehensive assessment of the dopamine release machinery. In aim two, we assess whether scaffolding proteins mediate dopamine secretion as soluble release factors, or whether they are assembled in clustered release sites to target dopamine release to specific membrane domains. The latter possibility is strongly supported by our preliminary data. We will combine superresolution microscopy, subcellular fractionation, electron microscopy and mouse genetics to study the existence and composition of dopamine release sites in the mouse striatum. We will assess how dopamine release sites are associated with vesicle clusters, with receptors for dopamine and for the co-transmitters GABA and glutamate, and with cholinergic innervation, which powerfully triggers dopamine release. These experiments will establish the existence, appearance and composition of dopamine release sites and their structural arrangement into striatal synaptic microcircuits. Our approach is the first comprehensive approach to dissect the secretory pathway for dopamine. We expect to identify new mechanisms that support dopamine release and to uncover general principles for neuromodulation.

概括 多巴胺是重要的神经调节剂，多巴胺信号传导中的病理是大脑的标志 神经退行性，药物滥用和精神分裂症等疾病。尽管有这些重要的角色 多巴胺对其释放的分子机制知之甚少。因为多巴胺的作用 作为音量发射器，尚不清楚多巴胺释放是否涉及保证的分子机械 空间和时间精度用于释放。或者，多巴胺释放可以分布在 轴突，与体积传输一致。经典发射器的释放依赖于活动 区域，一种高度组织的蛋白质结构，包含脚手架蛋白，例如边缘和麋鹿，以及 确定突触囊泡胞吐作用的精确定位，速度和准确性。活动区也 提供了调节可塑性释放的机制。我们的初步实验表明 在小鼠纹状体中释放多巴胺是绝对必需的突触前脚手架蛋白边缘的 麋鹿可用于多巴胺释放。这与经典快速突触不同，其中淘汰 任何一种蛋白质家族都会降低释放的50-80％。因此，我们假设多巴胺释放 需要机械专业的释放位点。该假设通过超分辨率加强 纹状体脑切片中的显微镜，这表明几种释放位点脚手架蛋白是聚集的 内部多巴胺轴突。我们采用两种义务的方法来解决这一中心假设。在一个目标中，我们 在小鼠纹状体的急性脑切片中使用严格的条件小鼠遗传学和电生理学 系统地解决脚手架蛋白，启动蛋白和Ca2+通道系的必要性 多巴胺释放以及来自多巴胺神经元的GABA和谷氨酸的共释放。这是第一个研究 分子支架对多巴胺分泌的要求，这将导致全面评估 多巴胺释放机械。在目标二，我们评估脚手架蛋白是否介导多巴胺 分泌作为可溶性释放因素，还是将它们组装在聚类的释放位点中以靶向 多巴胺释放到特定的膜结构域。后一种可能性得到了我们的初步支持 数据。我们将结合分辨率显微镜，亚细胞分馏，电子显微镜和小鼠 研究小鼠纹状体中多巴胺释放位点的存在和组成的遗传学。我们将 评估多巴胺释放位点与囊泡簇如何与多巴胺的受体相关 共同发射器GABA和谷氨酸，并具有胆碱能神经，这有力地触发了多巴胺 发布。这些实验将确定多巴胺释放的存在，外观和组成 位点及其结构排列成纹状体突触微电路。我们的方法是第一个 剖析多巴胺分泌途径的综合方法。我们希望确定新的 支持多巴胺释放并揭示神经调节的一般原理的机制。