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 多巴胺是一种重要的神经调节剂，多巴胺信号传导的病理学是大脑的标志 神经退行性疾病、药物滥用和精神分裂症等疾病。尽管发挥了这些重要作用 多巴胺，人们对其释放的分子机制知之甚少。因为多巴胺的作用 作为容量传送器，目前尚不清楚多巴胺的释放是否涉及分子机制，从而保证 发布的空间和时间精度。或者，多巴胺的释放可以分布在整个表面 轴突，这与体积传输一致。经典发射机的释放依赖于主动 zone，一种高度组织化的蛋白质结构，包含 RIM 和 ELKS 等支架蛋白 决定突触小泡胞吐作用的精确定位、速度和准确性。活动区域也 提供可塑性过程中释放调节的机制。我们的初步实验表明 突触前支架蛋白 RIM 对于小鼠纹状体中多巴胺的释放是绝对必需的，但是 ELKS 对于多巴胺的释放来说是可有可无的。这与经典的快速突触不同，在经典的快速突触中， 任一蛋白质家族都会导致释放量减少 50-80%。因此我们假设多巴胺释放 需要机械上专门的发布站点。这个假设得到了超分辨率的支持 纹状体脑切片的显微镜检查，显示几个释放位点支架蛋白聚集在一起 多巴胺轴突内部。我们采取双管齐下的方法来解决这一中心假设。在目标一中，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 系统地解决了支架蛋白、启动蛋白和 Ca2+ 通道系链的必要性 多巴胺释放以及 GABA 和谷氨酸从多巴胺神经元的共同释放。这是第一个研究 多巴胺分泌的分子支架的要求，并将进行全面的评估 多巴胺释放机制。在目标二中，我们评估支架蛋白是否介导多巴胺 作为可溶性释放因子分泌，或者它们是否组装在聚集的释放位点以靶向 多巴胺释放到特定的膜域。后一种可能性得到了我们初步的有力支持 数据。我们将结合超分辨率显微镜、亚细胞分离、电子显微镜和小鼠 遗传学研究小鼠纹状体中多巴胺释放位点的存在和组成。我们将 评估多巴胺释放位点如何与囊泡簇、多巴胺受体以及 共同递质 GABA 和谷氨酸，并具有胆碱能神经支配，可强烈触发多巴胺 发布。这些实验将确定多巴胺释放的存在、外观和成分 位点及其在纹状体突触微电路中的结构排列。我们的方法是第一个 剖析多巴胺分泌途径的综合方法。我们期望发现新的 支持多巴胺释放的机制并揭示神经调节的一般原理。