A Fast Proteometric Synapse Census Platform

描述(申请人提供)：哺乳动物中枢神经系统(CNS)的突触在分子和功能特性上都非常不同。遗憾的是，目前我们对这种多样性的了解还很初级，有关这一主题的量化数据很少。中枢神经系统突触的多样性对更好地了解大脑突触电路的发育、功能和紊乱构成了巨大的障碍。这种令人痛苦的无知状态持续存在的主要原因在于，在单个突触水平上探索突触群体的工具很少，而且能力有限。为了应对突触多样性给基础神经科学和临床神经科学带来的挑战，本项目旨在开发一个最先进的新的蛋白质计量学成像平台，能够以单一突触分辨率原位分析非常大的突触群体。该平台的部署和推广将有助于研究和治疗与特定突触亚群相关的许多神经发育、精神和神经退行性疾病，并为研究中枢神经系统记忆编码、存储和提取的分子机制、电路结构和障碍开辟新的视角。该平台将基于免疫荧光阵列断层扫描(IAT)，并涉及开发新的抗体标准化协议和新的图像采集硬件和软件。这些创新将大幅提高IAT的重复性、定量可靠性和速度，并克服迄今阻碍在单个突触水平上对大量突触群体进行蛋白质计量学分析的限制。该平台将能够对每小时100万个以上的突触进行蛋白质统计，每个突触有50个或更多的标记，同时保持每个突触的精确神经解剖学和分子坐标。新平台将通过对成年小鼠皮质桶状柱进行的7个标记的蛋白计量学调查来展示，该调查将列举该柱中数千万个突触中的每一个，并允许根据神经递质类型和一组神经元类型标记对每个突触进行分类。结果将通过开放源码的方法出版物传播

DESCRIPTION (provided by applicant): Synapses of the mammalian central nervous system (CNS) are very deeply diverse in both molecular and functional properties. At present, unfortunately, our understanding of this diversity is rudimentary, and quantitative data on the subject are very few. Left unfathomed, CNS synapse diversity poses formidable obstacles to better understanding of the development, function and disorders of the brain's synaptic circuitry. The major reason for the persistence of this distressing state of ignorance lies in the fact that tools for exploring synapse populations at the level of individual synapses are few and limited in their capabilities. To address the challenges synapse diversity poses to both basic and clinical neuroscience, this project aims to develop a superlative new proteometric imaging platform capable of analyzing very large synapse populations in situ with single- synapse resolution. Deployment and dissemination of this platform will facilitate study and treatment of the many neurodevelopmental, mental and neurodegenerative disorders linked to specific synapse subpopulations, as well as opening new perspectives on molecular mechanisms, circuit architectures and disorders of CNS memory encoding, storage and retrieval. The platform will be based on immunofluorescence array tomography (IAT) and involve development of novel antibody standardization protocols and novel image acquisition hardware and software. These innovations will improve the reproducibility, quantitative reliability, and speed of IAT by large margins and overcome limitations that have so far prevented proteometric analysis of large synapse populations at the single-synapse level. The platform would be capable of proteometric census of a million of more synapses per hour, at 50 or more markers per synapse, while maintaining precise neuroanatomical and molecular coordinates for each synapse. The new platform will be demonstrated by a 7-marker proteometric survey of an adult mouse cortex barrel column that would enumerate each of the tens of millions of synapses in the column and allow classification of each synapse based on neurotransmitter type and a set of neurons type markers. The results will be disseminated via methods publications, open-source