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多样化。不幸的是，目前我们对这种多样性的了解还很初级，关于这个问题的定量数据也很少。如果对中枢神经系统突触的多样性不了解，就会给更好地理解大脑突触回路的发育、功能和紊乱带来巨大障碍。这种令人沮丧的无知状态持续存在的主要原因在于，在单个突触的水平上探索突触数量的工具很少，而且它们的能力有限。为了解决突触多样性给基础和临床神经科学带来的挑战，该项目旨在开发一种最高级的新型蛋白质测量成像平台，能够以单突触分辨率原位分析非常大的突触群。该平台的部署和传播将促进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的研究和治疗，并为CNS记忆编码、存储和检索的分子机制、电路结构和疾病开辟新的视角。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。这些创新将在很大程度上提高IAT的重现性、定量可靠性和速度，并克服迄今为止阻碍在单突触水平上对大突触群体进行蛋白质计量分析的局限性。该平台将能够每小时对100万个以上的突触进行蛋白质计量普查，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柱的7个标记蛋白计量学调查来证明，该调查将枚举列中数千万个突触中的每一个，并允许基于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。结果将通过方法、出版物、开源传播

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