Tools and approaches for functional connectomics of dense neuropils

致密神经细胞功能连接组学的工具和方法

• 批准号: 9980918
• 负责人: Daniel Kerschensteiner
• 金额: $ 19.69万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980918
• 关键词: Address; Amacrine Cells; Anatomy; Architecture; Axon; Benchmarking; Calcium; Cells; Data Set; Disease; Electron Microscopy; Functional disorder; Future; Genetic; Goals; Image; Individual; Inner Plexiform Layer; Interneurons; Knowledge; Label; Logic; Maps; Measures; Methods; Multimodal Imaging; Nervous System Physiology; Nervous system structure; Neurites; Neurons; Neuropil; Pathway interactions; Pattern; Photoreceptors; Physiological; Process; Resolution; Retina; Retinal Degeneration; Shapes; Signal Transduction; Stimulus; Structure; Synapses; Techniques; Tissues; Viral; Vision; Visual; base; calcium indicator; cell type; computerized tools; density; design; insight; multimodality; novel strategies; performance tests; preference; prevent; response; spatiotemporal; tool; two-photon; visual information; visual processing

PROJECT SUMMARY Many computations in the nervous system occur at the level of individual neurites within large extensively branched arbors (i.e., subcellular processing). Most neurites operate in dense neuropils, in which processes of diverse cell types are tightly packed and abundantly interconnected. To understand subcellular processing, we need to measure neurite responses to physiological stimuli and relate them to local patterns of synaptic inputs. To delineate the functional architecture of neuropils and reveal the logic of their connectivity, we need to characterize neurite responses and synapse patterns at high density. Neurite responses can be observed by two-photon imaging, and synaptic inputs can be reconstructed in serial-section electron microscopy (ssEM). A number of technical obstacles have precluded the combination of these techniques (i.e., functional connectomics) to study subcellular processing in dense neuropils. Here, we develop new tools and approaches to overcome these obstacles. In Aim 1, we develop genetic, viral, and computational tools for multispectral two-photon calcium imaging and signal demixing to enable dense functional characterization of neuropils. In Aim 2, we devise a novel strategy for combining two-photon imaging and ssEM (i.e., multimodal imaging), and establish a high-throughput ssEM method for analyzing local synaptic connectivity patterns in the context of larger-scale circuit wiring (i.e., multiresolution imaging). We use our advances to study amacrine cells (ACs), a diverse class of retinal interneurons. The neurites of more than 50 AC types extract salient visual information in a dense neuropil the inner retina. We will acquire a complete functional connectomic dataset of ACs. This dataset, which will be made publicly available, will form the basis of a future R01 application to study the mechanisms of subcellular processing in ACs, the functional architecture of the AC neuropil, and the logic of its connectivity.

项目摘要 神经系统中的许多计算发生在大型的单个神经突的水平上 广泛的分支乔木（即亚细胞处理）。大多数神经突的密集作用 Neuropils，其中各种细胞类型的过程紧密堆积，并且丰富 互联。要了解亚细胞处理，我们需要测量对神经突的反应 生理刺激并将其与突触输入的局部模式相关联。描绘 神经胶质的功能架构并揭示其连通性的逻辑，我们需要 表征高密度的神经突反应和突触模式。神经突反应可能是 通过两光子成像观察到突触输入，可以在串行部分中重建 电子显微镜（SSEM）。许多技术障碍排除了 这些技术（即功能连接组学）用于研究密集的亚细胞处理 神经胶质。在这里，我们开发了克服这些障碍的新工具和方法。在AIM 1中，我们 开发用于多光谱两光子钙成像的遗传，病毒和计算工具 和信号解散以实现神经胶体的致密功能表征。在AIM 2中，我们 设计一种新颖的策略，用于结合两光子成像和SSEM（即多模式成像）， 并建立一个用于分析局部突触连接模式的高通量SSEM方法 在大型电路接线的背景下（即多解决成像）。我们利用我们的进步 研究一类视网膜中间神经元的无长熟细胞（ACS）。多于 50种交流类型的提取物在密集的神经胶体中的显着视觉信息。我们将 获取ACS的完整功能连接数据集。这个数据集将被制作 公开可用，将构成未来R01应用程序的基础，以研究 ACS中的亚细胞处理，AC神经的功能架构及其逻辑 连接性。