C4: Neuroanatomy

C4：神经解剖学

• 批准号: 10705971
• 负责人: Samuel Sheng-Hung Wang
• 金额: $ 50.22万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-08 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705971
• 关键词: Anatomy; Area; Atlases; Brain; Brain region; Cognition; Collaborations; Communities; Computer software; Data; Data Science; Data Set; Decision Making; Democracy; Electron Microscopy; Funding; Gene Expression; Gene Expression Profiling; Geometry; Goals; Histology; Hour; Human; Image; Image Analysis; Imaging technology; Immediate-Early Genes; Laboratories; Learning; Light; Maps; Methods; Microscopy; Monitor; Mus; Neuroanatomy; Neurons; Neurosciences; Optics; Pattern; Preparation; Process; Protocols documentation; Quality Control; Reproducibility; Research; Research Project Grants; Roentgen Rays; Sampling; Short-Term Memory; Site; Stains; Standardization; System; Techniques; Thick; Time; Tissue imaging; Tissues; Tracer; Transmission Electron Microscopy; Viral; Work; analysis pipeline; cell type; data exchange; high throughput technology; image processing; improved; in situ sequencing; in vivo imaging; microscopic imaging; millimeter; neural; neural circuit; neuroimaging; neuromechanism; programs; reconstruction; relational database; scale up; tool; transcriptomics

Project Summary/Abstract: Core 4, Neuroanatomy The overall goal of this U19 collaboration is to elucidate how working memory and decision-making are supported by interacting neurons and brain regions. To achieve this goal, our research projects will need cutting-edge neuroanatomy tools, which the Neuroanatomy Core will provide. As it has done in the first U19 funding period, this core will continue to support protocols, software, and standards for light-sheet microscopy, used with viral tracers to map long-range connectivity between brain regions. We will also add two state-of-the-art imaging technologies: light-sheet microscopy of cleared whole mouse brains to enable brainwide imaging of neural recording sites and immediate-early gene expression, and serial-section transmission electron microscopy (TEM) to provide rapid automated ultrastructural analysis. The first aim will be to support brainwide imaging of neural recording sites, activation patterns, and connectivity. We will support the automated image processing and brain-registration pipeline that we developed and extend it to new research aims. Our pipeline will identify recording sites registered to a standardized atlas, and how brain regions are activated throughout learning. We will provide transcriptional profiling of our imaged tissue for post-hoc identification of cell types in our imaging datasets. The second aim will be to optimize electron-microscopy sample preparation and serial sectioning. Datasets from the TEM system will be processed by our petascale image-analysis pipeline to search for sequential connectivity underlying sequential neural activity. In the long term, petascale connectomics may be applied to other projects in the U19 to investigate circuit mechanisms of cognition in various brain areas. Our TEM system has the highest raw throughput capacity of its kind in the world, but further work is needed to realize its full potential. This core will optimize sample preparation and serial sectioning for TEM, and complete software required to fully automate TEM imaging. We will optimize EM staining protocols for uniform, high contrast in cubic millimeters of tissue, using our new X-ray-assisted technique. We will also optimize our automated tape-collecting ultramicrotome system, to scale up serial sectioning from 4,000 ultrathin sections to tens of thousands of sections. The third aim will be to automate high-throughput, parallel TEM imaging. When complete, our high-throughput technology will enable imaging of cubic-millimeter datasets in a few weeks instead of the current 6-12 months. New functionalities will extend the duty cycle from the current eight hours with human monitoring to 24 hours automatically, enabling each TEM to produce over 20 TB of data in 24 hours. Software that can handle data throughput at this scale will be built to realize the full potential of our imaging pipeline. More broadly, we expect that our pioneering methods for automating light-sheet and electron microscopy, when shared with the broader community, will improve efficiency, rigor, and reproducibility in anatomical research across the field of neuroscience and democratize access to petascale connectomics.

项目摘要/摘要：核心4，神经解剖学 这项U19合作的总体目标是阐明工作记忆和决策是如何 由相互作用的神经元和大脑区域支持。为了实现这一目标，我们的研究项目将需要 尖端神经解剖学工具，神经解剖学核心将提供。就像它在第一个U19中所做的那样 在资助期间，该核心将继续支持光片显微镜的协议、软件和标准， 与病毒示踪剂一起使用，以绘制大脑区域之间的远程连接。我们还将添加两个 最先进的成像技术：对清除的整个小鼠大脑进行光片显微镜检查，以使 神经记录部位和即刻早期基因表达的全脑成像，以及连续切片 提供快速自动化超微结构分析的透射电子显微镜(TEM)。 第一个目标是支持对神经记录部位、激活模式和 连通性。我们将支持我们开发的自动图像处理和脑注册管道 并将其扩展到新的研究目标。我们的流程将确定登记在标准化地图集上的录音地点， 以及大脑区域在整个学习过程中是如何被激活的。我们将提供我们的图像的转录档案 用于在我们的成像数据集中识别细胞类型的组织。 第二个目标是优化电子显微镜样品的制备和连续切片。 来自瞬变电磁系统的数据集将由我们的千万亿级图像分析流水线处理，以搜索 时序神经活动背后的时序连接。从长远来看，千万亿级的连接术可能是 应用于U19的其他项目，以研究不同大脑区域的认知回路机制。我们的 TEM系统的原始吞吐能力是世界上最高的，但需要进一步的工作来实现 充分发挥其潜力。该岩芯将优化样品的制备和连续切片，并完成 全自动电子显微镜成像所需的软件。我们将优化EM染色方案，以实现均匀、高 使用我们的新X射线辅助技术，以立方毫米组织为单位进行对比。我们还将优化我们的 自动集带超薄切片机系统，将连续切片从4,000超薄切片扩大到 数以万计的部分。第三个目标将是自动化高通量、并行的瞬变电磁成像。什么时候 完成后，我们的高通量技术将在几周内实现立方毫米数据集的成像 而不是目前的6-12个月。新功能将从目前的8小时延长工作周期 人工监控到24小时自动，使每个瞬变电磁能够在24小时内产生超过20 TB的数据 几个小时。能够处理这种规模的数据吞吐量的软件将被构建来实现我们的 成像管道。更广泛地说，我们希望我们的自动化光片和电子的开创性方法 当与更广泛的社区共享时，显微镜将提高效率、严谨性和重复性 神经科学领域的解剖学研究，并使获得拍级连接的途径大众化。