面向多尺度全脑图谱绘制的神经环路显微成像新技术

The purpose of this study is to break through the bottleneck of microscopic imaging of neural circuits faced by the construction of a multi-scale whole-brain connectivity atlas, to develop new neural tracing and tissue optical clearing technology for rodents and nonhuman primates, to establish a comprehensive and practical procedure for tissue sample preparation, to achieve a new breakthrough in high-throughput fluorescence microscopic imaging technology at the micrometer scale, and to develop innovative and efficient image processing methods. Research contents: (1) To optimize the tracer combination strategy that is suitable for the construction of a macaque whole-brain connectivity atlas and for the new tissue optical clearing techniques, and to develop efficient new tracer tools, such as herpes simplex virus; (2) to elucidate the mechanism of long-term preservation of fluorescence in tissue optical clearing and to develop new tissue optical clearing techniques suitable for nonhuman primates; (3) to develop new imaging techniques such as polarization-sensitive optical coherence tomography; (4) to establish fast and reliable automated cell recognition and counting methods for retrograde neuronal tracing, as well as efficient and fast reconstruction of white matter tracts at the micrometer scale based on polarization-sensitive optical coherence tomography. This study will provide new technology and methodology for the construction of a whole-brain connectivity atlas that is a cell-specific and based on strategies such as immediately early gene expression and neurotropic virus tracing.

本项课题旨在突破多尺度全脑连接图谱绘制所面临的神经环路显微成像技术瓶颈，发展适合于啮齿类和非人灵长类的新型神经元标记技术和脑组织透明技术，建立完备和实用的样品制备流程，实现微米分辨率高通量荧光显微成像技术的新突破，提出高效、快速的图像处理新方法。研究内容：（1）优化适合于猕猴全脑连接图谱绘制和新型脑组织透明技术的示踪剂组合策略，并发展高效的单纯疱疹病毒等新型示踪工具；（2）阐明脑组织透明技术荧光长时间保持的机理，并发展适合于非人灵长类的新型脑组织透明技术；（3）发展偏振光敏感光学相干断层成像等新成像技术；（4）建立基于逆向示踪标记的快速、可靠的自动化细胞识别和计数方法，以及基于偏振光敏感光学相干断层成像的高效、快速的微米尺度脑白质纤维束重建新方法。为绘制细胞类型特异的、基于即早基因表达和嗜神经病毒示踪等策略的全脑连接图谱提供新技术和新方法学。

本项目是重大研究计划集成项目，集中了中国科学院自动化研究所、中国科学院武汉病毒所、浙江大学等三家单位在脑图谱绘制、病毒工具改造和构建以及在猴脑脑区定位、切片和染色等方面的研究经验，力图通过科研合作解决神经纤维示踪方面的重大科学问题和挑战。通过本项目2年的工作，我们已经取得了三个方面的研究进展，包括：第一，发展了一种新型利用单纯疱疹病毒H129装载编码储铁蛋白Ferritin磁共振报告基因并结合超高场磁共振成像技术进行活体神经纤维示踪的工具，并完成了大小鼠的测试；第二，发展和优化了适合于猕猴全脑连接图谱绘制和新型脑组织透明技术的示踪剂组合策略，阐明基于有机溶剂溶脂的脑组织透明技术荧光长时间保持的机理，在此基础上发展适合于啮齿类和非人灵长类以及多种标记技术的新型脑组织透明技术；第三，建立基于逆向示踪标记的快速、可靠的自动化细胞识别和计数方法，在鼠脑和猴脑标本上进行了测试和验证。这些技术进展将为神经纤维示踪提供重要工具。相关成果已发表学术论文5篇，申请国家发明专利1项。