全脑尺度获取小鼠情感与记忆神经环路三维精细图谱的方法研究

Advanced brain functions including emotion and memory are achieved by the cooperation of local circuits and long-projection circuits with different neuron types. To understand the complex brain functions, we need to accurately acquire the intact architectonic information of the relevant neural circuits in whole brain range. However, current optical microscopy imaging technologies have failed to provide a brain atlas of fine neural circuits in 3D continuous way. In this project, a new fast and accurate acquisition method is proposed to simultaneously obtain the fine structure of specific neural circuits and the coordinate information of the passing brain regions in the same mouse brain. Standard entire-brain fluorescence-labeled sample preparation methods will be established to provide 3D high-resolution coordinate atlas of mouse brain regions and nucleus for locating and indicating the neural circuits. A high-throughput block microscopic optical imaging technique with sub-micron-resolution is about to be developed to enable to acquire the whole-brain distribution of specific-type neurons associated to emotion and memory at cellular level. Further, a multi-color fast brain-wise fluorescence block microscopy is intended to be built, aiming at continuously imaging in the entire brain range in two channels with self registration at the voxel resolution of 0.32×0.32×2 µm³in 5 days. 3D mouse brain coordinate atlas as well as high-resolution and high-throughput imaging research platform built by this project will play a key role in various studies of brain science.

情感与记忆等脑高级功能往往由多种类型神经元的局部和长程环路协同作用，因此认识和理解脑功能需要在全脑范围对相关神经环路的精细结构信息进行准确完整的获取。然而，现有光学显微成像技术无法提供全脑尺度神经环路三维连续精细图谱。本项目拟建立在全脑范围内对同一只小鼠与特定功能相关的神经环路精细结构及环路所涉及脑区的定位信息进行同步快速准确获取的方法，包括：标准化的全脑荧光标记样本制备方法，发展亚微米分辨率高通量的立体显微光学成像方法，能在细胞分辨水平获取特定类型神经元全脑分布数据集；发展多色快速全脑荧光立体显微成像方法，以0.32×0.32×2µm³体素分辨率，达成全脑连续、双色、自配准的成像仅用时5天，领先国际水平的目标。本研究建立的小鼠脑立体高分辨率高通量成像研究平台，有望在脑科学的各类研究中发挥重大作用。

原有的光学显微成像技术难以在全脑尺度提供神经环路的三维连续精细形态和空间位置信息。本项目建立了标准化的小鼠全脑荧光标记样本的制备方法，发明了亚微米分辨率高通量的显微光学体积成像方法，能在单细胞分辨水平获取特定类型神经元全脑分布数据集；以0.32×0.32×2μm³体素分辨率，达成全脑连续、双色、自配准的图像获取仅用时3天，具有国际领先水平的目标。研制了一套实现自动化切片收集的快速全脑光学成像系统，可高效、准确地同时获取全脑三维解剖结构与分子表型数据集。建立了能够保持神经元精细结构的大体积塑性包埋方法和标准化实验流程，设计和开发了可处理TB级图像大数据的方法和软件。利用上述方法和新技术，建立了全脑特定类型神经元分布和统计分析研究平台，建立了完整神经元形态学分析的流程，并开展了相关的应用研究。共发表标注有项目标号的SCI期刊论文11篇，其中包括Nature Communications 1篇，PNAS 1篇等。已申请中国发明专利 4项。本研究建立的小鼠脑立体高分辨率高通量成像研究平台，有望在脑科学的各类研究中发挥重大作用。