三维电镜在脑干耳蜗核神经元形态学研究中的应用

doi:10.3969/j.issn.1674-8115.2022.02.002

上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (2): 142-149.doi: 10.3969/j.issn.1674-8115.2022.02.002

• 论著 · 基础研究 • 上一篇

三维电镜在脑干耳蜗核神经元形态学研究中的应用

周佳蕾¹^,²^,³^,⁴(), 盛海斌¹^,²(), 王皓煜³, 鲁岩³, 王方方³, 吴皓¹^,²^,³, 华云峰¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海市耳鼻疾病转化医学重点实验室，上海 200125
^3.上海精准医学研究院，上海 200125
^4.上海交通大学医学院附属儿童医院耳鼻咽喉头颈外科，上海 200062

收稿日期:2021-09-07 出版日期:2022-02-28 发布日期:2022-03-17
通讯作者: 盛海斌,华云峰 E-mail:zhoujl@shchildren.com.cn;shenghaibin2020@outlook.com;yunfeng.hua@shsmu.edu.cn
作者简介:周佳蕾（1985—），女，主管听力师，硕士生；电子信箱：zhoujl@shchildren.com.cn。
基金资助:
国家自然科学基金(82171133);上海市黄浦区产业扶持基金(XK2019011);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

Application of three-dimensional electron microscopy to morphological study of neurons in brainstem cochlear nucleus

Jialei ZHOU¹^,²^,³^,⁴(), Haibin SHENG¹^,²(), Haoyu WANG³, Yan LU³, Fangfang WANG³, Hao WU¹^,²^,³, Yunfeng HUA¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200125, China
^3.Shanghai Institute of Precision Medicine, Shanghai 200125, China
^4.Department of Otolaryngology-Head and Neck Surgery, Children's Hospital of Shanghai Jiao Tong University School of Medicine, Shanghai 200062, China

Received:2021-09-07 Online:2022-02-28 Published:2022-03-17
Contact: Haibin SHENG,Yunfeng HUA E-mail:zhoujl@shchildren.com.cn;shenghaibin2020@outlook.com;yunfeng.hua@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(82171133);Industrial Support Fund of Huangpu District in Shanghai(XK2019011);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

目的·探索利用新型三维电子显微成像的方法对小鼠耳蜗核组织开展跨尺度神经解剖学和连接组学研究的可行性。方法·成年CBA/Ca小鼠（2月龄）脑组织经固定、解剖后，获取其中完整耳蜗核组织，并对该组织行重金属块染（还原性锇酸扩增法）、梯度乙醇和无水丙酮脱水、低黏度树脂包埋。通过X射线显微成像技术对耳蜗核组织进行断层扫描成像及三维重构，并对其染色效果进行评估，再根据听神经特异性分布对其目标区域进行定位。采用扫描电镜快速获得该组织的低分辨预扫图像，并与X射线显微成像进行比对，确定目标层面后使用连续切片扫描电镜采集目标区域的图像进行三维重构，并对该目标区域丛细胞及其表面的突触等超微结构进行追踪标记和重构分析。结果·成功制备了CBA/Ca小鼠完整耳蜗核的三维电镜样品。X射线显微成像采集并重构了具有细胞分辨率的耳蜗核立体结构，实现了耳蜗核各亚区的解剖定位。连续切片扫描电镜成功采集耳蜗核目标区域丛细胞的三维电镜数据，完成了丛细胞胞体上根蕾状突触和其他非听神经来源突触的追踪、标记和重构。三维电镜的数据分析显示，投射至目标丛细胞表面的听神经根蕾状突触有5个，共形成了348个突触活跃区，而非听神经来源的突触有97个。结论·利用锇酸块染、树脂包埋等方法对成年小鼠完整耳蜗核进行三维电镜样品的制备具有可行性。X射线显微成像可用于耳蜗核组织各亚区以及目标区域的快速和精确定位，且所采集的三维电镜数据可用于耳蜗核神经元的形态学以及突触连接方式的研究。

关键词: 耳蜗核, 丛细胞, 根蕾状突触, 连续切片扫描电镜, X射线显微成像

Abstract:

Objective·To explore the feasibility of cross-scale neuroanatomy and connectomics of mouse cochlear nucleus (CN) by using a new three-dimensional (3D) electron microscopic imaging method.

Methods·The intact CN was obtained after the brain tissue of adult CBA/Ca mice (2 months old) was fixed and dissected, stained with heavy metals (reducing osmium amplification method) , dehydrated with gradient ethanol and anhydrous acetone and embedded with low viscosity resin. The CN tissue was imaged by X-ray microscopy and 3D reconstruction, and its staining quality was evaluated. Then targeted subdivision was located according to the specific distribution of auditory nerve fibers. The low-resolution pre-scan of the CN tissue was performed by scanning electron microscopy, and compared with X-ray microscopic dataset. After the target location was determined, the volume of interest was mapped by serial block-face scanning electron microscopy (SBEM) for 3D reconstruction, and the ultrastructures of bushy cells and the synapses projecting on its surface were tracked, annotated and reconstructed.

Results·3D electron microscopic samples of intact CN of CBA/Ca mice were successfully prepared. The 3D structure of CN with cell resolution was collected and reconstructed by X-ray microscopic imaging, and the anatomical localization of subregions of CN was achieved. The 3D electron microscopic data of bushy cells in the target area of CN were successfully collected by SBEM. The tracking, labeling and reconstruction of endbulb of Held synapses and other non-auditory synapses on the cell body of target bushy cells were completed. The data of 3D electron microscopy showed that there were 5 endbulb of Held synapses projecting to the surface of the target bushy cell, forming a total of 348 synaptic active zones, while there were 97 synapses from non-auditory nerve.

Conclusion·It is feasible to prepare 3D electron microscopic samples of adult mouse intact CN by Osmium-based enbloc staining and resin embedding. X-ray microscopic imaging can be used for rapid and accurate localization of subregions and target volume of CN, and the acquired 3D electron microscopic data can be used to study the neuronal morphology and synaptic connections in the CN.

Key words: cochlear nucleus (CN), bushy cell, endbulb of Held synapase, serial block-face scanning electron microscopy (SBEM), X-ray microscopic imaging

中图分类号:

R764.4

周佳蕾, 盛海斌, 王皓煜, 鲁岩, 王方方, 吴皓, 华云峰. 三维电镜在脑干耳蜗核神经元形态学研究中的应用[J]. 上海交通大学学报(医学版), 2022, 42(2): 142-149.

Jialei ZHOU, Haibin SHENG, Haoyu WANG, Yan LU, Fangfang WANG, Hao WU, Yunfeng HUA. Application of three-dimensional electron microscopy to morphological study of neurons in brainstem cochlear nucleus[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(2): 142-149.

图/表 5

图1 显微镜下观察小鼠CN的解剖位置Note：The anatomically separated mouse brain tissue is shown (side view). After removing the upper cerebellar tissue (red dotted line), the CN (green area) and the stump of AN (black arrow) can be clearly seen. D—dorsal; A—anterior; Cx—cerebral cortex; AN—auditory nerve; Cb—cerebellum; Med—medulla oblongata.

Fig 1 Observation of anatomical location of CN in mice under microscope

表1 小鼠CN样品的三维成像信息

Tab 1 Three-dimensional imaging information of mouse CN samples

Item	Sample
Item	1^#	2^#	3^#	4^#	5^#
Mouse species	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca	CBA/Ca
Gender	male	male	male	male	male
Age/week	7	7	7	8	8
Sample size/mm³	1.3×1.7×2.5	1.4×1.6×2.8	1.3×1.5×2.6	1.3×1.5×2.5	1.2×1.4×2.6
X-ray microscopic imaging resolution/μm³	1.6	1.5	0.7	1.4	2.8
SBEM imaging
Pixel/nm³	10×10×50	15×15×50	‒	12×12×35	15×15×50
Data size/μm³	114.3×105.0×105.0	754.6×192.2×258.1	‒	443.0×141.1×91.0	193.1×505.1×126.7
Accelerating voltage/kV	2.0	2.0	‒	2.0	2.0
Exposure time/μs	1.5	1.0	‒	1.0	1.5

图2 CN样品的X射线显微成像Note：A. Three-dimensional reconstruction of CN sample by X-ray microscopic imaging. B/C. Sagittal (B) and coronal (C) images of the reconstructed CN. Scale bar: 100 μm. ICP—inferior cerebellar peduncle.

Fig 2 X-ray microscopic imaging of CN samples

图3 CN样品表面的X射线显微图像与电镜预扫描图像的比对Note：A. Scanning electron micrograph of surface-section of CN sample. Scale bar: 100 μm. B. X-ray microscopic imaging of the corresponding layer of CN sample. Scale bar: 100 μm. A1—the content shown in the red box of Fig A; A2—the content shown in the green box of Fig A; B1—the content shown in the red box of Fig B; B2—the content shown in the green box of Fig B. Scale bar: 50 μm. Red—neuronal cell body; Green—auditory nerve fiber.

Fig 3 Comparision of X-ray microscopic image of CN sample surface with electron microscopic pre-scanning image

图4 VCN丛细胞簇的三维电镜图像数据集及相关神经结构的标记与重构Note：A. The volume of CN acquired by three-dimensional electron microscopic imaging. B. Local high-resolution electron microscopic image of VCN. AZ—active zone; ax—axon; syn—synapse. Scale bar: 1 μm. C. Tracing of endbulb of Held synapses on bushy cell surface. Blue—bushy cell dendrites; Green—endbulb of Held synapses; Red—synaptic active zones. D. Tracing of non-auditory nerve synapses on bushy cell surface. Blue—bushy cell dendrites; Purple—non-auditory nerve terminals. E. Three-dimensional reconstruction of endbulb of Held synapses on bushy cells. F. Three-dimensional reconstruction of non-auditory nerve synapses on bushy cells. Scale bar: 10 μm.

Fig 4 Labeling and reconstruction of three-dimensional electron microscopic image dataset of VCN bushy cell cluster and related neural structures

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三维电镜在脑干耳蜗核神经元形态学研究中的应用

Application of three-dimensional electron microscopy to morphological study of neurons in brainstem cochlear nucleus

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