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

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。

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.