Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo.
Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo.
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DOI：
10.1038/s41598-018-32820-5
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发表时间：
2018-09-27
影响因子：
4.6
通讯作者：
Polleux F
中科院分区：
文献类型：
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作者：
Hirabayashi Y;Tapia JC;Polleux F
神经科学的一个具有挑战性的方面是围绕着在跨越几个数量级（纳米到米）的尺度上映射神经回路（连接组学）内的突触连接。尽管连续切片电子显微镜（SSEM）技术有了显着的改进，但几个主要的障碍已经削弱了其对哺乳动物神经回路的普遍适用性。在本研究中，我们引入了一种新的方法，通过将遗传编码的抗坏血酸过氧化物酶（APEX 2）作为融合蛋白与膜靶向荧光报告基因（CAAX-Venus）相结合，并将其引入体内单个锥体神经元中，使用非常稀疏的子宫内皮层电穿孔来规避这些障碍。这种方法使我们能够对单个神经元进行相关光-SSEM（CoLSSEM），相关光-EM（CLEM）的变体，通过高分辨率共聚焦显微镜的组合，以有针对性的方式重建其树突和轴突分支，随后用ATUM-SEM（自动磁带收集超微切片机-扫描电子显微镜）技术对其超微结构特征和突触连接进行成像。我们的方法将显着提高电路内的神经元的大规模重建的可行性，并允许识别的神经元的功能和/或结构的连接，连接组学的主要目标之一的一些超微结构特征的描述。
A challenging aspect of neuroscience revolves around mapping the synaptic connections within neural circuits (connectomics) over scales spanning several orders of magnitude (nanometers to meters). Despite significant improvements in serial section electron microscopy (SSEM) technologies, several major roadblocks have impaired its general applicability to mammalian neural circuits. In the present study, we introduce a new approach that circumvents some of these roadblocks by adapting a genetically-encoded ascorbate peroxidase (APEX2) as a fusion protein to a membrane-targeted fluorescent reporter (CAAX-Venus), and introduce it in single pyramidal neurons in vivo using extremely sparse in utero cortical electroporation. This approach allows us to perform Correlated Light-SSEM (CoLSSEM), a variant of Correlated Light-EM (CLEM), on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequent imaging of its ultrastructural features and synaptic connections with ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly will improve the feasibility of large-scale reconstructions of neurons within a circuit, and permits the description of some ultrastructural features of identified neurons with their functional and/or structural connectivity, one of the main goal of connectomics.