Reconstruction of genetically identified neurons imaged by serial-section electron microscopy
Reconstruction of genetically identified neurons imaged by serial-section electron microscopy
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DOI：
10.7554/elife.15015
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发表时间：
2016-07-25
期刊：
影响因子：
7.7
通讯作者：
Sanes, Joshua R.
中科院分区：
文献类型：
--
作者：
Joesch, Maximilian;Mankus, David;Sanes, Joshua R.
解析神经回路中突触连接的模式目前需要连续切片电子显微镜。然而，完整的电路重建是非常缓慢的，并且对于许多目的可能不是必需的，例如比较多个动物之间的神经元结构和连接。在这里，我们提出了一种替代策略，有针对性地重建特定的神经元类型。我们使用病毒载体传递过氧化物酶衍生物，催化电子密度示踪剂的产生，以遗传识别神经元，并开发了一种方案，在保持超微结构质量的同时增强标记细胞的电子密度。标记神经元的高对比度实现了两项加速数据采集的创新：从快速获取的低分辨率重建中选择区域的目标高分辨率重新成像，以及无监督分割算法。该管道将成像和重建时间减少了两个数量级，促进了对电路图案的直接查询。
Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs.