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bioRxiv · 10.1101/148585

Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo

Hirabayashi, Y.·Tapia, J. C.·Polleux, F.
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Abstract

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 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 (IUCE). This approach allows to perform Correlated Light-SSEM (CoLSSEM) on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequently imaging of its ultrastuctural features and synaptic connections with the ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly improves the the feasibility of large-scale reconstructions of neurons within a circuit, and bridges the description of ultrastructural features of genetically-identified neurons with their functional and/or structural connectivity, one of the main goal of connectomics.

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Hirabayashi, Y., Tapia, J. C., Polleux, F.. 2017-06-10. Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo. https://doi.org/10.1101/148585

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