Reconstruction of genetically identified neurons imaged by serial-section electron microscopy Journal Article


Author(s): Jösch, Maximilian; Mankus, David; Yamagata, Masahito; Shahbazi, Ali; Schalek, Richard L; Suissa-Peleg, Adi; Meister, Markus; Lichtman, Jeff W; Scheirer, Walter J; Sanes, Joshua R
Article Title: Reconstruction of genetically identified neurons imaged by serial-section electron microscopy
Affiliation
Abstract: 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.
Keywords: Immunohistochemistry; protein expression; Electron microscopy; Ultrastructure; Confocal microscopy; connectome; gene transfer; magnitude estimation method; motoneuron; nerve reconstruction; synaptic membrane
Journal Title: eLife
Volume: 5
Issue 2016JULY
ISSN: 2050-084X
Publisher: eLife Sciences Publications  
Date Published: 2016-07-07
Start Page: Article number: e15015
Copyright Statement: CC BY 4.0
Sponsor: This work was supported by NIH grant NS76467 to MM, JL and JRS, an HHMI Collaborative Innovation Award to JRS, an IARPA contract #D16PC00002 to WJS and by The International Human Frontier Science Program Organization fellowship to MJ.
DOI: 10.7554/eLife.15015
Open access: yes (OA journal)