Format

Send to

Choose Destination
Elife. 2016 Jul 7;5. pii: e15015. doi: 10.7554/eLife.15015.

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

Author information

1
Center for Brain Science, Harvard University, Cambridge, United States.
2
Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States.
3
University of Notre Dame, Notre Dame, United States.
4
School of Engineering and Applied Sciences, Harvard University, Cambridge, United States.
5
Division of Biology, California Institute of Technology, Pasadena, United States.

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:

D. melanogaster; cell biology; connectomics; electron microscopy; mouse; neuroscience; peroxidase; reconstruction

PMID:
27383271
PMCID:
PMC4959841
DOI:
10.7554/eLife.15015
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for eLife Sciences Publications, Ltd Icon for PubMed Central
Loading ...
Support Center