Extracellular space preservation aids the connectomic analysis of neural circuits

  1. Marta Pallotto
  2. Paul V Watkins
  3. Boma Fubara
  4. Joshua H Singer
  5. Kevin L Briggman  Is a corresponding author
  1. National Institutes of Health, United States
  2. University of Maryland, United States

Abstract

Dense connectomic mapping of neuronal circuits is limited by the time and effort required to analyze 3D electron microscopy (EM) datasets. Algorithms designed to automate image segmentation suffer from substantial error rates and require significant manual error correction. Any improvement in segmentation error rates would therefore directly reduce the time required to analyze 3D EM data. We explored preserving extracellular space (ECS) during chemical tissue fixation to improve the ability to segment neurites and to identify synaptic contacts. ECS preserved tissue is easier to segment using machine learning algorithms, leading to significantly reduced error rates. In addition, we observed that electrical synapses are readily identified in ECS preserved tissue. Finally, we determined that antibodies penetrate deep into ECS preserved tissue with only minimal permeabilization, thereby enabling correlated light microscopy (LM) and EM studies. We conclude that preservation of ECS benefits multiple aspects of the connectomic analysis of neural circuits.

Article and author information

Author details

  1. Marta Pallotto

    Circuit Dynamics and Connectivity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Paul V Watkins

    Circuit Dynamics and Connectivity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Boma Fubara

    Circuit Dynamics and Connectivity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Joshua H Singer

    Department of Biology, University of Maryland, College Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Kevin L Briggman

    Circuit Dynamics and Connectivity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    For correspondence
    briggmankl@mail.nih.gov
    Competing interests
    The authors declare that no competing interests exist.

Ethics

Animal experimentation: We fixed and examined tissue from a variety of brain regions of C57BL/6 mice, aged 9 to 12 weeks in accordance with NIH animal ethics guidelines. All of the animals were handled according to an approved institutional animal care and use committee (IACUC) protocol. The protocol was approved by the NINDS Animal Care and Use Committee (#1340-15).

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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  1. Marta Pallotto
  2. Paul V Watkins
  3. Boma Fubara
  4. Joshua H Singer
  5. Kevin L Briggman
(2015)
Extracellular space preservation aids the connectomic analysis of neural circuits
eLife 4:e08206.
https://doi.org/10.7554/eLife.08206

Share this article

https://doi.org/10.7554/eLife.08206

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