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.

Reviewing Editor

  1. Michael Hausser, University College London, United Kingdom

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).

Version history

  1. Received: April 18, 2015
  2. Accepted: October 27, 2015
  3. Accepted Manuscript published: December 9, 2015 (version 1)
  4. Accepted Manuscript updated: December 24, 2015 (version 2)
  5. Version of Record published: February 10, 2016 (version 3)

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.

Metrics

  • 4,024
    views
  • 1,055
    downloads
  • 75
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Further reading

    1. Cell Biology
    2. Neuroscience
    Marcos Moreno-Aguilera, Alba M Neher ... Carme Gallego
    Research Article Updated

    Alternative RNA splicing is an essential and dynamic process in neuronal differentiation and synapse maturation, and dysregulation of this process has been associated with neurodegenerative diseases. Recent studies have revealed the importance of RNA-binding proteins in the regulation of neuronal splicing programs. However, the molecular mechanisms involved in the control of these splicing regulators are still unclear. Here, we show that KIS, a kinase upregulated in the developmental brain, imposes a genome-wide alteration in exon usage during neuronal differentiation in mice. KIS contains a protein-recognition domain common to spliceosomal components and phosphorylates PTBP2, counteracting the role of this splicing factor in exon exclusion. At the molecular level, phosphorylation of unstructured domains within PTBP2 causes its dissociation from two co-regulators, Matrin3 and hnRNPM, and hinders the RNA-binding capability of the complex. Furthermore, KIS and PTBP2 display strong and opposing functional interactions in synaptic spine emergence and maturation. Taken together, our data uncover a post-translational control of splicing regulators that link transcriptional and alternative exon usage programs in neuronal development.

    1. Genetics and Genomics
    2. Neuroscience
    Kenneth Chiou, Noah Snyder-Mackler
    Insight

    Single-cell RNA sequencing reveals the extent to which marmosets carry genetically distinct cells from their siblings.