Extracellular space preservation aids the connectomic analysis of neural circuits
- National Institutes of Health, United States
- 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.
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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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Extracellular space preservation aids the connectomic analysis of neural circuits
eLife 4:e08206.
https://doi.org/10.7554/eLife.08206
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