Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform

Abstract

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously.

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Fig. 1: Different assays for assessing movement capacity in C. elegans.
Fig. 2: WF-NTP platform and software.
Fig. 3: Measurements of the eccentricity of the particles and of the coilers.
Fig. 4: Speed–bend and cut-off filters improve the accuracy of the WF-NTP analysis.
Fig. 5: Maximal velocity can be analyzed with the WF-NTP.
Fig. 6: Tracking accuracy at different worm densities and time intervals in 9-cm plates.
Fig. 7: Aldicarb and chemotactic assays can be performed with the WF-NTP.
Fig. 8: Experimental workflow and outline.
Fig. 9: Background subtraction example files.
Fig. 10: Examples of color-coded tracks made by the WF-NTP.
Fig. 11: Examples of faulty parameters.
Fig. 12: Guidance images for platform assembly.
Fig. 13: Examples of analyses carried out with the WF-NTP.
Fig. 14: Effect of sample size on statistical significance.

Data availability

The associated raw data from Figs. 3, 4, 5, 6, 7, 13 and 14 can be accessed via https://doi.org/10.17863/CAM.48480 and demo-data can be accessed via https://doi.org/10.17863/CAM.46983. All other images and movies can be requested via the corresponding authors.

Code availability

The WF-NTP software and plugins can be downloaded from https://github.com/impact27/WF_NTP or https://doi.org/10.5281/zenodo.3630199 (to ensure the version described in this paper: v.3.3.3). We highly recommend downloading the software from GitHub to ensure the latest updates and improvements. The software runs under the license of Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0): https://creativecommons.org/licenses/by-nc-sa/4.0/. The code in this protocol has been peer reviewed.

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Acknowledgements

We thank the Houtkooper lab for feedback and questions on the WF-NTP software. This project was funded by a European Research Council (ERC) starting grant (281622 PDControl to E.A.A.N.); the Alumni chapter Gooische Groningers, facilitated by the Ubbo Emmius Fonds (to E.A.A.N.); and an Aspasia Fellowship from NWO (015.014.005 to E.A.A.N.).

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Contributions

M.K. performed most experiments, optimized the final experimental pipeline, adjusted the current WF-NTP software and added new applications and wrote the manuscript together with R.I.S. and E.A.A.N., with contributions from all authors. Q.P. was extensively involved in rewriting, problem solving, editing and optimization of the WF-NTP software. R.I.S. performed experiments on the neurodegenerative strains and helped to verify parameters of the WF-NTP software. M.P., M.V., C.M.D. and T.P.J.K. pioneered the use of the WF-NTP and developed the initial software and platform. M.K., R.I.S. and E.A.A.N. were extensively involved in discussions and interpretations of results.

Corresponding authors

Correspondence to Mandy Koopman or Ellen A. A. Nollen.

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Peer review information Nature Protocols thanks Christophe Restif and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

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Delivoria, D. C. et al. Sci. Adv. 5, eaax5108 (2019): https://doi.org/10.1126/sciadv.aax5108

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Koopman, M., Peter, Q., Seinstra, R.I. et al. Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform. Nat Protoc 15, 2071–2106 (2020). https://doi.org/10.1038/s41596-020-0321-9

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