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Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications

Nature Protocols volume 9, pages 1533–1548 (2014)Cite this article

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Abstract

This article describes a method to quantify the movements of larval zebrafish in multiwell plates, using the open-source MATLAB applications LSRtrack and LSRanalyze. The protocol comprises four stages: generation of high-quality, flatly illuminated video recordings with exposure settings that facilitate object recognition; analysis of the resulting recordings using tools provided in LSRtrack to optimize tracking accuracy and motion detection; analysis of tracking data using LSRanalyze or custom MATLAB scripts; and implementation of validation controls. The method is reliable, automated and flexible, requires <1 h of hands-on work for completion once optimized and shows excellent signal:noise characteristics. The resulting data can be analyzed to determine the following: positional preference; displacement, velocity and acceleration; and duration and frequency of movement events and rest periods. This approach is widely applicable to the analysis of spontaneous or stimulus-evoked zebrafish larval neurobehavioral phenotypes resulting from a broad array of genetic and environmental manipulations, in a multiwell plate format suitable for high-throughput applications.

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Figure 1: Experimental design.
Figure 2: Equipment.
Figure 3: LSRtrack user interface.
Figure 4: Troubleshooting LSRtrack.
Figure 5: LSRanalyze user interface.
Figure 6: Typical results from the protocol.
Figure 7: LSRtrack error reports.
Figure 8: Stability of group motor activity.

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Acknowledgements

Development of LSRtrack and the current protocol was supported by research grants from the National Institute of Neurological Disorders and Stroke (NINDS) (NS080881 and NS058369), the National Institute of Environmental Health Sciences (NIEHS) (ES022644), the Pittsburgh Foundation (M2005-0071) and the Society for Progressive Supranuclear Palsy (468-08). Y.Z. is a Tsinghua Scholar at the University of Pittsburgh.

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Authors and Affiliations

  1. Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Yangzhong Zhou, Richard T Cattley, Clinton L Cario, Qing Bai & Edward A Burton

  2. Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Yangzhong Zhou, Richard T Cattley, Clinton L Cario, Qing Bai & Edward A Burton

  3. Tsinghua University Medical School, Beijing, China

    Yangzhong Zhou

  4. Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Edward A Burton

  5. Geriatric Research, Education and Clinical Center, Pittsburgh Veterans' Affairs Healthcare System, Pittsburgh, Pennsylvania, USA

    Edward A Burton

  6. Department of Neurology, Pittsburgh Veterans' Affairs Healthcare System, Pittsburgh, Pennsylvania, USA

    Edward A Burton

Authors
  1. Yangzhong Zhou
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  2. Richard T Cattley
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  3. Clinton L Cario
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  4. Qing Bai
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  5. Edward A Burton
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Contributions

Y.Z., C.L.C. and E.A.B. designed and wrote the software; Y.Z., R.T.C., Q.B. and E.A.B. developed the experimental protocol; Y.Z., R.T.C. and Q.B. carried out the experiments; and Y.Z., R.T.C. and E.A.B. wrote the manuscript.

Corresponding author

Correspondence to Edward A Burton.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Instructions and drawings are shown for construction of optional equipment used in the protocol. (PDF 508 kb)

Supplementary Software

Software is adapted from Cario, C.L., Farrell, T.C., Milanese, C. & Burton, E.A. Automated measurement of zebrafish larval movement. J. Physiol. 589 3703–3708 (2011) with permission from Wiley (© 2011 The Authors. Journal compilation © 2011 The Physiological Society). (ZIP 46 kb)

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Zhou, Y., Cattley, R., Cario, C. et al. Quantification of larval zebrafish motor function in multiwell plates using open-source MATLAB applications. Nat Protoc 9, 1533–1548 (2014). https://doi.org/10.1038/nprot.2014.094

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