Published: April 4th, 2021
This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.
Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.
Caenorhabiditis elegans is widely recognized as an outstanding model in neuroscience based on it having 302 neurons that coordinate all worm behaviors, including mating, feeding, egg-laying, defecation, swimming, and locomotion on solid media1. These hermaphroditic nematodes are also widely used to understand a wide array of human disease mechanisms, made possible by its well-characterized genome and high homology of ~80% genes between C. elegans and humans2,3,4. C. elegans have long been used to interrogate human mitochondri....
1. Worm locomotor activity analysis in liquid media on glass slides using ZebraLab software
Analysis of C. elegans locomotor activity in the liquid media could easily capture an integrated phenotype of mitochondrial disease worm models that may not be easily quantifiable on solid media. ZebraLab was used to quantify locomotor activity of the well-established mitochondrial complex I disease gas-1(fc21) strain relative to WTworms in liquid media at the L4 larval stage. The activity of 5 worms in a single liquid drop was recorded over 1 min, with a total of 19 videos (technical replicate.......
Here, the study summarized detailed information and rationales for studying C. elegans neuromuscular activity at the level of diverse outcomes, including worm thrashing, locomotion, pharyngeal pumping, and chemotaxis. The comparison of 16 different activity analysis methodologies was performed in terms of the relative throughput, advantages, and limitations of quantify nematode activities in a single worm or worm populations at different ages and experimental durations. Among these, two novel adaptations and.......
We are grateful to Anthony Rosner, PhD., with his organizational support for the early preparation of this project, and to Erin Haus for contributing to protocol analysis. This work was funded by the Juliet's Cure FBXL4 Mitochondrial Disease Research Fund, the Jaxson Flynt C12ORF65 Research Fund, and the National Institutes of Health (R01-GM120762, R01-GM120762-08S1, R35-GM134863, and T32-NS007413). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders or the National Institutes of Health.....
|C. elegans wild isolate
|Caenorhabditis Genetics Center (CGC)
|Nematode Growth Medium (NGM)
|Research Products International Corp.
|OP50 Escherichia coli
|Uracil auxotroph E. coli strain
|Petri dishes (60 mm)
|VWR 5.85 g NaCl, 1 g K2 HPO4, 6 g KH2PO4, and 5 mg cholesterol, in 1 l H2O
|VWR 101175-162, 103467-156, EM1.09828.1000, 97061-660
|96-well flat bottom
|Mathew et al. 45
|S1 Standalone Java platform
|Software for automation of difference image of scanned plates
|Software for automated quantization and tracking of zebrafish behavior, designed by ViewPoint (http://www.viewpoint.fr/en/p/software/zebralab-zebrafish-behavior-screening) and here applied to C. elegans. This system is applicable for high-throughput behavioral analysis
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