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High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments

Nature Methods volume 8, pages 599605 (2011) | Download Citation

Abstract

To quantitatively understand chemosensory behaviors, it is desirable to present many animals with repeatable, well-defined chemical stimuli. To that end, we describe a microfluidic system to analyze Caenorhabditis elegans behavior in defined temporal and spatial stimulus patterns. A 2 cm × 2 cm structured arena allowed C. elegans to perform crawling locomotion in a controlled liquid environment. We characterized behavioral responses to attractive odors with three stimulus patterns: temporal pulses, spatial stripes and a linear concentration gradient, all delivered in the fluid phase to eliminate variability associated with air-fluid transitions. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. We identified both expected and unexpected responses in wild-type worms and sensory mutants by quantifying dozens of behavioral parameters. The devices are inexpensive, easy to fabricate, reusable and suitable for delivering any liquid-borne stimulus.

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Acknowledgements

We thank S. Leibler for the use of his clean-room facility, J. Larsch for control experiments and assistance with manual scoring of behavior, and members of the Bargmann laboratory for critical help, insight and advice. This work was supported by the Howard Hughes Medical Institute and by the G. Harold and Leila Y. Mathers Foundation. C.I.B. is supported by the Howard Hughes Medical Institute. D.R.A. is supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

Author information

Affiliations

  1. Howard Hughes Medical Institute and Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, New York, USA.

    • Dirk R Albrecht
    •  & Cornelia I Bargmann

Authors

  1. Search for Dirk R Albrecht in:

  2. Search for Cornelia I Bargmann in:

Contributions

D.R.A. designed and fabricated the devices, wrote the analysis code, performed the experiments and analyzed data. D.R.A. and C.I.B. designed the experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dirk R Albrecht.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12, Supplementary Table 1, Supplementary Notes 1–4

  2. 2.

    Supplementary Data 1

    Microfluidic channel pattern file.

Videos

  1. 1.

    Supplementary Video 1

    Worm locomotion in the structured microfluidic environment. Real-time video shows a wild-type young adult worm crawling forward through the arena, pausing briefly (at 8 s), then performing two pirouettes: long reversals (at 12 s and 20 s) followed by a sharp turns (at 17 s and 25 s). Video shows a 2.3 mm × 1.7 mm region. All videos are encoded with the Xvid codec (available at http://www.xvidmovies.com/codec).

  2. 2.

    Supplementary Video 2

    Loading worms into a device. Video shows 18 wild-type (right arena) and 22 tax-4 (left arena) worms loaded simultaneously into a single device containing two 16 mm A 15 mm arenas. Wild-type worms paused and reversed frequently for about 20 min, whereas tax-4 mutants quickly dispersed. tax-4 worms did not respond to the central stripe containing 0.92 μM isoamyl alcohol (IAA) odor and dye for visualization. Video is accelerated 15×.

  3. 3.

    Supplementary Video 3

    Annotated video of an odor pulse assay. Video shows tracking and analysis of 23 wild-type worms responding to repeated 0.92 μM IAA odor stimulation according to the pulse sequence in Figure 3a. Each worm is numbered in the arena and labeled with its current centroid (circle and green +) and a line indicating 30-s of centroid history. Line color indicates behavioral history according to the legend, for example, white indicates forward movement and red indicates the pirouette state. The ethogram (right) shows a scrolling 45-s window of worm behavior, time-adjusted such that each worm's response lines up with the odor pulse pattern. Green arrowheads on the ethogram indicate the position of each worm at the current video frame. Tick marks represent 15 s. Video is accelerated 15×.

  4. 4.

    Supplementary Video 4

    Dynamic odor stripe assay. Video shows the locomotor responses of 25 worms to two attractive odor stripes (0.92 μM and 1.84 μM IAA, top to bottom) separated by odor-free buffer. Flow is left to right, and odor stripes contain dye for visualization. Worms experience a sharp odor gradient at the stripe edges but no mechanical or flow rate discontinuity. Near the end of the video, odor stripes are turned off by closing external valves. Video is accelerated 15×.

  5. 5.

    Supplementary Video 5

    Example behavioral responses upon odor stripe exit. Odor (0.92 μM IAA) is present in the lower half of the video (gray). Three outward encounters of the odor stripe edge are shown; each response is shown twice. The first worm responds with a correct forward turn, the second with a correct pirouette and the third with an incorrect pirouette. 'Correct' refers to the worm remaining in the attractive odor stripe after the response. Video shows a 3.9 mm × 3.3 mm region and is accelerated 15×.

Zip files

  1. 1.

    Supplementary Software 1

    Software to identify instantaneous behavioral states from video, and to summarize behavior and speed data over space and time.

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DOI

https://doi.org/10.1038/nmeth.1630

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