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Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

Nature Methods volume 8, pages 153–158 (2011)Cite this article

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Abstract

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.

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Figure 1: Illumination system for live animal tracking and optogenetic stimulation and quantification of behavior elicited by targeted illumination.
Figure 2: Optical stimulation of anterior/posterior mechanosensory neurons or forward/backward command interneurons.
Figure 3: Quantification of behavioral responses elicited by different anterior illumination intensities.
Figure 4: Illumination patterns used to explore the integration of anterior and posterior signals and behavior generated from the stimulation.
Figure 5: Simultaneous two-color illumination.

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Acknowledgements

We thank members of the Caenorhabditis Genetic Center, W. Schafer and Y. Tanizawa (Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK) and E. Boyden (Massachusetts Institute of Technology) for reagents; the US National Institutes of Health (H.L.), Alfred P. Sloan Foundation (H.L.), the Human Frontier Science Program Organization—HFSPO (S.J.H.), the Deutsche Forschungsgemeinschaft, grants GO1011/2-1, SFB807-P11, FOR1279-P1 and Cluster of Excellence Frankfurt, Macromolecular Complexes (A.G.) for funding; and K. Erbguth for discussions. We also thank J. Andrews and B. Parker in our machine shop.

Author information

Authors and Affiliations

  1. School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    Jeffrey N Stirman & Hang Lu

  2. Interdisciplinary Program in Bioengineering, Institute of Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    Jeffrey N Stirman, Matthew M Crane & Hang Lu

  3. Johann Wolfgang Goethe University, Institute of Biochemistry, Biocenter N220, Frankfurt, Germany

    Steven J Husson, Sebastian Wabnig, Christian Schultheis & Alexander Gottschalk

  4. Frankfurt Institute for Molecular Life Sciences, Johann Wolfgang Goethe University, Frankfurt, Germany

    Steven J Husson, Sebastian Wabnig, Christian Schultheis & Alexander Gottschalk

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  1. Jeffrey N Stirman
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Contributions

J.N.S., M.M.C., A.G. and H.L. designed the experiments. J.N.S. and M.M.C. wrote the software. J.N.S. constructed the illumination system, performed experiments and analyzed the data. S.J.H., S.W. and C.S. contributed to reagents and provided valuable discussions. J.N.S., M.M.C., S.J.H., A.G. and H.L. prepared the manuscript.

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Correspondence to Alexander Gottschalk or Hang Lu.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Notes 1–2 (PDF 779 kb)

Supplementary Video 1

Using the dorsal coiling effect and head illumination to dictate the locomotive path of an animal expressing ChR2 in the cholinergic motor neurons. False color recreation was overlaid to show stimulation location and timing. (MOV 175 kb)

Supplementary Video 2

Direct muscular control of a paralyzed worm using light. Worms expressing ChR2 in the muscle cells were paralyzed with an ivermectin solution (0.01 mg ml-1) and were controlled using structured illumination. False color recreation was overlaid to show stimulation location and timing. (MOV 663 kb)

Supplementary Video 3

Illumination line scans performed on freely behaving pmec-4::ChR2 animals eliciting acceleration or reversal behavior depending on the location of illumination. The line scan travels from posterior to anterior and, in separate experiments, anterior to posterior at ~100 μm s-1. Simultaneous blue and red light was used in order to visualize the location of illumination. (MOV 1194 kb)

Supplementary Video 4

Using localized light stimulation to excite anterior or posterior gentle touch neurons (in animals carrying pmec-4::ChR2), and anterior or posterior command interneurons (in animals carrying pglr-1::ChR2). False color recreation was overlaid to show stimulation location and timing. (MOV 2109 kb)

Supplementary Video 5

Examples of four response states ('NR', 'Sl/P', 'r', 'R') to optical stimulation of the anterior gentle touch neurons (pmec-4::ChR2). False color recreation was overlaid to show stimulation location and timing. (MOV 1820 kb)

Supplementary Video 6

Demonstration of complex illumination patterns to investigate sensory integration in pmec-4::ChR2 animals: light pulses of gradually increasing intensity were delivered to the anterior touch neurons, and in a separate experiment the same pulses delivered anteriorly while the posterior touch neurons were continuously illuminated. False color recreation was overlaid to show stimulation location and timing. (MOV 1399 kb)

Supplementary Video 7

At certain anterior and posterior illumination intensities the behavior response (forward and reverse locomotion) alternate (in animals carrying pmec-4::ChR2). False color recreation was overlaid to show stimulation location and timing. (MOV 393 kb)

Supplementary Video 8

Using two-color, spatially distinct optical stimuli to rapidly curtail touch avoidance behavior in animals carrying pmec-4::ChR2 and plgr-1::MAC::mCherry. Also shown is the inhibition of a spontaneous reversal, demonstrating that natural, and not merely optogenetically generated reversals, can be inhibited. False color recreation was overlaid to show stimulation location and timing. (MOV 2325 kb)

Supplementary Software

Software used for tracking and illumination control. (ZIP 3483 kb)

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Stirman, J., Crane, M., Husson, S. et al. Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans. Nat Methods 8, 153–158 (2011). https://doi.org/10.1038/nmeth.1555

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