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Optogenetic inhibition of behavior with anion channelrhodopsins

Nature Methods volume 14, pages 271–274 (2017)Cite this article

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Abstract

Optogenetics uses light exposure to manipulate physiology in genetically modified organisms. Abundant tools for optogenetic excitation are available, but the limitations of current optogenetic inhibitors present an obstacle to demonstrating the necessity of neuronal circuits. Here we show that anion channelrhodopsins can be used to specifically and rapidly inhibit neural systems involved in Drosophila locomotion, wing expansion, memory retrieval and gustation, thus demonstrating their broad utility in the circuit analysis of behavior.

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Figure 1: G. theta anion channelrhodopsins are inhibitors of motor function and neuronal spiking.
Figure 2: Flies avoid optogenetic suppression of sweet-taste receptors.

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Acknowledgements

We thank J. Spudich (University of Texas Health Science Center at Houston) for providing the GtACR1 and GtACR2 sequences and plasmids; J.A. Veenstra (Université de Bordeaux) for the anti-Crz antibody; G. Rubin (Howard Hughes Medical Institute) and J. Park (University of Tennessee, Knoxville) for providing materials; S. Aw (Institute of Molecular and Cell Biology) for loan of the high-speed camera; G. Augustine for reading the manuscript; L. Robinson (Insight Editing London) for editing of the manuscript; and S.Y.H. Tan for drawing the behavior rig diagrams. F.M., S.O., J.Y.C. and A.C.-C. were supported by grant MOE-2013-T2-2-054 from the Ministry of Education; J.C.S. and A.C.-C. were supported by grants 1231AFG030 and 1431AFG120 from the A*STAR Joint Council Office. J.H. was supported by the A*STAR Scientific Scholars Fund. T.-W.K. was supported by the National Research Foundation Fellowship NRF-NRFF2015-06 and a block grant from the Temasek Life Sciences Laboratory. The authors were supported by a Biomedical Research Council block grant to the Institute of Molecular and Cell Biology. F.M., S.O., K.C. and A.C.-C. received support from the Duke-NUS Medical School, including the Integrated Biology and Medicine doctoral program (to K.C.).

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Authors and Affiliations

  1. Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, Singapore

    Farhan Mohammad, Stanislav Ott, Katarina Chlebikova, Jia Yi Chua & Adam Claridge-Chang

  2. Institute for Molecular and Cell Biology, Agency for Science Technology and Research, Singapore

    James C Stewart, Joses Ho & Adam Claridge-Chang

  3. Temasek Life Sciences Laboratory, Singapore

    Tong-Wey Koh

  4. Department of Physiology, National University of Singapore, Singapore

    Adam Claridge-Chang

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  1. Farhan Mohammad
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Contributions

Conceptualization, F.M. and A.C.-C.; methodology, F.M., J.C.S. and A.C.-C.; software, J.C.S. (CRITTA, LabView) and J.H. (Python); investigation, F.M. (transgenic design, genetics, falling, walking immobilization, wing expansion, eye toxicity, valence, PER and neuroanatomy), J.C.S. (falling and high-frame rate paralysis), S.O. (learning and survival), J.Y.C. (brain dissection, immunohistochemistry and microscopy), K.C. (PER) and T.-W.K. (electrophysiology); resources, J.C.S. (instrumentation); data analysis, J.H. (valence, electrophysiology), K.C. (PER), J.C.S. (paralysis, falling and walking immobilization), S.O. (STM) and F.M. (paralysis, valence and anatomy); writing original draft, F.M. and A.C.-C. with contributions from all authors; writing revision, F.M., S.O., J.C.S. and A.C.-C.; visualization, F.M., J.H., J.C.S., S.O., K.C. and A.C.-C.; supervision, A.C.-C.; project administration, A.C.-C.; funding acquisition, A.C.-C.

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Correspondence to Adam Claridge-Chang.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 24927 kb)

GtACR flies fall from a vertical surface when illuminated

Flies expressing one of three optogenetic inhibitors in their cholinergic neurons (Cha-Gal4>UAS-GtACR1, Cha-Gal4>UAS-GtACR2 and ChaGal4>UAS-eNpHR) were illuminated with light from a projector. Cha>GtACR1 and Cha>GtACR2 flies fell from the vertical acrylic surface upon exposure to green or blue light respectively, and were immobilized. Cha>GtACR2 flies retained some motor activity while illuminated with blue light. Cha>eNpHR flies did not fall upon exposure to red light and remained mobile. (MP4 9806 kb)

GtACR flies are immobilized by illumination

A. Green light at 38 μW/mm2 rendered a Cha>GtACR1 fly immobile, though it regained some motor control during illumination. Green dot indicates when light was turned on. B. Illumination of a GtACR1/+ fly with 38 μW/mm2 green light had no effect. C. A Cha>GtACR2 fly was rendered completely paralyzed by illumination with 391 μW/mm2 blue light. Blue dot indicates when light was turned on. D. A GtACR2/+ fly was unaffected by illumination with 391 μW/mm2 blue light. E. While positioned 3 mm above an amber LED (approximately 1.9 mW/mm2), a Cha>eNpHR fly retained mobility, though it was paralyzed transiently when passing directly above the emitter. Light was on throughout this recording. F. A Cha>eNpHR fly was unaffected by amber illumination at 495 μW/mm2. Amber dot indicates when light was on. (MP4 25723 kb)

Supplementary Video 3

Cha>GtACR flies adopt a static pose during illumination (indicated by colored dots), but Cha>Chrimson flies have active seizures and adopt a tetanic pose with extended wings. Control animals were unaffected by projector light (green 92 μW/mm2; blue 67 μW/mm2; red 70 μW/mm2). (MP4 25469 kb)

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Mohammad, F., Stewart, J., Ott, S. et al. Optogenetic inhibition of behavior with anion channelrhodopsins. Nat Methods 14, 271–274 (2017). https://doi.org/10.1038/nmeth.4148

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