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Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications

Alexej Dawydow, Ronnie Gueta, Dmitrij Ljaschenko, Sybille Ullrich, Moritz Hermann, Nadine Ehmann, Shiqiang Gao, André Fiala, Tobias Langenhan, Georg Nagel, and Robert J. Kittel
PNAS published ahead of print September 8, 2014 https://doi.org/10.1073/pnas.1408269111
Alexej Dawydow
aDepartment of Neurophysiology, Institute of Physiology, Julius-Maximilians-University of Würzburg, D-97070 Würzburg, Germany;
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Ronnie Gueta
bInstitute for Molecular Plant Physiology and Biophysics, Biocenter, Julius-Maximilians-University of Würzburg, D-97082 Würzburg, Germany; and
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Dmitrij Ljaschenko
aDepartment of Neurophysiology, Institute of Physiology, Julius-Maximilians-University of Würzburg, D-97070 Würzburg, Germany;
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Sybille Ullrich
bInstitute for Molecular Plant Physiology and Biophysics, Biocenter, Julius-Maximilians-University of Würzburg, D-97082 Würzburg, Germany; and
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Moritz Hermann
cDepartment of Molecular Neurobiology of Behaviour, Georg-August-University of Göttingen, D-37077 Göttingen, Germany
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Nadine Ehmann
aDepartment of Neurophysiology, Institute of Physiology, Julius-Maximilians-University of Würzburg, D-97070 Würzburg, Germany;
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Shiqiang Gao
bInstitute for Molecular Plant Physiology and Biophysics, Biocenter, Julius-Maximilians-University of Würzburg, D-97082 Würzburg, Germany; and
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André Fiala
cDepartment of Molecular Neurobiology of Behaviour, Georg-August-University of Göttingen, D-37077 Göttingen, Germany
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Tobias Langenhan
aDepartment of Neurophysiology, Institute of Physiology, Julius-Maximilians-University of Würzburg, D-97070 Würzburg, Germany;
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Georg Nagel
bInstitute for Molecular Plant Physiology and Biophysics, Biocenter, Julius-Maximilians-University of Würzburg, D-97082 Würzburg, Germany; and
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  • For correspondence: robert.kittel@uni-wuerzburg.denagel@uni-wuerzburg.de
Robert J. Kittel
aDepartment of Neurophysiology, Institute of Physiology, Julius-Maximilians-University of Würzburg, D-97070 Würzburg, Germany;
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  • For correspondence: robert.kittel@uni-wuerzburg.denagel@uni-wuerzburg.de
  1. Edited by Hartmut Michel, Max Planck Institute of Biophysics, Frankfurt, Germany, and approved August 12, 2014 (received for review May 8, 2014)

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Significance

Controlling neuronal activity in live tissue is a long sought-after goal in the neurosciences. Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that can be genetically expressed to depolarize neurons with light. Thereby, this “optogenetic tool” delivers cellular specificity and elegant options for studying the neuronal basis of behavior in intact organisms. Unfortunately, low-light transmission through pigmented tissue greatly complicates light delivery to target cells and curtails experiments in freely moving animals. This study introduces a ChR mutant, ChR2-XXL, that gives rise to the largest photocurrents of all ChRs published so far and increases light sensitivity more than 10,000-fold over wild-type ChR2 in Drosophila larvae. As a result, behavioral photostimulation is evoked in freely moving flies using diffuse, ambient light.

Abstract

Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.

Footnotes

  • ↵1A.D. and R.G. contributed equally to this work.

  • ↵2To whom correspondence may be addressed. Email: robert.kittel{at}uni-wuerzburg.de or nagel{at}uni-wuerzburg.de.
  • Author contributions: A.F., T.L., G.N., and R.J.K. designed research; A.D., R.G., D.L., S.U., M.H., N.E., S.G., G.N., and R.J.K. performed research; A.D., R.G., D.L., S.U., M.H., N.E., S.G., A.F., T.L., G.N., and R.J.K. analyzed data; and A.F., G.N., and R.J.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1408269111/-/DCSupplemental.

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Introducing Channelrhodopsin-2-XXL
Alexej Dawydow, Ronnie Gueta, Dmitrij Ljaschenko, Sybille Ullrich, Moritz Hermann, Nadine Ehmann, Shiqiang Gao, André Fiala, Tobias Langenhan, Georg Nagel, Robert J. Kittel
Proceedings of the National Academy of Sciences Sep 2014, 201408269; DOI: 10.1073/pnas.1408269111

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Introducing Channelrhodopsin-2-XXL
Alexej Dawydow, Ronnie Gueta, Dmitrij Ljaschenko, Sybille Ullrich, Moritz Hermann, Nadine Ehmann, Shiqiang Gao, André Fiala, Tobias Langenhan, Georg Nagel, Robert J. Kittel
Proceedings of the National Academy of Sciences Sep 2014, 201408269; DOI: 10.1073/pnas.1408269111
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