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Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications
Edited by Hartmut Michel, Max Planck Institute of Biophysics, Frankfurt, Germany, and approved August 12, 2014 (received for review May 8, 2014)

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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