Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system–mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.
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We thank A. Karpova (Janelia Farm) for technical advice, reagents and generous assistance with construct preparation for Drosophila; K. Hibbard and members of the Janelia Fly Core for fly husbandry and assistance with fly crosses; and J. Pulver for technical advice and assistance with data analysis software. We thank Y. Aso, W. Ming and G. Rubin (Janelia Farm) for kindly allowing us to use their circular light arena and for useful discussion. We thank I. Negrashov, S. Sawtelle and J. Liu for arena-related development and support. We thank J.R. Carlson (Yale University) for Gr64f-Gal4 flies, W.D. Tracey Jr. (Duke University) for UAS-Chr2 flies, G.M. Rubin (Janelia Farm) for pBDP-Gal4 flies and B.J. Dickson (IMP, Vienna and Janelia Farm) for VT031497-Gal4 flies.
S.S.K., S.R.P. and V.J. were supported by the Howard Hughes Medical Institute. The 1000 Plants (1KP) initiative, led by G.K.-S.W., is funded by the Alberta Ministry of Enterprise and Advanced Education, Alberta Innovates Technology Futures (AITF) Innovates Centre of Research Excellence (iCORE), Musea Ventures, and BGI-Shenzhen. B.Y.C. and E.S.B. were funded by Defense Advanced Research Projects Agency (DARPA) Living Foundries HR0011-12-C-0068. B.Y.C. was funded by the US National Science Foundation (NSF) Biophotonics Program. M.C.-P. was funded by US National Institutes of Health (NIH) grant 5R01EY014074-18. E.S.B. was funded by the MIT Media Lab, Office of the Assistant Secretary of Defense for Research and Engineering, Harvard/MIT Joint grants in Basic Neuroscience, NSF (especially CBET 1053233 and EFRI 0835878), NIH (especially 1DP2OD002002, 1R01NS067199, 1R01DA029639, 1R01GM104948, 1RC1MH088182 and 1R01NS075421), Wallace H. Coulter Foundation, Alfred P. Sloan Foundation, Human Frontiers Science Program, New York Stem Cell Foundation Robertson Neuroscience Investigator Award, Institution of Engineering and Technology A.F. Harvey Prize, and Skolkovo Institute of Science and Technology.
B.Y.C., E.S.B., G.K.-S.W., N.C.K. and Y.K.C. are inventors on pending patents covering the described work. E.S.B. is an equity holder in Eos Neuroscience.
Supplementary Figures 1–22 and Supplementary Tables 1–4 (PDF 5903 kb)
The fly was tethered and centered in the visual arena. In this movie, a flowing random dot pattern is shown. The visual arena was removed from the setup in other conditions. Fly behavior was recorded using a camera with 850 nm IR illuminator. (AVI 3506 kb)
A fly with Chrimson expression in sugar receptors shows PER to deep red light stimulation. (AVI 2519 kb)
A control fly without Chrimson expression shows clear startle response to deep red light. (AVI 3276 kb)
PER of a fly with Chrimson expression in sugar receptors is not affected by visual distractors. (AVI 2326 kb)
The startle response of a control fly without Chrimson expression is effectively inhibited. (AVI 1975 kb)
Top: Light-induced CO2 avoidance behavior (VT031497-Gal4 x UAS-Chrimson in attP18). Bottom: A control group (WTB x UAS-Chrimson in attP18). Circles show raw video images with false color red background indicating the illuminated quadrants. The effect of light is quantified (see Methods) and plotted as a single blue line corresponding to the presented examples and a plot representing the mean of all 9 sessions (±SEM error bars). Plots will be in red region if more than 50% of flies are in illuminated quadrants. Replay speed: 4x. (MP4 6110 kb)
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Klapoetke, N., Murata, Y., Kim, S. et al. Independent optical excitation of distinct neural populations. Nat Methods 11, 338–346 (2014). https://doi.org/10.1038/nmeth.2836
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