Independent optical excitation of distinct neural populations

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  • An Addendum to this article was published on 28 August 2014
  • A Corrigendum to this article was published on 28 August 2014


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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Figure 1: Novel channelrhodopsin spectral classes discovered through algal transcriptome sequencing.
Figure 2: Comparison of optical spiking in cultured neurons expressing different channelrhodopsins.
Figure 3: Chrimson evokes action potentials in larval Drosophila motor neurons and triggers stereotyped behavior in adult Drosophila.
Figure 4: Characterization of channelrhodopsin blue light (470-nm) sensitivities for two-color excitation in cultured neurons.
Figure 5: Independent optical excitation of neural populations in mouse cortical slice using Chrimson and Chronos.

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  1. 1

    Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

  2. 2

    Han, X. & Boyden, E.S. Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS ONE 2, e299 (2007).

  3. 3

    Chow, B.Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).

  4. 4

    Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

  5. 5

    Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

  6. 6

    Boyden, E.S. A history of optogenetics: the development of tools for controlling brain circuits with light. F1000 Biol. Rep. 3, 11 (2011).

  7. 7

    Zhang, F. et al. Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat. Neurosci. 11, 631–633 (2008).

  8. 8

    Erbguth, K., Prigge, M., Schneider, F., Hegemann, P. & Gottschalk, A. Bimodal activation of different neuron classes with the spectrally red-shifted channelrhodopsin chimera C1V1 in Caenorhabditis elegans. PLoS ONE 7, e46827 (2012).

  9. 9

    Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).

  10. 10

    Prigge, M. et al. Color-tuned channelrhodopsins for multiwavelength optogenetics. J. Biol. Chem. 287, 31804–31812 (2012).

  11. 11

    Wang, W. et al. Tuning the electronic absorption of protein-embedded all-trans-retinal. Science 338, 1340–1343 (2012).

  12. 12

    Waddell, W.H., Schaffer, A.M. & Becker, R.S. Visual pigments. 3. Determination and interpretation of the fluorescence quantum yields of retinals, Schiff bases, and protonated Schiff bases. J. Am. Chem. Soc. 95, 8223–8227 (1973).

  13. 13

    Govorunova, E.G., Spudich, E.N., Lane, C.E., Sineshchekov, O.A. & Spudich, J.L. New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride. mBio 2, e00115–e00111 (2011).

  14. 14

    Lin, J.Y., Knutsen, P.M., Muller, A., Kleinfeld, D. & Tsien, R.Y. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat. Neurosci. 16, 1499–1508 (2013).

  15. 15

    Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh. Nat. Neurosci. 14, 513–518 (2011).

  16. 16

    Berndt, A., Yizhar, O., Gunaydin, L.A., Hegemann, P. & Deisseroth, K. Bi-stable neural state switches. Nat. Neurosci. 12, 229–234 (2009).

  17. 17

    Bamann, C., Gueta, R., Kleinlogel, S., Nagel, G. & Bamberg, E. Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond. Biochemistry 49, 267–278 (2010).

  18. 18

    Mattis, J. et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods 9, 159–172 (2012).

  19. 19

    Govorunova, E.G., Sineshchekov, O.A., Li, H., Janz, R. & Spudich, J.L. Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis. J. Biol. Chem. 288, 29911–29922 (2013).

  20. 20

    Johnson, M.T. et al. Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes. PLoS ONE 7, e50226 (2012).

  21. 21

    Lin, J.Y. A user's guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 96, 19–25 (2011).

  22. 22

    Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 2279–2284 (2005).

  23. 23

    de Vries, S.E. & Clandinin, T.R. Loom-sensitive neurons link computation to action in the Drosophila visual system. Curr. Biol. 22, 353–362 (2012).

  24. 24

    Schroll, C. et al. Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr. Biol. 16, 1741–1747 (2006).

  25. 25

    Gaudry, Q., Hong, E.J., Kain, J., de Bivort, B.L. & Wilson, R.I. Asymmetric neurotransmitter release enables rapid odour lateralization in Drosophila. Nature 493, 424–428 (2013).

  26. 26

    Honjo, K., Hwang, R.Y. & Tracey, W.D. Jr. Optogenetic manipulation of neural circuits and behavior in Drosophila larvae. Nat. Protoc. 7, 1470–1478 (2012).

  27. 27

    Zhang, W., Ge, W. & Wang, Z. A toolbox for light control of Drosophila behaviors through Channelrhodopsin 2-mediated photoactivation of targeted neurons. Eur. J. Neurosci. 26, 2405–2416 (2007).

  28. 28

    Xiang, Y. et al. Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468, 921–926 (2010).

  29. 29

    Inagaki, H.K. et al. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat. Methods 10.1038/nmeth.2765 (22 December 2013).

  30. 30

    Claridge-Chang, A. et al. Writing memories with light-addressable reinforcement circuitry. Cell 139, 405–415 (2009).

  31. 31

    Pulver, S.R., Pashkovski, S.L., Hornstein, N.J., Garrity, P.A. & Griffith, L.C. Temporal dynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determine behavioral output in Drosophila larvae. J. Neurophysiol. 101, 3075–3088 (2009).

  32. 32

    Bernstein, J.G., Garrity, P.A. & Boyden, E.S. Optogenetics and thermogenetics: technologies for controlling the activity of targeted cells within intact neural circuits. Curr. Opin. Neurobiol. 22, 61–71 (2012).

  33. 33

    Hwang, R.Y. et al. Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr. Biol. 17, 2105–2116 (2007).

  34. 34

    Dahanukar, A., Lei, Y.T., Kwon, J.Y. & Carlson, J.R. Two Gr genes underlie sugar reception in Drosophila. Neuron 56, 503–516 (2007).

  35. 35

    Minke, B. & Kirschfeld, K. The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin. J. Gen. Physiol. 73, 517–540 (1979).

  36. 36

    Salcedo, E. et al. Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins. J. Neurosci. 19, 10716–10726 (1999).

  37. 37

    Lin, H.H., Chu, L.A., Fu, T.F., Dickson, B.J. & Chiang, A.S. Parallel neural pathways mediate CO2 avoidance responses in Drosophila. Science 340, 1338–1341 (2013).

  38. 38

    Atasoy, D., Aponte, Y., Su, H.H. & Sternson, S.M. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008).

  39. 39

    Zorzos, A.N., Scholvin, J., Boyden, E.S. & Fonstad, C.G. Three-dimensional multiwaveguide probe array for light delivery to distributed brain circuits. Opt. Lett. 37, 4841–4843 (2012).

  40. 40

    Kim, T.I. et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics. Science 340, 211–216 (2013).

  41. 41

    Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).

  42. 42

    Stockklausner, C. & Klocker, N. Surface expression of inward rectifier potassium channels is controlled by selective Golgi export. J. Biol. Chem. 278, 17000–17005 (2003).

  43. 43

    Stockklausner, C., Ludwig, J., Ruppersberg, J.P. & Klocker, N. A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K+ channels. FEBS Lett. 493, 129–133 (2001).

  44. 44

    Gradinaru, V., Thompson, K.R. & Deisseroth, K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 36, 129–139 (2008).

  45. 45

    Ma, D. et al. Role of ER export signals in controlling surface potassium channel numbers. Science 291, 316–319 (2001).

  46. 46

    Pfeiffer, B.D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).

  47. 47

    Mahr, A. & Aberle, H. The expression pattern of the Drosophila vesicular glutamate transporter: a marker protein for motoneurons and glutamatergic centers in the brain. Gene Expr. Patterns 6, 299–309 (2006).

  48. 48

    Pfeiffer, B.D. et al. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105, 9715–9720 (2008).

  49. 49

    Guo, A. et al. Conditioned visual flight orientation in Drosophila: dependence on age, practice, and diet. Learn. Mem. 3, 49–59 (1996).

  50. 50

    Reiser, M.B. & Dickinson, M.H. A modular display system for insect behavioral neuroscience. J. Neurosci. Methods 167, 127–139 (2008).

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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.

Author information

N.C.K., E.S.B., M.C.-P. and V.J. contributed to the study design and data analysis. G.K.-S.W. and B.Y.C. oversaw transcriptomic sequencing. E.S.B. and M.C.-P. supervised mammalian opto/electrophysiological parts of the project. N.C.K. coordinated all experiments and data analysis. N.C.K., Y.K.C., A.S.C. and T.K.M. conducted and analyzed all in vitro electrophysiology. M.M., B.S., N.C.K., T.K.M., E.J.C., Z.T., J.W., Y.X., Z.Y. and Y.Z. conducted algal RNA experiments or transcriptome sequencing and analysis. N.C.K., Y.M. and A.B.-B. performed and analyzed all slice electrophysiology. V.J. prepared Chrimson for injection into Drosophila. S.S.K. and V.J. designed adult fly behavior experiments. S.S.K. performed all fly behavior experiments and data analysis. S.R.P. designed, performed and analyzed all larval Drosophila experiments. Correspondence should be addressed to V.J. ( for Chrimson flies. All authors contributed to the discussions and writing of the manuscript.

Correspondence to Gane Ka-Shu Wong or Edward S Boyden.

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Competing interests

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 information

Supplementary Text and Figures

Supplementary Figures 1–22 and Supplementary Tables 1–4 (PDF 5903 kb)

Experimental setup with a visual arena

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)

PER of a Gr64f X Chrimson fly to 720 nm light in darkness

A fly with Chrimson expression in sugar receptors shows PER to deep red light stimulation. (AVI 2519 kb)

Startle response to 720 nm light in darkness

A control fly without Chrimson expression shows clear startle response to deep red light. (AVI 3276 kb)

PER of a Gr64f X Chrimson fly to 720 nm light in a blue random dot arena

PER of a fly with Chrimson expression in sugar receptors is not affected by visual distractors. (AVI 2326 kb)

Inhibited startle response to 720 nm light in a blue random dot arena

The startle response of a control fly without Chrimson expression is effectively inhibited. (AVI 1975 kb)

Optogenetics in freely behaving intact flies

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) doi:10.1038/nmeth.2836

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