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Scanless two-photon excitation of channelrhodopsin-2

Abstract

Light-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2–expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials.

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Figure 1: TF-GPC design.
Figure 2: Two-photon photoactivation of ChR2 by TF-GPC in HEK 293 cells.
Figure 3: Action potential generation by two-photon TF-GPC in primary neuronal culture.
Figure 4: Two-photon photoactivation by TF-GPC in cortical brain slices.
Figure 5: TF-GPC provides lateral and axial precision in ChR2 activation in brain slices.
Figure 6: Multispot photoactivation in cortical slices.

References

  1. Penfield, W. & Rasmussen, T. The cerebral cortex of man: a clinical study of localization of function. J. Am. Med. Assoc. 144, 1412–1700 (1950).

    Google Scholar 

  2. Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940–13945 (2003).

    Article  CAS  Google Scholar 

  3. Gunaydin, L.A. et al. Ultrafast optogenetic control. Nat. Neurosci 13, 387–392 (2010).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).

    Article  CAS  Google Scholar 

  6. Grossman, N. et al. Multi-site optical excitation using ChR2 and micro-LED array. J. Neural Eng. 7, 16004 (2010).

    Article  Google Scholar 

  7. Aravanis, A.M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4, S143–S156 (2007).

    Article  Google Scholar 

  8. Gradinaru, V. et al. Targeting and readout strategies for fast optical neural control in vitro and in vivo. J. Neurosci. 27, 14231–14238 (2007).

    Article  CAS  Google Scholar 

  9. Gradinaru, V., Mogri, M., Thompson, K.R., Henderson, J.M. & Deisseroth, K. Optical deconstruction of parkinsonian neural circuitry. Science 324, 354–359 (2009).

    Article  CAS  Google Scholar 

  10. Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 61–64 (2008).

    Article  CAS  Google Scholar 

  11. Lagali, P.S. et al. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat. Neurosci. 11, 667–675 (2008).

    Article  CAS  Google Scholar 

  12. Feldbauer, K. et al. Channelrhodopsin-2 is a leaky proton pump. Proc. Natl. Acad. Sci. USA 106, 12317–12322 (2009).

    Article  CAS  Google Scholar 

  13. Rickgauer, J.P. & Tank, D.W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl. Acad. Sci. USA 106, 15025–15030 (2009).

    Article  CAS  Google Scholar 

  14. Andrasfalvy, B.K., Zemelman, B.V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. USA 107, 11981–11986 (2010).

    Article  CAS  Google Scholar 

  15. Curtis, J.E., Koss, B.A. & Grier, D.G. Dynamic holographic optical tweezers. Opt. Commun. 207, 169 (2002).

    Article  CAS  Google Scholar 

  16. Lutz, C. et al. Holographic photolysis of caged neurotransmitters. Nat. Methods 5, 821–827 (2008).

    Article  CAS  Google Scholar 

  17. Zahid, M. et al. Holographic photolysis for multiple cell stimulation in mouse hippocampal slices. PLoS ONE 5, e9431 (2010).

    Article  Google Scholar 

  18. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).

    Article  Google Scholar 

  19. Papagiakoumou, E., de Sars, V., Oron, D. & Emiliani, V. Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses. Opt. Express 16, 22039–22047 (2008).

    Article  CAS  Google Scholar 

  20. Papagiakoumou, E., de Sars, V., Emiliani, V. & Oron, D. Temporal focusing with spatially modulated excitation. Opt. Express 17, 5391–5401 (2009).

    Article  CAS  Google Scholar 

  21. Golan, L., Reutsky, I., Farah, N. & Shoham, S. Design and characteristics of holographic neural photo-stimulation systems. J. Neural Eng. 6, 66004 (2009).

    Article  CAS  Google Scholar 

  22. Glückstad, J. Phase contrast image synthesis. Opt. Commun. 130, 225 (1996).

    Article  Google Scholar 

  23. Rodrigo, P.J., Daria, V.R. & Glückstad, J. Real-time three-dimensional optical micromanipulation of multiple particles and living cells. Opt. Lett. 29, 2270–2272 (2004).

    Article  Google Scholar 

  24. Rodrigo, P.J., Palima, D. & Glückstad, J. Accurate quantitative phase imaging using generalized phase contrast. Opt. Express 16, 2740–2751 (2008).

    Article  Google Scholar 

  25. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

    Article  CAS  Google Scholar 

  26. Wang, S. et al. All optical interface for parallel, remote, and spatiotemporal control of neuronal activity. Nano Lett. 7, 3859–3863 (2007).

    Article  CAS  Google Scholar 

  27. Guo, Z.V., Hart, A.C. & Ramanathan, S. Optical interrogation of neural circuits in Caenorhabditis elegans. Nat. Methods 6, 891–896 (2009).

    Article  CAS  Google Scholar 

  28. Shoham, S., O'Connor, D.H., Sarkisov, D.V. & Wang, S.S. Rapid neurotransmitter uncaging in spatially defined patterns. Nat. Methods 2, 837–843 (2005).

    Article  CAS  Google Scholar 

  29. Losavio, B.E., Iyer, V., Patel, S. & Saggau, P. Acousto-optic laser scanning for multi-site photo-stimulation of single neurons in vitro. J. Neural Eng. 7, 045002 (2010).

    Article  Google Scholar 

  30. Kirkby, P.A., Srinivas Nadella, K.M. & Silver, R.A. A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt. Express 18, 13721–13745 (2010).

    Article  Google Scholar 

  31. Glückstad, J. & Mogensen, P.C. Optimal phase contrast in common-path interferometry. Appl. Opt. 40, 268–282 (2001).

    Article  Google Scholar 

  32. Palima, D. & Glückstad, J. Multi-wavelength spatial light shaping using generalized phase contrast. Opt. Express 16, 1331–1342 (2008).

    Article  Google Scholar 

  33. 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).

    Article  CAS  Google Scholar 

  34. Arenkiel, B.R. et al. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54, 205–218 (2007).

    Article  CAS  Google Scholar 

  35. Otsu, Y. et al. Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope. J. Neurosci. Methods 173, 259–270 (2008).

    Article  Google Scholar 

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Acknowledgements

We thank I. Perch-Nielsen for the phase-contrast filter layout design, E. Schwartz for genotyping ChR2-YFP mice, S. Wiese, Z. Fu and M. Viesel for generating cDNA constructs, A. Triller, T. Gally, K. Spence, A. Burgo and K. Zylbersztejn for cell culture preparation, all members of the Neurophysiology and New Microscopy Laboratory for comments and technical help, D. Oron, D. Palima, S. Dieudonné, M. Diana and G. Fortin for helpful discussions, J. Feldmann for critical reading of the paper, Spectra-Physics, Inc. for loan of the high-power laser, and Phasics S.A. for providing the phase-analyzer software. V.E. was supported by the European Science Foundation and the Centre National de la Recherche Scientifique through the European Young Investigator program and by the European Network of Neuroscience Institutes (LSHM-CT-2005-19063). E.P. and V.E. were supported by the European Commission FP6 Specific Targeted Project Photolysis (LSHM-CT-2007-037765). E.P. was supported by the Fondation pour la Recherche Médicale. F.A. was supported by the European doctoral school Frontières du Vivant. A.B. was supported by Paris School of Neuroscience. J.G. was supported by the Danish Technical Scientific Research Councils (09-060742), E.Y.I. was supported by the US National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (PN2EY018241) and the Paris School of Neuroscience. E.Y.I. and V.E. were supported by Human Frontier Science Program (RGP0013/2010).

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Authors and Affiliations

Authors

Contributions

E.P. set up and characterized the optical properties of the TF-GPC microscope; F.A. and A.B. implemented the optical microscope with electrophysiological recording; E.P., F.A. and A.B. performed the experiments on cell cultures and brain slices; F.A. and A.B. analyzed the experiments on cell cultures and brain slices; V.d.S. developed the software; J.G. contributed to the set up of the GPC microscope; E.Y.I. contributed in conceiving the experiments in cultured cells and brain slices and discussed the results; E.Y.I. and V.E. prepared the manuscript; and V.E. conceived and supervised the project.

Corresponding author

Correspondence to Valentina Emiliani.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Note 1 (PDF 1042 kb)

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Papagiakoumou, E., Anselmi, F., Bègue, A. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat Methods 7, 848–854 (2010). https://doi.org/10.1038/nmeth.1505

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