Technical Report

Temporally precise single-cell-resolution optogenetics

Received:
Accepted:
Published online:

Abstract

Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in mouse cortical brain slices with single-cell resolution and <1-ms temporal precision. We used soCoChR to perform connectivity mapping on intact cortical circuits.

  • Subscribe to Nature Neuroscience for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

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

  2. 2.

    Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L. & Tank, D. W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci. 13, 1433–1440 (2010).

  3. 3.

    Li, C.-Y. T., Poo, M.-M. & Dan, Y. Burst spiking of a single cortical neuron modifies global brain state. Science 324, 643–646 (2009).

  4. 4.

    Houweling, A. R. & Brecht, M. Behavioural report of single neuron stimulation in somatosensory cortex. Nature 451, 65–68 (2008).

  5. 5.

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

  6. 6.

    Ronzitti, E. et al. Recent advances in patterned photostimulation for optogenetics, J. Opt. 19, 113001 (2017).

  7. 7.

    Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).

  8. 8.

    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).

  9. 9.

    Bègue, A. et al. Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation. Biomed. Opt. Express 4, 2869–2879 (2013).

  10. 10.

    Chaigneau, E. et al. Two-photon holographic stimulation of ReaChR. Front. Cell. Neurosci. 10, 234 (2016).

  11. 11.

    Ronzitti, E. et al. Sub-millisecond optogenetic control of neuronal firing with two-photon holographic photoactivation of Chronos. J. Neurosci. https://doi.org/10.1523/JNEUROSCI.1246-17.2017 (2017).

  12. 12.

    Hernandez, O. et al. Three-dimensional spatiotemporal focusing of holographic patterns. Nat. Commun. 7, 11928 (2016).

  13. 13.

    Papagiakoumou, E. et al. Functional patterned multiphoton excitation deep inside scattering tissue. Nat. Photonics 7, 274–278 (2013).

  14. 14.

    Packer, A. M. et al. Two-photon optogenetics of dendritic spines and neural circuits. Nat. Methods 9, 1202–1205 (2012).

  15. 15.

    Anselmi, F., Ventalon, C., Bègue, A., Ogden, D. & Emiliani, V. Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning. Proc. Natl. Acad. Sci. USA 108, 19504–19509 (2011).

  16. 16.

    Dal Maschio, M., Donovan, J. C., Helmbrecht, T. O. & Baier, H. Linking neurons to network function and behavior by two-photon holographic optogenetics and volumetric imaging. Neuron 94, 774–789.e5 (2017).

  17. 17.

    Nicholson, C. & Syková, E. Extracellular space structure revealed by diffusion analysis. Trends Neurosci. 21, 207–215 (1998).

  18. 18.

    Valluru, L. et al. Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors. J. Biol. Chem. 280, 6085–6093 (2005).

  19. 19.

    Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).

  20. 20.

    Jacobs, E. C., Bongarzone, E. R., Campagnoni, C. W., Kampf, K. & Campagnoni, A. T. Soma-restricted products of the myelin proteolipid gene are expressed primarily in neurons in the developing mouse nervous system. Dev. Neurosci. 25, 96–104 (2003).

  21. 21.

    Lim, S. T., Antonucci, D. E., Scannevin, R. H. & Trimmer, J. S. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons. Neuron 25, 385–397 (2000).

  22. 22.

    Garrido, J. J. et al. A targeting motif involved in sodium channel clustering at the axonal initial segment. Science 300, 2091–2094 (2003).

  23. 23.

    Schäfer, M. K. E. et al. L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms. Neurobiol. Dis. 40, 222–237 (2010).

  24. 24.

    Bianco, A., Dienstbier, M., Salter, H. K., Gatto, G. & Bullock, S. L. Bicaudal-D regulates fragile X mental retardation protein levels, motility, and function during neuronal morphogenesis. Curr. Biol. 20, 1487–1492 (2010).

  25. 25.

    Ran, B., Bopp, R. & Suter, B. Null alleles reveal novel requirements for Bic-D during Drosophila oogenesis and zygotic development. Development 120, 1233–1242 (1994).

  26. 26.

    Zhang, X. & Bennett, V. Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains. J. Cell Biol. 142, 1571–1581 (1998).

  27. 27.

    Wu, C., Ivanova, E., Zhang, Y. & Pan, Z.-H. rAAV-mediated subcellular targeting of optogenetic tools in retinal ganglion cells in vivo. PLoS One 8, e66332 (2013).

  28. 28.

    Greenberg, K. P., Pham, A. & Werblin, F. S. Differential targeting of optical neuromodulators to ganglion cell soma and dendrites allows dynamic control of center-surround antagonism. Neuron 69, 713–720 (2011).

  29. 29.

    Marchler-Bauer, A. et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 43, D222–D226 (2015).

  30. 30.

    Ren, Z. et al. Multiple trafficking signals regulate kainate receptor KA2 subunit surface expression. J. Neurosci. 23, 6608–6616 (2003).

  31. 31.

    Grubb, M. S. & Burrone, J. Channelrhodopsin-2 localised to the axon initial segment. PLoS One 5, e13761 (2010).

  32. 32.

    Zhang, Z., Feng, J., Wu, C., Lu, Q. & Pan, Z.-H. Targeted expression of channelrhodopsin-2 to the axon initial segment alters the temporal firing properties of retinal ganglion cells. PLoS One 10, e0142052 (2015).

  33. 33.

    Baker, C. A., Elyada, Y. M., Parra, A. & Bolton, M. M. Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin. Elife 5, e14193 (2016).

  34. 34.

    Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  35. 35.

    Szabo, V., Ventalon, C., De Sars, V., Bradley, J. & Emiliani, V. Spatially selective holographic photoactivation and functional fluorescence imaging in freely behaving mice with a fiberscope. Neuron 84, 1157–1169 (2014).

  36. 36.

    Brill, J., Mattis, J., Deisseroth, K. & Huguenard, J. R. LSPS/optogenetics to improve synaptic connectivity mapping: unmasking the role of basket cell-mediated feedforward inhibition. eNeuro 3, ENEURO.0142–15.2016 (2016).

  37. 37.

    Kohara, K. et al. Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits. Nat. Neurosci. 17, 269–279 (2014).

  38. 38.

    Little, J. P. & Carter, A. G. Subcellular synaptic connectivity of layer 2 pyramidal neurons in the medial prefrontal cortex. J. Neurosci. 32, 12808–12819 (2012).

  39. 39.

    Pala, A. & Petersen, C. C. In vivo measurement of cell-type-specific synaptic connectivity and synaptic transmission in layer 2/3 mouse barrel cortex. Neuron 85, 68–75 (2015).

  40. 40.

    Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).

  41. 41.

    Xu, C. & Webb, W. W. Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B 13, 481 (1996).

  42. 42.

    Chuong, A. S. et al. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat. Neurosci. 17, 1123–1129 (2014).

  43. 43.

    Song, S., Sjöström, P. J., Reigl, M., Nelson, S. & Chklovskii, D. B. Highly nonrandom features of synaptic connectivity in local cortical circuits. PLoS Biol. 3, e68 (2005).

  44. 44.

    Yoshimura, Y., Dantzker, J. L. M. & Callaway, E. M. Excitatory cortical neurons form fine-scale functional networks. Nature 433, 868–873 (2005).

  45. 45.

    Ikegaya, Y. et al. Synfire chains and cortical songs: temporal modules of cortical activity. Science 304, 559–564 (2004).

  46. 46.

    Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

  47. 47.

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

  48. 48.

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

  49. 49.

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

  50. 50.

    Hofherr, A., Fakler, B. & Klöcker, N. Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers. J. Cell Sci. 118, 1935–1943 (2005).

  51. 51.

    Ducros, M. et al. Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing. J. Neurosci. Methods 198, 172–180 (2011).

  52. 52.

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

  53. 53.

    Gerchberg, R. & Saxton, W. A practical algorithm for the determination of the phase from image and diffraction plane pictures. Optik 35, 237 (1972).

  54. 54.

    Haist, T., Schönleber, M. & Tiziani, H. J. Computer-generated holograms from 3D-objects written on twisted-nematic liquid crystal displays. Opt. Commun. 140, 299–308 (1997).

  55. 55.

    Hernandez, O., Guillon, M., Papagiakoumou, E. & Emiliani, V. Zero-order suppression for two-photon holographic excitation. Opt. Lett. 39, 5953–5956 (2014).

  56. 56.

    Golan, L., Reutsky, I., Farah, N. & Shoham, S. Design and characteristics of holographic neural photo-stimulation systems. J. Neural Eng. 6, 066004 (2009).

  57. 57.

    Yang, S. et al. Three-dimensional holographic photostimulation of the dendritic arbor. J. Neural Eng. 8, 046002 (2011).

  58. 58.

    Conti, R., Assayag, O., de Sars, V., Guillon, M. & Emiliani, V. Computer generated holography with intensity-graded patterns. Front. Cell. Neurosci. 10, 236 (2016).

  59. 59.

    Mütze, J. et al. Excitation spectra and brightness optimization of two-photon excited probes. Biophys. J. 102, 934–944 (2012).

  60. 60.

    Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photonics 7, 205–209 (2013).

  61. 61.

    Dell, R. B., Holleran, S. & Ramakrishnan, R. Sample size determination. ILAR J. 43, 207–213 (2002).

Download references

Acknowledgements

We thank M. Gajowa for participating in preliminary opsin screening, F. Simony and M. Gajowa for help with viral injections, C. Tourain for technical support in building the holographic system, V. de Sars for software development and J. Cécile for help with CHO cell culture and transfection. V.E. thanks the Agence Nationale de la Recherche (ANR-10-INBS-04-01, France-BioImaging Infrastructure network; ANR-14-CE13-0016, Holohub), the National Institutes of Health (NIH 1-U01-NS090501-01), the FRC and the Rotary Club through the program Espoir en Tete and the Getty Lab. O.A.S. thanks the Simons Foundation for the Social Brain Fellowship and the ISEF (International Sephardic Educational Foundation) for an ISEF postdoctoral fellowship. This research was also developed with funding from the Defense Advanced Research Projects Agency (DARPA), contract No. N66001-17-C-4015. The views, opinions and/or findings expressed are those of the author and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government. V.E. and E.S.B. thank the Human Frontiers Science Program (Grant RGP0015/2016) for financial support. E.S.B. additionally acknowledges, for funding, John Doerr, the Open Philanthropy Project, the HHMI-Simons Faculty Scholars Program, NIH R44EB021054, the MIT Media Lab, NIH 1R24MH106075, NIH 2R01DA029639, NIH 1R01NS087950, NIH 1R01MH103910, NIH Director’s Pioneer Award 1DP1NS087724 and NIH 1R01GM104948.

Author information

Author notes

  1. Or A. Shemesh, Dimitrii Tanese and Valeria Zampini contributed equally to this work.

Affiliations

  1. Media Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA

    • Or A. Shemesh
    • , Changyang Linghu
    • , Kiryl Piatkevich
    •  & Edward S. Boyden
  2. Department of Biological Engineering, MIT, Cambridge, MA, USA

    • Or A. Shemesh
    • , Changyang Linghu
    • , Kiryl Piatkevich
    •  & Edward S. Boyden
  3. Center for Neurobiological Engineering, MIT, Cambridge, MA, USA

    • Or A. Shemesh
    • , Changyang Linghu
    • , Kiryl Piatkevich
    •  & Edward S. Boyden
  4. Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA

    • Or A. Shemesh
    • , Changyang Linghu
    • , Kiryl Piatkevich
    •  & Edward S. Boyden
  5. McGovern Institute for Brain Research, MIT, Cambridge, MA, USA

    • Or A. Shemesh
    • , Changyang Linghu
    • , Kiryl Piatkevich
    •  & Edward S. Boyden
  6. Neurophotonics Laboratory, Wave Front Engineering Microscopy Group, CNRS UMR8250, Université Paris Descartes, Paris, France

    • Dimitrii Tanese
    • , Valeria Zampini
    • , Emiliano Ronzitti
    • , Eirini Papagiakoumou
    •  & Valentina Emiliani
  7. Institut de la Vision, UM 80, UPMC, Paris, France

    • Valeria Zampini
    •  & Emiliano Ronzitti
  8. Institut national de la santé et de la recherche médicale (Inserm), Paris, France

    • Eirini Papagiakoumou

Authors

  1. Search for Or A. Shemesh in:

  2. Search for Dimitrii Tanese in:

  3. Search for Valeria Zampini in:

  4. Search for Changyang Linghu in:

  5. Search for Kiryl Piatkevich in:

  6. Search for Emiliano Ronzitti in:

  7. Search for Eirini Papagiakoumou in:

  8. Search for Edward S. Boyden in:

  9. Search for Valentina Emiliani in:

Contributions

O.A.S. designed, screened and tested soma-targeted opsins. O.A.S., C.L. and K.P. performed and analyzed 1P experiments in cultured cells. V.Z. and D.T. performed and analyzed 2P experiments in cultured cells and brain slices. V.Z. performed and optimized virus injection and implemented electrophysiological recording on the 2P rigs. E.R. and E.P. designed and built up setup 1. D.T. designed and built up setup 2, optimized multicell stimulation and designed calibration procedures. O.A.S., E.S.B., D.T., V.Z. and V.E. interpreted data, designed experiments, and wrote the paper with contributions from all authors. E.S.B. and V.E. conceived and supervised the project.

Competing interests

O.A.S., E.S.B. and C.L. are inventors on pending patents covering the described work.

Corresponding authors

Correspondence to Edward S. Boyden or Valentina Emiliani.

Integrated supplementary information

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–13, Supplementary Tables 1–5, and Supplementary Note

  2. Life Sciences Reporting Summary