Technical Report

Temporally precise single-cell-resolution optogenetics

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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.

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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.


  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


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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.

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