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All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

Nature Methods volume 11, pages 825–833 (2014)Cite this article

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Abstract

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

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Figure 1: Nonpumping Arch-derived voltage indicators with improved speed, sensitivity and brightness.
Figure 2: CheRiff is a fast and sensitive blue-shifted channelrhodopsin.
Figure 3: Optopatch enables high-fidelity optical stimulation and recording in cultured neurons.
Figure 4: Homeostatic plasticity of intrinsic excitability in hiPSC-derived neurons probed via Optopatch2.
Figure 5: Optopatch2 in organotypic brain slice.

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Acknowledgements

We thank N. Anand, L. Rosenbaum, T. Shen and V. Nathan for technical assistance; A. Douglass, A. Ting, F. Zhang, L. Looger and D. Kim for helpful discussions; and Cellular Dynamics Inc. for technical assistance with hiPSC neurons. This work was supported by the Harvard Center for Brain Science, PECASE award N00014-11-1-0549, US National Institutes of Health (NIH) grant 1-R01-EB012498-01 and New Innovator grant 1-DP2-OD007428, a National Science Foundation (NSF) Graduate Fellowship (D.R.H. and S.L.F.), the Natural Sciences and Engineering Research Council of Canada (Discovery grants to R.E.C. and D.J.H.), the Canadian Institutes of Health Research (R.E.C.) and graduate scholarships from the University of Alberta and Alberta Innovates (Y.Z.). E.S.B. was supported by Defense Advanced Research Projects Agency (DARPA) Living Foundries HR0011-12-C-0068, a New York Stem Cell Foundation (NYSCF) Robertson Neuroscience Investigator Award, an Institution of Engineering and Technology (IET) A.F. Harvey Prize, NIH grants 1R01NS075421, 1R01NS067199, 1DP2OD002002 and 1R01DA029639, the Human Frontiers Science Program, and NSF CAREER Award CBET 1053233 and EFRI 0835878. Work in V.N.M.'s lab was supported by NIH grants R01DC011291 and R01DC013329.

Author information

Author notes

  1. Daniel R Hochbaum and Yongxin Zhao: These authors contributed equally to this work.

  2. Edward S Boyden and Robert E Campbell: These authors jointly directed this work.

Authors and Affiliations

  1. Applied Physics Program, School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, USA

    Daniel R Hochbaum

  2. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Yongxin Zhao, D Jed Harrison & Robert E Campbell

  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Samouil L Farhi, Christopher A Werley, Peng Zou, Joel M Kralj, Niklas Smedemark-Margulies & Adam E Cohen

  4. The MIT Media Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA

    Nathan Klapoetke, Yong Ku Cho & Edward S Boyden

  5. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA

    Nathan Klapoetke, Yong Ku Cho & Edward S Boyden

  6. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA

    Nathan Klapoetke, Yong Ku Cho & Edward S Boyden

  7. McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA

    Nathan Klapoetke, Yong Ku Cho & Edward S Boyden

  8. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Vikrant Kapoor & Venkatesh N Murthy

  9. Department of Physics, Harvard University, Cambridge, Massachusetts, USA

    Dougal Maclaurin & Adam E Cohen

  10. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Jessica L Saulnier, Gabriella L Boulting, Christoph Straub & Bernardo L Sabatini

  11. Institute of Botany, Cologne Biocenter, University of Cologne, Cologne, Germany

    Michael Melkonian

  12. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

    Gane Ka-Shu Wong

  13. Department of Medicine, University of Alberta, Edmonton, Alberta, Canada

    Gane Ka-Shu Wong

  14. Beijing Genomics Institute–Shenzhen, Shenzhen, China

    Gane Ka-Shu Wong

  15. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Bernardo L Sabatini & Adam E Cohen

Authors
  1. Daniel R Hochbaum
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  2. Yongxin Zhao
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Contributions

D.R.H. designed the Optopatch construct and system. Y.Z. and D.R.H. engineered the QuasArs. D.R.H., N.K. and Y.K.C. engineered CheRiff. D.R.H. and S.L.F. acquired the optical electrophysiology data. C.A.W. developed the low-magnification imaging system. V.K. and J.L.S. designed and prepared the slice experiments. D.R.H., D.M. and A.E.C. analyzed data. P.Z. assisted with measurements of rhodopsin photophysics. J.M.K. screened ion channel blockers on hiPSC-derived neurons. N.S.-M. performed cell culture and sample preparation. G.L.B. performed immunostaining in hiPSC-derived neurons. C.S. assisted with measurements of ArcLight in slice. M.M. and G.K.-S.W. provided the transcriptomic data from which sdChR was mined and phenotyped. D.R.H. and A.E.C. wrote the paper with input from S.L.F., Y.Z. and R.E.C. D.J.H., V.N.M., B.L.S., E.S.B. and R.E.C. supervised aspects of the research. Correspondence regarding directed evolution of Arch should be addressed to R.E.C. (robert.e.campbell@ualberta.ca). A.E.C. conceived of and oversaw the project.

Corresponding author

Correspondence to Adam E Cohen.

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Competing interests

A.E.C. and J.M.K. are founders of Q-State Biosciences, a company focused on combining all-optical electrophysiology with cell-based models of disease. D.R.H., Y.Z., S.L.F., N.K., J.M.K., D.M., Y.K.C., D.J.H., R.E.C., E.S.B. and A.E.C. have filed patents related to Optopatch.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–23 and Supplementary Tables 1–6 (PDF 3634 kb)

Supplementary Software

The Sub-Nyquist Action Potential Timing (SNAPT) algorithms enable pixel-by-pixel determination of the timing of an action potential. The input is a movie of a neuron repetitively triggered to fire, where the neuron contains a high-speed fluorescent voltage reporter. The output is a movie at a higher frame-rate showing the propagation of the action potential through the neuron. The implementation given here is written for Matlab R2012a, and has been confirmed to run on a personal computer with 14 GB of RAM, running Windows 7 (64-bit). Several of the sub-routines are memory intensive. There are many adjustable parameters in the code, so it is important to run the code one piece at a time, looking at the output at each step and checking that it makes sense. (ZIP 368 kb)

Optopatch single neuron raw data

Raw QuasAr2 fluorescence from a neuron expressing Optopatch2, imaged at 1 kHz frame rate. Fluorescence was excited by whole-field red illumination (640 nm, 300 W/cm2). Activity was induced via whole-field blue light illumination (488 nm, 20 mW/cm2, 10 ms pulses, repeated at 5 Hz). Portions of the intervals between action potentials have been elided to maintain manageable movie size. Movie acquired on an sCMOS camera. (AVI 2020 kb)

Optopatch single neuron trial averaged AP

Same data as in Movie 1, averaged over 98 temporally registered action potentials. The average fluorescence intensity is shown in grayscale in the background, and the change in fluorescence is shown as a colormap. Movie acquired on an sCMOS camera. (AVI 288 kb)

Movie of AP initiation and propagation 1

Movie of an action potential (averaged over n = 203 optically evoked action potentials) in a neuron expressing Optopatch1. Region receiving blue light stimulus is shown in blue. The subthreshold voltage spreads during stimulation, followed by a spike in cell-wide fluorescence which peaked within two frames. Movie acquired on an EMCCD camera. (AVI 836 kb)

Sub-frame movie of AP initiation and propagation 1

Sub-frame interpolated movie showing action potential initiation and propagation in the neuron shown in Movie 3. Region receiving blue light stimulus is shown in blue. Action potential initiation occurs at the distal end of the axon initial segment. The movie was constructed from an average of n = 203 optically evoked action potentials. Movie acquired on an EMCCD camera. (AVI 2208 kb)

Sub-frame movie of AP initiation and propagation 2

Sub-frame interpolated movie showing action potential initiation and propagation in a neuron expressing Optopatch1, with blue light stimulation targeted to the soma. The movie was acquired using the same parameters as in Supplementary Movie 3. The movie was constructed from an average of n = 383 optically evoked action potentials. Movie acquired on an EMCCD camera. (AVI 992 kb)

Sub-frame movie of AP initiation and propagation 3

Sub-frame interpolated movie showing action potential initiation and propagation in a neuron expressing Optopatch1, with blue light stimulation targeted to a small group of dendrites. The movie was acquired using the same parameters as in Supplementary Movie 3. The movie was constructed from an average of n = 338 optically evoked action potentials. Movie acquired on an EMCCD camera. (AVI 1605 kb)

Wide-field spontaneous activity

Spontaneous network activity in neurons expressing Optopatch2, monitored with red illumination (640 nm, 100 W/cm2) in a field of view 1 mm × 3 mm. The movie was acquired at a 500 Hz frame rate. Movie acquired on an sCMOS camera. (AVI 11247 kb)

Wide-field evoked activity

Simultaneous excitation and recording of action potentials in many neurons expressing Optopatch2. A pulse of blue light (500 ms, 5 mW/cm2) was applied to stimulate neural activity via excitation of CheRiff, and activity was monitored with red illumination (640 nm, 100 W/cm2) in a field of view 1 mm × 3 mm. The blue dot next to the time stamp indicates when the blue light is on. Synaptic blockers were added to prevent network activity. The movie was acquired at a 500 Hz frame rate. Movie acquired on an sCMOS camera. (AVI 3205 kb)

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Hochbaum, D., Zhao, Y., Farhi, S. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat Methods 11, 825–833 (2014). https://doi.org/10.1038/nmeth.3000

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