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Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Abstract

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between −150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin–based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

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Figure 1: Arch is a fluorescent voltage indicator.
Figure 2: Optical recording of action potentials with Arch.
Figure 3: Arch(D95N) shows voltage-dependent fluorescence but no photocurrent.
Figure 4: Optical recording of action potentials with Arch(D95N).
Figure 5: Optical indicators of membrane potential classified by speed and sensitivity.

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Acknowledgements

We thank F. Engert (Harvard University) for generously providing support to A.D.D., providing laboratory space during the early stages of this work, and for facilitating this collaboration, E. Boyden (Massachusetts Institute of Technology), K. Rothschild (Boston University) and A. Ting (Massachusetts Institute of Technology) for discussions and contributions of equipment and reagents, and G. Lau, B. Lilley and H. Inada for technical assistance. This work was supported by the Harvard Center for Brain Science, US National Institutes of Health grants 1-R01-EB012498-01 and New Innovator grant 1-DP2-OD007428, the Harvard–Massachusetts Institute of Technology Joint Research Grants Program in Basic Neuroscience, an Intelligence Community postdoctoral fellowship (J.M.K.), a National Science Foundation Graduate Fellowship (D.R.H.), a Helen Hay Whitney Postdoctoral Fellowship (A.D.D.) and Charles A. King Trust Postdoctoral Fellowship (A.D.D.).

Author information

Authors and Affiliations

Authors

Contributions

A.E.C. conceived the project. J.M.K., A.D.D. and D.R.H. carried out experiments. D.M. designed and built the imaging system used in Figure 2. All authors designed experiments, analyzed data and wrote the paper.

Corresponding author

Correspondence to Adam E Cohen.

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Competing interests

A.E.C., J.M.K. and A.D.D. filed a patent application (PCT/US11/48793) on microbial rhodopsin–based voltage sensors.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–2 (PDF 477 kb)

Supplementary Video 1

Fluorescence from an HEK cell expressing Arch. The cell was subjected to steps in voltage from −100 mV to 100 mV at 1 Hz. The apparent voltage-sensitive pixels inside the cell are due to out-of-focus fluorescence from the upper and lower surfaces of the plasma membrane. Images are unmodified raw data. Movie is shown in real time. (AVI 663 kb)

Supplementary Video 2

Fluorescence from a rat hippocampal neuron expressing Arch, showing single-trial detection of action potentials. The field on the left shows the time-averaged fluorescence; the field on the right shows deviations from the time average. Movie has been slowed 25-fold. (AVI 5866 kb)

Supplementary Video 3

Fluorescence from a rat hippocampal neuron expressing Arch, averaged over n = 98 action potentials. Note the delayed rise and fall of the action potential in the small protrusion coming from the process at 7 o'clock relative to the cell body. The time-averaged fluorescence from the cell has been subtracted to highlight the change in fluorescence during an action potential. The background, in gray, shows the time-averaged image. (AVI 695 kb)

Supplementary Video 4

Fluorescence from a HEK cell expressing Arch(D95N) subjected to steps in voltage from −100 mV to 100 mV at 1 Hz. The apparent voltage-sensitive pixels inside the cell are due to out-of-focus fluorescence from the upper and lower surfaces of the plasma membrane. Images are unmodified raw data. Movie is shown in real time. (AVI 2009 kb)

Supplementary Video 5

Fluorescence from a HEK cell expressing Arch(D95N) subjected to a voltage-clamp triangle wave from −150 mV to 150 mV. The apparent voltage-sensitive pixels inside the cell are due to out-of-focus fluorescence from the upper and lower surfaces of the plasma membrane. The movie is sped up threefold. Images are unmodified raw data. (AVI 1110 kb)

Supplementary Software

extractV.m and ApplyWeights.m Matlab routines for analyzing voltage indicator image data. (ZIP 4 kb)

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Kralj, J., Douglass, A., Hochbaum, D. et al. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods 9, 90–95 (2012). https://doi.org/10.1038/nmeth.1782

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