Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors

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Genetically encoded fluorescence voltage sensors offer the possibility of directly visualizing neural spiking dynamics in cells targeted by their genetic class or connectivity. Sensors of this class have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics and limited signalling dynamic range in response to action potentials. Here we describe sensors that use fluorescence resonance energy transfer (FRET) to combine the rapid kinetics and substantial voltage-dependence of rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores. These FRET-opsin sensors significantly improve upon the spike detection fidelity offered by the genetically encoded voltage sensor, Arclight, while offering faster kinetics and higher brightness. Using FRET-opsin sensors we imaged neural spiking and sub-threshold membrane voltage dynamics in cultured neurons and in pyramidal cells within neocortical tissue slices. In live mice, rates and optical waveforms of cerebellar Purkinje neurons’ dendritic voltage transients matched expectations for these cells’ dendritic spikes.

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Figure 1: Design and membrane localization of FRET-opsin sensor constructs in cultured neurons.
Figure 2: MacQ-based sensors exhibit transient but not steady-state photocurrents.
Figure 3: FRET-opsin sensors report voltage depolarization via decreases in emission intensity from the fluorescence donor.
Figure 4: The rapid kinetics of MacQ sensors provide a superior frequency response curve as compared with Arclight.
Figure 5: Mac voltage sensors report single action potentials with higher spike detection fidelity than Arclight.
Figure 6: MacQ-mCitrine reports spikes from excitatory neurons in brain slices.
Figure 7: The MacQ-mCitrine sensor reports voltage transients in the dendrites of Purkinje neurons in live mice.


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We thank Yanping Zhang for help with protein extraction, Jesse Marshall for help with the electroporation protocol, Jane Li for maintenance of the Parv-Cre mouse line, Amy Lam for help with spectroscopy measurements, Michael Z. Lin for providing Arclight, mCitrine, and mOrange2 plasmids, and Larry Zweifel of University of Washington for providing the CAG-DIO backbone plasmid. We gratefully acknowledge research funding from DARPA, the Stanford CNC program, the Stanford BioX Interdisciplinary Initiatives Program (IIP), the NIH-Stanford Neuroscience Graduate Training Grant and the National Academies Keck Futures Initiative (NAKFI) research grant.

Author information

Y.G. designed, cloned and characterized the various sensor constructs in vitro. J.Z.L. produced the viruses. Y.G. and M.J.W. performed the experiments in vivo and analysed the data. M.J.S. supervised the research. Y.G. and M.J.S. wrote the manuscript. All authors edited the manuscript.

Correspondence to Yiyang Gong or Mark J. Schnitzer.

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Gong, Y., Wagner, M., Zhong Li, J. et al. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat Commun 5, 3674 (2014) doi:10.1038/ncomms4674

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