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Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation

Abstract

Optogenetics with microbial opsin genes has enabled high-speed control of genetically specified cell populations in intact tissue. However, it remains a challenge to independently control subsets of cells within the genetically targeted population. Although spatially precise excitation of target molecules can be achieved using two-photon laser-scanning microscopy (TPLSM) hardware, the integration of two-photon excitation with optogenetics has thus far required specialized equipment or scanning and has not yet been widely adopted. Here we take a complementary approach, developing opsins with custom kinetic, expression and spectral properties uniquely suited to scan times typical of the raster approach that is ubiquitous in TPLSM laboratories. We use a range of culture, slice and mammalian in vivo preparations to demonstrate the versatility of this toolbox, and we quantitatively map parameter space for fast excitation, inhibition and bistable control. Together these advances may help enable broad adoption of integrated optogenetic and TPLSM technologies across experimental fields and systems.

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Figure 1: Two-photon control of spiking with C1V1 variants in culture.
Figure 2: Two-photon control of spiking with C1V1 variants in acute brain slices.
Figure 3: Spatial resolution of C1V1-mediated two-photon excitation in acute brain slices.
Figure 4: Two-photon optogenetic control of spike firing in vivo in adult mammals.
Figure 5: Bistable two-photon optogenetic control in culture and slice with novel ChR variants.
Figure 6: Two-photon optogenetic inhibition.

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Acknowledgements

We thank R. Pashaie and the Deisseroth laboratory members for helpful discussions. We thank Prairie Technologies (T. Keifer, M. Szulczewsk and A. Statz) for discussions and work with the two-photon microscope. R.P. is supported by the US National Institute of Mental Health (F30 MH095468). K.D. is supported by the US National Institutes of Health, the Gatsby Foundation and the Defense Advanced Research Program Agency REPAIR Program.

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Authors

Contributions

R.P., B.G. and K.D. contributed to study design. C.R., O.Y. and R.P. contributed to cloning of constructs. C.R. cultured primary neurons, performed transfections and managed viral production. R.P. and N.W. contributed to viral injections. R.P. performed all two-photon experiments in both culture and slice preparations. R.P. and B.G. contributed to in vivo two-photon experiments supervised by K.D. and M.J.S. I.G. performed histological processing and fluorescence imaging. R.P. performed all data analysis. A.M.P., D.S.P. and R.Y. contributed to design, analysis and interpretation. K.D. supervised all aspects of the work. R.P. and K.D. wrote the paper.

Corresponding author

Correspondence to Karl Deisseroth.

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The authors declare no competing financial interests.

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Prakash, R., Yizhar, O., Grewe, B. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat Methods 9, 1171–1179 (2012). https://doi.org/10.1038/nmeth.2215

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