Holographic two-photon activation for synthetic optogenetics

Abstract

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca2+ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.

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Fig. 1: Blueprint for applying synthetic optogenetics.
Fig. 2: Two-photon-compatible photoswitch.
Fig. 3: Rational design of LiGluR.
Fig. 4: Sequence homology.
Fig. 5: Ranking photocurrent.
Fig. 6: Two-photon digital holography setup.
Fig. 7: Reaction scheme describing the synthesis of 6 and l-MAG0460.
Fig. 8
Fig. 9: LiGluR photocurrents stimulated by 1P and 2P excitation of l-MAG0460 in cultured neurons.
Fig. 10: The 2P-DH photoswitching of l-MAG0460 is compatible with red Ca2+ imaging on a spinning-disk confocal microscope.

Data availability

The authors declare that most data supporting the findings of this study are available within the paper; however, data are also available from the corresponding author upon request.

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Acknowledgements

Support was provided by the Israel Science Foundation (S.B.; 1096/17 and 438/18). The research submitted was in partial fulfillment of the degree of master of science for I.C. and M.D.B.

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E.C.C. developed the 2P protocols; M.A.K. designed and synthesized all photoswitches. S.B. performed genetic modifications. S.B. and E.C.C. performed electrophysiology and Ca2+-imaging experiments; S.B., E.C.C. and M.A.K. designed the research project. I.C., M.D.B., L.M., E.C.C., M.A.K. and S.B. wrote the paper.

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Correspondence to Shai Berlin.

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Key references using this protocol

Levitz, J. et al. Nat. Neurosci. 16, 507–516: https://doi.org/10.1038/nn.3346 (2013)

Berlin, S. et al. Elife 5, e12040: https://doi.org/10.7554/eLife.12040 (2016)

Key data used in this protocol

Carroll, E. C. et al. Proc. Natl. Acad. Sci. USA 112, E776–E785: https://doi.org/10.1073/pnas.1416942112 (2015)

Kienzler, M. A. et al. J. Am. Chem. Soc. 135, 17683–17686: https://doi.org/10.1021/ja408104w (2013)

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Carmi, I., De Battista, M., Maddalena, L. et al. Holographic two-photon activation for synthetic optogenetics. Nat Protoc 14, 864–900 (2019). https://doi.org/10.1038/s41596-018-0118-2

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