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Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina

Nature Protocols volume 5, pages 1347–1352 (2010)Cite this article

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Abstract

Cell type–specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon–excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.

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Figure 1: Setup for two-photon targeted recording of light response from GFP+ retinal ganglion cells.
Figure 2: The landmarks in the choroid for marking the orientation of the retina.
Figure 3: Examples of successful and unsuccessful experiments of two-photon targeted recording from GFP-expressing cells.

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Acknowledgements

We thank R. Eifert at Cold Spring Harbor Laboratory for the OLED holder schematics, and J. LeDue at the University of California, Berkeley for optical assistance. Support was contributed by NIH Grants RO1EY013528 and RO1EY019498.

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Authors and Affiliations

  1. Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA

    Wei Wei, Justin Elstrott & Marla B Feller

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  1. Wei Wei
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Contributions

W.W. was involved in conducting the experiments and preparing the article. J.E. assisted in conducting the experiments, wrote the visual stimulation and analysis code, constructed Figure 1a and assisted in writing the article. M.B.F. was involved in the experimental design and assisted in the preparation of the article.

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Correspondence to Marla B Feller.

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The authors declare no competing financial interests.

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Wei, W., Elstrott, J. & Feller, M. Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina. Nat Protoc 5, 1347–1352 (2010). https://doi.org/10.1038/nprot.2010.106

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