Skip to main content
SpringerLink
Log in

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

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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.

Similar content being viewed by others

References

  1. Münch, T. et al. Approach sensitivity in the retina processed by a multifunctional neural circuit. Nat. Neurosci. 12, 1308–1316 (2009).

    Article  Google Scholar 

  2. Huberman, A.D. et al. Genetic identification of an On–Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion. Neuron 62, 327–334 (2009).

    Article  CAS  Google Scholar 

  3. Dedek, K. et al. A novel type of interplexiform amacrine cell in the mouse retina. Eur. J. Neurosci. 30, 217–228 (2009).

    Article  Google Scholar 

  4. Chen, M., Weng, S., Deng, Q., Xu, Z. & He, S. Physiological properties of direction-selective ganglion cells in early postnatal and adult mouse retina. J. Physiol. (London) 587, 819–828 (2009).

    Article  CAS  Google Scholar 

  5. Van Wyk, M., Wässle, H. & Taylor, W.R. Receptive field properties of ON- and OFF-ganglion cells in the mouse retina. Vis. Neurosci. 26, 1–12 (2009).

    Article  Google Scholar 

  6. Huberman, A.D. et al. Architecture and activity-mediated refinement of axonal projections from a mosaic of genetically identified retinal ganglion cells. Neuron 59, 425–438 (2008).

    Article  CAS  Google Scholar 

  7. Kim, I.-J., Zhang, Y., Meister, M. & Sanes, J.R. Laminar restriction of retinal ganglion cell dendrites and axons: subtype-specific developmental patterns revealed with transgenic markers. J. Neurosci. 30, 1452–1462 (2010).

    Article  CAS  Google Scholar 

  8. Kim, I.-J., Zhang, Y., Yamagata, M., Meister, M. & Sanes, J.R. Molecular identification of a retinal cell type that responds to upward motion. Nature 452, 478–482 (2008).

    Article  CAS  Google Scholar 

  9. Siegert, S. et al. Genetic address book for retinal cell types. Nat. Neurosci. 12, 1197–1204 (2009).

    Article  CAS  Google Scholar 

  10. Yonehara, K. et al. Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse. PLoS ONE 3, e1533 (2008).

    Article  Google Scholar 

  11. Dhingra, A. et al. Probing neurochemical structure and function of retinal ON bipolar cells with a transgenic mouse. J. Comp. Neurol. 510, 484–496 (2008).

    Article  CAS  Google Scholar 

  12. Xu, C., Zipfel, W., Shear, J.B., Williams, R.M. & Webb, W.W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl Acad. Sci. USA 93, 10763–10768 (1996).

    Article  CAS  Google Scholar 

  13. Euler, T. et al. Eyecup scope—optical recordings of light stimulus-evoked fluorescence signals in the retina. Pflugers Arch. 457, 1393–1414 (2009).

    Article  CAS  Google Scholar 

  14. Euler, T., Detwiler, P.B. & Denk, W. Directionally selective calcium signals in dendrites of starburst amacrine cells. Nature 418, 845–852 (2002).

    Article  CAS  Google Scholar 

  15. Huckfeldt, R.M., et al. Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions. Nat. Neurosci. 12 35–43 (2009).

  16. Mumm, J.S. et al. In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells. Neuron 52, 609–621 (2006).

    Article  CAS  Google Scholar 

  17. Schroeter, E.H., Wong, R.O.L. & Gregg, R.G. In vivo development of retinal ON-bipolar cell axonal terminals visualized in nyx∷MYFP transgenic zebrafish. Vis. Neurosci. 23, 833–843 (2006).

    Article  Google Scholar 

  18. Morgan, J., Huckfeldt, R.M. & Wong, R.O.-L. Imaging techniques in retinal research. Exp. Eye Res. 80, 297–306 (2005).

    Article  CAS  Google Scholar 

  19. Majewska, A., Yiu, G. & Yuste, R. A custom-made two-photon microscope and deconvolution system. Pflugers Arch. 441, 398–408 (2000).

    Article  CAS  Google Scholar 

  20. Kerschensteiner, D. et al. Genetic control of circuit function: Vsx1 and Irx5 transcription factors regulate contrast adaptation in the mouse retina. J. Neurosci. 28, 2342–2352 (2008).

    Article  CAS  Google Scholar 

  21. Pelli, D.G. The Video ToolBox software for visual psychophysics: transforming numbers into movies. Spat. Vis. 10, 437–442 (1997).

    Article  CAS  Google Scholar 

  22. Brainard, D.H. The Psychophysics Toolbox. Spat. Vis. 10, 433–436 (1997).

    Article  CAS  Google Scholar 

  23. Sakmann, B. & Neher, E. Geometric Parameters of Pipettes and Membrane Patches 637–645 (Plenum Press, 1983).

  24. The Axon Guide: A Guide to Electrophysiology & Biophysics Laboratory Techniques 3rd edn, 27–84 (MDS Analytical Technologies, 2008).

  25. Perkins, K.L. Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices. J. Neurosci. Methods 154, 1–18 (2006).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Justin Elstrott
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marla B Feller
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Marla B Feller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.106

  • Springer Nature Limited

This article is cited by

Search

Navigation