Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins

Nature Methods volume 7, pages 643–649 (2010)Cite this article

Subjects

Abstract

Cortical information processing relies on synaptic interactions between diverse classes of neurons with distinct electrophysiological and connection properties. Uncovering the operational principles of these elaborate circuits requires the probing of electrical activity from selected populations of defined neurons. Here we show that genetically encoded voltage-sensitive fluorescent proteins (VSFPs) provide an optical voltage report from targeted neurons in culture, acute brain slices and living mice. By expressing VSFPs in pyramidal cells of mouse somatosensory cortex, we also demonstrate that these probes can report cortical electrical responses to single sensory stimuli in vivo. These protein-based voltage probes will facilitate the analysis of cortical circuits in genetically defined cell populations and are hence a valuable addition to the optogenetic toolbox.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: VSFP2.3 reports membrane voltage transients through differential two-color fluorescence.
Figure 2: Distribution and voltage responses of VSFP2.3 in the mouse cerebral cortex.
Figure 3: VSFP2.3 reports synaptic responses in individual pyramidal neurons from acute brain slices in single trials.
Figure 4: VSFP2.3 imaging in the somatosensory cortex of living mice.
Figure 5: VSFP2.42 reports cortical responses to deflections of a single whisker.
Figure 6: Construction of receptive field maps using in vivo VSFP2 imaging.

Similar content being viewed by others

References

  1. Grinvald, A. & Hildesheim, R. VSDI: a new era in functional imaging of cortical dynamics. Nat. Rev. Neurosci. 5, 874–885 (2004).

    Article  CAS  Google Scholar 

  2. Knöpfel, T., Diez-Garcia, J. & Akemann, W. Optical probing of neuronal circuit dynamics. Trends Neurosci. 29, 4602–4612 (2006).

    Article  Google Scholar 

  3. Dimitrov, D. et al. Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLoS One 2, e440 (2007).

    Article  Google Scholar 

  4. Perron, A. et al. Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential. Front. Mol. Neurosci. 2, 5 (2009).

    Article  Google Scholar 

  5. Sakai, R., Repunte-Canonigo, V., Raj, C.D. & Knöpfel, T. Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur. J. Neurosci. 13, 2314–2318 (2001).

    Article  CAS  Google Scholar 

  6. Murata, Y., Iwasaki, H., Sasaki, M., Inaba, K. & Okamura, Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435, 1239–1243 (2005).

    Article  CAS  Google Scholar 

  7. Mutoh, H. et al. Spectrally-resolved response properties of the three most advanced FRET based fluorescent protein voltage probes. PLoS One 4, e4555 (2009).

    Article  Google Scholar 

  8. Hatanaka, Y., Hisanaga, S.I., Heizmann, C.W. & Murakami, F. Distinct migratory behavior of early- and late-born neurons derived from the cortical ventricular zone. J. Comp. Neurol. 479, 1–14 (2004).

    Article  Google Scholar 

  9. Akemann, W., Lundby, A., Mutoh, H. & Knopfel, T. Effect of voltage sensitive fluorescent proteins on neuronal excitability. Biophys. J. 96, 3959–3976 (2009).

    Article  CAS  Google Scholar 

  10. Petersen, C.C., Grinvald, A. & Sakmann, B. Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage-sensitive dye imaging combined with whole-cell voltage recordings and neuron reconstructions. J. Neurosci. 23, 1298–1309 (2003).

    Article  CAS  Google Scholar 

  11. Ferezou, I., Bolea, S. & Petersen, C.C. Visualizing the cortical representation of whisker touch: voltage-sensitive dye imaging in freely moving mice. Neuron 50, 617–629 (2006).

    Article  CAS  Google Scholar 

  12. Ferezou, I. et al. Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice. Neuron 56, 907–923 (2007).

    Article  CAS  Google Scholar 

  13. Palmer, A.E. et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006).

    Article  CAS  Google Scholar 

  14. Mennerick, S. et al. Diverse voltage-sensitive dyes modulate GABAA receptor function. J. Neurosci. 30, 2871–2879 (2010).

    Article  CAS  Google Scholar 

  15. Chanda, B. et al. A hybrid approach to measuring electrical activity in genetically specified neurons. Nat. Neurosci. 8, 1619–1626 (2005).

    Article  CAS  Google Scholar 

  16. Sjulson, L. & Miesenbock, G. Rational optimization and imaging in vivo of a genetically encoded optical voltage reporter. J. Neurosci. 28, 5582–5593 (2008).

    Article  CAS  Google Scholar 

  17. Perron, A., Mutoh, H., Launey, T. & Knöpfel, T. Red-shifted voltage-sensitive fluorescent proteins. Chem. Biol. 16, 1268–1277 (2009).

    Article  CAS  Google Scholar 

  18. Manns, I.D., Sakmann, B. & Brecht, M. Sub- and suprathreshold receptive field properties of pyramidal neurones in layers 5A and 5B of rat somatosensory barrel cortex. J. Physiol. (Lond.) 556, 601–622 (2004).

    Article  CAS  Google Scholar 

  19. Crochet, S. & Petersen, C.C. Correlating whisker behavior with membrane potential in barrel cortex of awake mice. Nat. Neurosci. 9, 608–610 (2006).

    Article  CAS  Google Scholar 

  20. Katayama, H., Yamamoto, A., Mizushima, N., Yoshimori, T. & Miyawaki, A. GFP-like proteins stably accumulate in lysosomes. Cell Struct. Funct. 33, 1–12 (2008).

    Article  CAS  Google Scholar 

  21. Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat. Methods 5, 683–685 (2008).

    Article  CAS  Google Scholar 

  22. Sauer, B. & Henderson, N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166–5170 (1988).

    Article  CAS  Google Scholar 

  23. Akagi, K. et al. Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 25, 1766–1773 (1997).

    Article  CAS  Google Scholar 

  24. Cossart, R., Ikegaya, Y. & Yuste, R. Calcium imaging of cortical networks dynamics. Cell Calcium 37, 451–457 (2005).

    Article  CAS  Google Scholar 

  25. Lutz, C. et al. Holographic photolysis of caged neurotransmitters. Nat. Methods 5, 821–827 (2008).

    Article  CAS  Google Scholar 

  26. Nikolenko, V. et al. SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators. Front. Neural Circuits. 2, 5 (2008).

    Article  Google Scholar 

  27. Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W. & Knöpfel, T. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements. PLoS One 3, e2514 (2008).

    Article  Google Scholar 

  28. Matsuda, T. & Cepko, C.L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. USA 101, 16–22 (2004).

    Article  CAS  Google Scholar 

  29. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins devrived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  30. Shimogori, T. & Ogawa, M. Gene application with in utero electroporation in mouse embryonic brain. Dev. Growth Differ. 50, 499–506 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank all members of the Knöpfel laboratory for comments and invaluable technical help, T. Shimogori and R. Yoshida for in utero electroporation of VSFP plasmids, and Thomas Behnisch for his participation in initial in vivo experiments. This work was funded by grants from RIKEN Brain Science Institute (T.K.), the RIKEN Brain Science Institute director's fund (T.K.), US National Institutes of Health grant NS057631 (T.K.), a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT, H.M.), the Japanese Society for the Promotion of Science–Canadian Institutes of Health Research postdoctoral fellowship program (A.P.), a grant-in-aid for Japanese Society for the Promotion of Science fellows (A.P.) and La Fondation pour les Sciences du Cerveau (J.R.).

Author information

Author notes

  1. Walther Akemann and Hiroki Mutoh: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Brain Science Institute, Wako City, Saitama, Japan.,

    Walther Akemann, Hiroki Mutoh, Amélie Perron, Jean Rossier & Thomas Knöpfel

  2. Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherché 7637, Ecole Supérieure de Physique et de Chimie Industrielles, Paris, France

    Jean Rossier

Authors
  1. Walther Akemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroki Mutoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amélie Perron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean Rossier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas Knöpfel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.A., H.M., A.P. and T.K. conceived and designed the experiments. A.P. developed VSFP2.42. H.M. performed recordings in cultured cells. W.A. performed experiments in acute brain slices. W.A., H.M. and T.K. performed in vivo imaging experiments. W.A., H.M., A.P., J.R. and T.K. contributed to data analysis, preparation of figures and writing of the manuscript.

Corresponding author

Correspondence to Thomas Knöpfel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 399 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Akemann, W., Mutoh, H., Perron, A. et al. Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins. Nat Methods 7, 643–649 (2010). https://doi.org/10.1038/nmeth.1479

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1479

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing