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.

A hybrid approach to measuring electrical activity in genetically specified neurons

Nature Neuroscience volume 8, pages 1619–1626 (2005)Cite this article

Abstract

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: The hVOS system in HEK 293 cells.
Figure 2: Speed and recovery of hVOS.
Figure 3: Toxicity of hVOS system.
Figure 4: Recording of action potentials with hVOS in GT1 cells.
Figure 5: Primary neurons transfected with farnesylated eGFP.
Figure 6: Optical recordings of electrical activity in primary neuronal cultures.
Figure 7: Fluorescence recordings of membrane potential using DPA in conjunction with fluorescently labeled anti-CD8 antibodies.

References

  1. Salzberg, B.M., Grinvald, A., Cohen, L.B., Davila, H.V. & Ross, W.N. Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons. J. Neurophysiol. 40, 1281–1291 (1977).

    Article  CAS  Google Scholar 

  2. Djurisic, M. et al. Optical monitoring of neural activity using voltage-sensitive dyes. Methods Enzymol. 361, 423–451 (2003).

    Article  CAS  Google Scholar 

  3. Ikegaya, Y. et al. Synfire chains and cortical songs: temporal modules of cortical activity. Science 304, 559–564 (2004).

    Article  CAS  Google Scholar 

  4. Lieke, E.E., Frostig, R.D., Arieli, A., Ts'o, D.Y., Hildesheim, R. & Grinvald, A. Optical imaging of cortical activity: real-time imaging using extrinsic dye-signals and high resolution imaging based on slow intrinsic-signals. Annu. Rev. Physiol. 51, 543–559 (1989).

    Article  CAS  Google Scholar 

  5. Grinvald, A., Salzberg, B.M., Lev-Ram, V. & Hildesheim, R. Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophys. J. 51, 643–651 (1987).

    Article  CAS  Google Scholar 

  6. Miyawaki, A. Fluorescence imaging of physiological activity in complex systems using GFP-based probes. Curr. Opin. Neurobiol. 13, 591–596 (2003).

    Article  CAS  Google Scholar 

  7. Miesenbock, G. Genetic methods for illuminating the function of neural circuits. Curr. Opin. Neurobiol. 14, 395–402 (2004).

    Article  CAS  Google Scholar 

  8. Siegel, M.S. & Isacoff, E.Y. A genetically encoded optical probe of membrane voltage. Neuron 19, 735–741 (1997).

    Article  CAS  Google Scholar 

  9. Ataka, K. & Pieribone, V.A. A genetically targetable fluorescent probe of channel gating with rapid kinetics. Biophys. J. 82, 509–516 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Gonzalez, J.E. & Tsien, R.Y. Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys. J. 69, 1272–1280 (1995).

    Article  CAS  Google Scholar 

  12. Cacciatore, T.W. et al. Identification of neural circuits by imaging coherent electrical activity with FRET-based dyes. Neuron 23, 449–459 (1999).

    Article  CAS  Google Scholar 

  13. Bedlack, R.S., Jr ., Wei, M.D., Fox, S.H., Gross, E. & Loew, L.M. Distinct electric potentials in soma and neurite membranes. Neuron 13, 1187–1193 (1994).

    Article  CAS  Google Scholar 

  14. Rohr, S. & Salzberg, B.M. Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale. Biophys. J. 67, 1301–1315 (1994).

    Article  CAS  Google Scholar 

  15. Jiang, W. & Hunter, T. Analysis of cell-cycle profiles in transfected cells using a membrane-targeted GFP. Biotechniques 24, 349–354 (1998).

    Article  CAS  Google Scholar 

  16. Loew, L.M. et al. A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. J. Membr. Biol. 130, 1–10 (1992).

    Article  CAS  Google Scholar 

  17. Chanda, B., Asamoah, O.K., Blunck, R., Roux, B. & Bezanilla, F. Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement. Nature 436, 852–856 (2005).

    Article  CAS  Google Scholar 

  18. Fernandez, J.M., Taylor, R.E. & Bezanilla, F. Induced capacitance in the squid giant axon. Lipophilic ion displacement currents. J. Gen. Physiol. 82, 331–346 (1983).

    Article  CAS  Google Scholar 

  19. de Ruyter van Steveninck, RR., Lewen, G.D., Strong, S.P., Koberle, R. & Bialek, W. Reproducibility and variability in neural spike trains. Science 275, 1805–1808 (1997).

    Article  CAS  Google Scholar 

  20. Kalyanaraman, B., Feix, J.B., Sieber, F., Thomas, J.P. & Girotti, A.W. Photodynamic action of merocyanine 540 on artificial and natural cell membranes: involvement of singlet molecular oxygen. Proc. Natl. Acad. Sci. USA 84, 2999–3003 (1987).

    Article  CAS  Google Scholar 

  21. Hales, T.G., Sanderson, M.J. & Charles, A.C. GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1–7) neurons. Neuroendocrinology 59, 297–308 (1994).

    Article  CAS  Google Scholar 

  22. Bezanilla, F., Perozo, E. & : Stefani, E. Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. Biophys. J. 66, 1011–1021 (1994).

    Article  CAS  Google Scholar 

  23. Salter-Cid, L. et al. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc. Natl. Acad. Sci. USA 96, 5434–5439 (1999).

    Article  CAS  Google Scholar 

  24. Metzger, F. et al. Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter. Eur. J. Neurosci. 15, 40–50 (2002).

    Article  Google Scholar 

  25. Oliva, A.A., Jr ., Jiang, M., Lam, T., Smith, K.L. & Swann, J.W. Novel hippocampal interneuronal subtypes identified using transgenic mice that express green fluorescent protein in GABAergic interneurons. J. Neurosci. 20, 3354–3368 (2000).

    Article  CAS  Google Scholar 

  26. Sippy, T., Cruz-Martin, A., Jeromin, A. & Schweizer, F.E. Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor-1. Nat. Neurosci. 6, 1031–1038 (2003).

    Article  CAS  Google Scholar 

  27. Gordey, M., Mekmanee, L. & Mody, I. Altered effects of ethanol in NR2A(DeltaC/DeltaC) mice expressing C-terminally truncated NR2A subunit of NMDA receptor. Neuroscience 105, 987–997 (2001).

    Article  CAS  Google Scholar 

  28. Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M. & Mody, I. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. USA 100, 14439–14444 (2003).

    Article  CAS  Google Scholar 

  29. Blunck, R., Starace, D.M., Correa, A.M. & Bezanilla, F. Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells. Biophys. J. 86, 3966–3980 (2004).

    Article  CAS  Google Scholar 

  30. Knopfel, T., Tomita, K., Shimazaki, R. & Sakai, R. Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. Methods 30, 42–48 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank W. Hubbell (UCLA) for the gift of DPA and A. Charles (UCLA) for the GT1 cells. Thanks to F. Chow for her assistance with primary neuronal cultures and transfections. We also thank T. Otis and M. Pratap for preparing viruses for slice transfections and for access to their setup. This work was supported by grants from the US National Institutes of Health (GM30376 to F.B., NS30549 to I.M., NS41317 to F.E.S.), the American Heart Association (0225006Y and 0535214N to B.C.) and Deutschen Forschungsgemeinschaft (BL538-1/1 to R.B.).

Author information

Author notes

  1. Baron Chanda and Rikard Blunck: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Physiology and Anesthesiology, David Geffen School of Medicine at the University of California Los Angeles (UCLA), Los Angeles, 90095, California, USA

    Baron Chanda, Rikard Blunck & Francisco Bezanilla

  2. Department of Neurology, David Geffen School of Medicine at the University of California Los Angeles (UCLA), Los Angeles, 90095, California, USA

    Leonardo C Faria & Istvan Mody

  3. Department of Neurobiology, David Geffen School of Medicine at the University of California Los Angeles (UCLA), Los Angeles, 90095, California, USA

    Felix E Schweizer

Authors
  1. Baron Chanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rikard Blunck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leonardo C Faria
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Felix E Schweizer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Istvan Mody
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Francisco Bezanilla
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francisco Bezanilla.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chanda, B., Blunck, R., Faria, L. et al. A hybrid approach to measuring electrical activity in genetically specified neurons. Nat Neurosci 8, 1619–1626 (2005). https://doi.org/10.1038/nn1558

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn1558

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