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.

Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics

Nature Neuroscience volume 14, pages 527–532 (2011)Cite this article

Subjects

Abstract

Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomical receptive field of a neuron.

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

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Recording methodology and biocytin recovery rates.
Figure 2: Physiological characterization and genetic manipulation.
Figure 3: Multiple gene delivery.
Figure 4: Synaptic receptive mapping and connectivity of a visual cortical neuron.

References

  1. Kleinfeld, D. & Griesbeck, O. From art to engineering? The rise of in vivo mammalian electrophysiology via genetically targeted labeling and nonlinear imaging. PLoS Biol. 3, e355 (2005).

    Article  Google Scholar 

  2. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    Article  CAS  Google Scholar 

  3. Luo, L., Callaway, E.M. & Svoboda, K. Genetic dissection of neural circuits. Neuron 57, 634–660 (2008).

    Article  CAS  Google Scholar 

  4. Arenkiel, B.R. & Ehlers, M.D. Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature 461, 900–907 (2009).

    Article  CAS  Google Scholar 

  5. Geschwind, D.H. & Konopka, G. Neuroscience in the era of functional genomics and systems biology. Nature 461, 908–915 (2009).

    Article  CAS  Google Scholar 

  6. O'Connor, D.H., Huber, D. & Svoboda, K. Reverse engineering the mouse brain. Nature 461, 923–929 (2009).

    Article  CAS  Google Scholar 

  7. Scanziani, M. & Hausser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).

    Article  CAS  Google Scholar 

  8. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  CAS  Google Scholar 

  9. Lichtman, J.W., Livet, J. & Sanes, J.R. A technicolour approach to the connectome. Nat. Rev. Neurosci. 9, 417–422 (2008).

    Article  CAS  Google Scholar 

  10. Margrie, T.W., Sakmann, B. & Urban, N.N. Action potential propagation in mitral cell lateral dendrites is decremental and controls recurrent and lateral inhibition in the mammalian olfactory bulb. Proc. Natl. Acad. Sci. USA 98, 319–324 (2001).

    Article  CAS  Google Scholar 

  11. Wehr, M. & Zador, A.M. Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex. Nature 426, 442–446 (2003).

    Article  CAS  Google Scholar 

  12. Waters, J., Larkum, M., Sakmann, B. & Helmchen, F. Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo. J. Neurosci. 23, 8558–8567 (2003).

    Article  CAS  Google Scholar 

  13. Loewenstein, Y. et al. Bistability of cerebellar Purkinje cells modulated by sensory stimulation. Nat. Neurosci. 8, 202–211 (2005).

    Article  CAS  Google Scholar 

  14. Margrie, T.W., Brecht, M. & Sakmann, B. In vivo whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflügers Arch. 444, 491–498 (2002).

    Article  CAS  Google Scholar 

  15. Brecht, M., Schneider, M., Sakmann, B. & Margrie, T.W. Whisker movements evoked by stimulation of single pyramidal cells in rat motor cortex. Nature 427, 704–710 (2004).

    Article  CAS  Google Scholar 

  16. Arenz, A., Silver, R.A., Schaefer, A.T. & Margrie, T.W. The contribution of single synapses to sensory representation in vivo. Science 321, 977–980 (2008).

    Article  CAS  Google Scholar 

  17. Harvey, C.D., Collman, F., Dombeck, D.A. & Tank, D.W. Intracellular dynamics of hippocampal place cells during virtual navigation. Nature 461, 941–946 (2009).

    Article  CAS  Google Scholar 

  18. Epsztein, J., Lee, A.K., Chorev, E. & Brecht, M. Impact of spikelets on hippocampal CA1 pyramidal cell activity during spatial exploration. Science 327, 474–477 (2010).

    Article  CAS  Google Scholar 

  19. Hromádka, T., Deweese, M.R. & Zador, A.M. Sparse representation of sounds in the unanesthetized auditory cortex. PLoS Biol. 6, e16 (2008).

    Article  Google Scholar 

  20. Lee, A.K., Epsztein, J. & Brecht, M. Head-anchored whole-cell recordings in freely moving rats. Nat. Protoc. 4, 385–392 (2009).

    Article  CAS  Google Scholar 

  21. Zhang, F., Aravanis, A.M., Adamantidis, A., de Lecea, L. & Deisseroth, K. Circuit-breakers: optical technologies for probing neural signals and systems. Nat. Rev. Neurosci. 8, 577–581 (2007).

    Article  CAS  Google Scholar 

  22. Zhang, F., Wang, L.-P., Boyden, E.S. & Deisseroth, K. Channelrhodopsin-2 and optical control of excitable cells. Nat. Methods 3, 785–792 (2006).

    Article  CAS  Google Scholar 

  23. Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).

    Article  CAS  Google Scholar 

  24. Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 61–64 (2008).

    Article  CAS  Google Scholar 

  25. Komai, S. et al. Postsynaptic excitability is necessary for strengthening of cortical sensory responses during experience-dependent development. Nat. Neurosci. 9, 1125–1133 (2006).

    Article  CAS  Google Scholar 

  26. Marshel, J.H., Mori, T., Nielsen, K.J. & Callaway, E.M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

    Article  CAS  Google Scholar 

  27. Douglas, R.J. & Martin, K.A. Mapping the matrix: the ways of neocortex. Neuron 56, 226–238 (2007).

    Article  CAS  Google Scholar 

  28. Eilers, J.K. & Konnerth, A. A practical guide: dye loading with patch pipettes. in Imaging in Neuroscience and Development (eds. Yuste, R.K. & Konnerth, A.) 277–281 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005).

  29. Brecht, M. et al. Organization of rat vibrissa motor cortex and adjacent areas according to cytoarchitectonics, microstimulation, and intracellular stimulation of identified cells. J. Comp. Neurol. 479, 360–373 (2004).

    Article  Google Scholar 

  30. Margrie, T.W. et al. Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39, 911–918 (2003).

    Article  CAS  Google Scholar 

  31. Ugolini, G. Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups. J. Comp. Neurol. 356, 457–480 (1995).

    Article  CAS  Google Scholar 

  32. Ugolini, G. Advances in viral transneuronal tracing. J. Neurosci. Methods 194, 2–20 (2010).

    Article  Google Scholar 

  33. Mebatsion, T., Konig, M. & Conzelmann, K.K. Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 84, 941–951 (1996).

    Article  CAS  Google Scholar 

  34. Mebatsion, T. Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein. J. Virol. 75, 11496–11502 (2001).

    Article  CAS  Google Scholar 

  35. Ferster, D. Origin of orientation-selective EPSPs in simple cells of cat visual cortex. J. Neurosci. 7, 1780–1791 (1987).

    Article  CAS  Google Scholar 

  36. Mariño, J. et al. Invariant computations in local cortical networks with balanced excitation and inhibition. Nat. Neurosci. 8, 194–201 (2005).

    Article  Google Scholar 

  37. Conchello, J.-A. & Lichtman, J.W. Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).

    Article  CAS  Google Scholar 

  38. Le Bé, J.V. & Markram, H. Spontaneous and evoked synaptic rewiring in the neonatal neocortex. Proc. Natl. Acad. Sci. USA 103, 13214–13219 (2006).

    Article  Google Scholar 

  39. de Kock, C.P. & Sakmann, B. Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific. Proc. Natl. Acad. Sci. USA 106, 16446–16450 (2009).

    Article  CAS  Google Scholar 

  40. Nevian, T. & Helmchen, F. Calcium indicator loading of neurons using single-cell electroporation. Pflügers Arch. 454, 675–688 (2007).

    Article  CAS  Google Scholar 

  41. Kitamura, K., Judkewitz, B., Kano, M., Denk, W. & Hausser, M. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat. Methods 5, 61–67 (2008).

    Article  CAS  Google Scholar 

  42. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    Article  CAS  Google Scholar 

  43. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  Google Scholar 

  44. Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).

    Article  CAS  Google Scholar 

  45. Lutcke, H. et al. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice. Front. Neural Circuits 4, 9 (2010).

    PubMed  PubMed Central  Google Scholar 

  46. Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    Article  CAS  Google Scholar 

  47. Rochefort, N.L. et al. Sparsification of neuronal activity in the visual cortex at eye-opening. Proc. Natl. Acad. Sci. USA 106, 15049–15054 (2009).

    Article  CAS  Google Scholar 

  48. Kügler, S., Kilic, E. & Bahr, M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347 (2003).

    Article  Google Scholar 

  49. Brainard, D.H. The psychophysics toolbox. Spat. Vis. 10, 433–436 (1997).

    Article  CAS  Google Scholar 

  50. Cetin, A., Komai, S., Eliava, M., Seeburg, P.H. & Osten, P. Stereotaxic gene delivery in the rodent brain. Nat. Protoc. 1, 3166–3173 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

K.M.F. thanks S. Siegelbaum and R. Axel for support and encouragement and A. Mulligan for technical assistance. We also thank P.H. Seeburg for support; D. Castro, B. Matynoga and D. Drechsel for advice; K.-K. Conzelmann for rabies virus SAD-ΔG-mCherry; and P. Boross, E. Callaway, H. Wildner and F. Guillemot for discussions and reagents. E.A.R. is a Sir Henry Wellcome Postdoctoral Fellow. This work was supported by The Robert Leet and Clara Guthrie Patterson Trust and a National Institute on Deafness and Other Communication Disorders (NIDCD) K99 grant (K.M.F.), The Max-Planck Gesellschaft (A.T.S., M.K.S.), the Medical Research Council (MRC) and The Alexander Von Humboldt Foundation (T.W.M.).

Author information

Author notes

  1. Ede A Rancz and Kevin M Franks: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Ede A Rancz, Kevin M Franks, Andreas T Schaefer & Troy W Margrie

  2. Division of Neurophysiology, The National Institute for Medical Research, Mill Hill, UK

    Ede A Rancz, Bruno Pichler & Troy W Margrie

  3. Department of Neuroscience, Columbia University, New York, New York, USA

    Kevin M Franks

  4. Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg, Germany

    Martin K Schwarz

  5. SNWG Behavioural Neurophysiology, Max Planck Institute for Medical Research, Heidelberg, Germany

    Andreas T Schaefer

Authors
  1. Ede A Rancz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kevin M Franks
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin K Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruno Pichler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas T Schaefer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Troy W Margrie
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.A.R., K.M.F., A.T.S. and T.W.M. conceived experiments. K.M.F., A.T.S. and T.W.M. performed original proof-of-principle and multi-plasmid experiments. E.A.R. collected the biocytin, GFP and viral tracing data. M.K.S. co-designed and generated plasmids and provided virus. B.P. provided customized visual stimulation and compiled large-scale imaging data. E.A.R., K.M.F. and T.W.M. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Troy W Margrie.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 2036 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rancz, E., Franks, K., Schwarz, M. et al. Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics. Nat Neurosci 14, 527–532 (2011). https://doi.org/10.1038/nn.2765

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2765

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