Targeted single-cell electroporation of mammalian neurons in vivo

Abstract

In order to link our knowledge of single neurons with theories of network function, it has been a long-standing goal to manipulate the activity and gene expression of identified subsets of mammalian neurons within the intact brain in vivo. This protocol describes a method for delivering plasmid DNA into single identified mammalian neurons in vivo, by combining two-photon imaging with single-cell electroporation. Surgery, mounting of a chronic recording chamber and targeted electroporation of identified neurons can be performed within 1–2 h. Stable transgene expression can reliably be induced with high success rates both in single neurons as well as in small, spatially defined networks of neurons in the cerebral cortex of rodents.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Schematic diagram of targeted electroporation.
Figure 2: Recording chamber for combining chronic imaging with chronic electrophysiology.
Figure 3: Electroporation of single neurons and groups of neurons.
Figure 4: Strategy for targeted recording from an earlier electroporated neuron.
Figure 5: Activation of cortical neurons with light following targeted electroporation of channelrhodopsin-2.

References

  1. 1

    Albright, T.D., Jessell, T.M., Kandel, E.R. & Posner, M.I. Neural science: a century of progress and the mysteries that remain. Neuron 25 (Suppl.): S1–S55 (2000).

    Article  Google Scholar 

  2. 2

    Picciotto, M.R. & Wickman, K. Using knockout and transgenic mice to study neurophysiology and behavior. Physiol. Rev. 78, 1131–1163 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Mayford, M., Mansuy, I.M., Muller, R.U. & Kandel, E.R. Memory and behavior: a second generation of genetically modified mice. Curr. Biol. 7, R580–R589 (1997).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Aronoff, R. & Petersen, C.C. Layer, column and cell-type specific genetic manipulation in mouse barrel cortex. Front. Neurosci. 2, 64–71 (2008).

    Article  Google Scholar 

  6. 6

    Brickley, S.G., Revilla, V., Cull-Candy, S.G., Wisden, W. & Farrant, M. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409, 88–92 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Matthaei, K.I. Genetically manipulated mice: a powerful tool with unsuspected caveats. J. Physiol. 582, 481–488 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo . Proc. Natl. Acad. Sci. USA 101, 18206–18211 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Saito, T. & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237–246 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Hatanaka, Y., Hisanaga, S., 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 

  12. 12

    Barlow, H.B. Single units and sensation: a neuron doctrine for perceptual psychology? Perception 1, 371–394 (1972).

    CAS  Article  Google Scholar 

  13. 13

    Newsome, W.T., Britten, K.H. & Movshon, J.A. Neuronal correlates of a perceptual decision. Nature 341, 52–54 (1989).

    CAS  Article  Google Scholar 

  14. 14

    Parker, A.J. & Newsome, W.T. Sense and the single neuron: probing the physiology of perception. Annu. Rev. Neurosci. 21, 227–277 (1998).

    CAS  Article  Google Scholar 

  15. 15

    Romo, R. & Salinas, E. Touch and go: decision-making mechanisms in somatosensation. Annu. Rev. Neurosci. 24, 107–137 (2001).

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    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).

    CAS  Article  Google Scholar 

  18. 18

    Houweling, A.R. & Brecht, M. Behavioural report of single neuron stimulation in somatosensory cortex. Nature 451, 65–68 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Haas, K., Sin, W.C., Javaherian, A., Li, Z. & Cline, H.T. Single-cell electroporation for gene transfer in vivo . Neuron 29, 583–591 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Rathenberg, J., Nevian, T. & Witzemann, V. High-efficiency transfection of individual neurons using modified electrophysiology techniques. J. Neurosci. Methods 126, 91–98 (2003).

    Article  Google Scholar 

  21. 21

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

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

    Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  Article  Google Scholar 

  24. 24

    Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Iino, M. et al. Glia-synapse interaction through Ca2+-permeable AMPA receptors in Bergmann glia. Science 292, 926–929 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Van Tendeloo, V.F., Ponsaerts, P. & Berneman, Z.N. mRNA-based gene transfer as a tool for gene and cell therapy. Curr. Opin. Mol. Ther. 9, 423–431 (2007).

    CAS  PubMed  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    Komai, S., Denk, W., Osten, P., Brecht, M. & Margrie, T.W. Two-photon targeted patching (TPTP) in vivo . Nat. Protoc. 1, 647–652 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    CAS  Article  Google Scholar 

  31. 31

    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).

    CAS  Article  Google Scholar 

  32. 32

    Callaway, E.M. A molecular and genetic arsenal for systems neuroscience. Trends Neurosci. 28, 196–201 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Tsai, P.S. et al. eds. Principles, Design, and Construction of a Two-Photon Laser-Scanning Microscope for In Vitro and In Vivo Imaging (CRC Press, Boca Raton, FL, 2002).

    Google Scholar 

  34. 34

    Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    Article  Google Scholar 

  35. 35

    Bestman, J.E., Ewald, R.C., Chiu, S.L. & Cline, H.T. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat. Protoc. 1, 1267–1272 (2006).

    Article  Google Scholar 

  36. 36

    Mostany, R. & Portera-Cailliau, C. A craniotomy surgery procedure for chronic brain imaging. J. Vis. Exp. 12 (2008).

  37. 37

    Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Winfried Denk and Bill Richardson for helpful discussions, to Karl Deisseroth for the generous gift of channelrhodopsin plasmids, to Duncan Farquharson and Arifa Naeem for expert assistance, and to James Cottam, Spencer Smith and Christian Wilms for their comments on the manuscript. This work was supported by the Gatsby Foundation and the Wellcome Trust. B.J. is supported by a PhD Fellowship of the Boehringer Ingelheim Foundation and the Medical Research Council.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Michael Häusser.

Supplementary information

Supplementary Fig. 1

Schematic drawings of the recording chamber used for chronic imaging and electrophysiological recordings in rodents. All measurements in mm. (PDF 61 kb)

Supplementary Video 1: Single-cell electroporation guided by shadowimaging.

A neocortical layer 2/3 pyramidal neuron (same as in Fig. 3a) is identified by its fluorescent “shadow”. Once the pipette is in the correct position, successful electroporation is confirmed visually as the cell is filled with fluorescent dye and plasmid DNA. Width of frame: 75µm. (MOV 673 kb)

Supplementary Video 2: Successive electroporation of multiple neurons in a defined spatial pattern.

Multiple neocortical layer 2/3 pyramidal neurons are successively targeted for electroporation. (MOV 9678 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Judkewitz, B., Rizzi, M., Kitamura, K. et al. Targeted single-cell electroporation of mammalian neurons in vivo. Nat Protoc 4, 862–869 (2009). https://doi.org/10.1038/nprot.2009.56

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

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