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Targeted single-cell electroporation of mammalian neurons in vivo

Nature Protocols volume 4, pages 862–869 (2009)Cite this article

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.

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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.

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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.

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Author notes

  1. Kazuo Kitamura

    Present address: Present address: Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.,

Authors and Affiliations

  1. Wolfson Institute for Biomedical Research and Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Benjamin Judkewitz, Matteo Rizzi, Kazuo Kitamura & Michael Häusser

Authors
  1. Benjamin Judkewitz
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  2. Matteo Rizzi
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  3. Kazuo Kitamura
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  4. Michael Häusser
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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)

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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

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