Abstract
Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Koch, C. & Segev, I. The role of single neurons in information processing. Nat. Neurosci. 3 (suppl.), 1171–1177 (2000).
Häusser, M. & Mel, B. Dendrites: bug or feature? Curr. Opin. Neurobiol. 13, 372–383 (2003).
Jagadeesh, B., Gray, C.M. & Ferster, D. Visually evoked oscillations of membrane potential in cells of cat visual cortex. Science 257, 552–554 (1992).
Margrie, T.W., Brecht, M. & Sakmann, B. In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch. 444, 491–498 (2002).
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).
Chadderton, P., Margrie, T.W. & Häusser, M. Integration of quanta in cerebellar granule cells during sensory processing. Nature 428, 856–860 (2004).
Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).
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).
Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).
Margrie, T.W. et al. Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39, 911–918 (2003).
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).
Denk, W. & Detwiler, P.B. Optical recording of light-evoked calcium signals in the functionally intact retina. Proc. Natl. Acad. Sci. USA 96, 7035–7040 (1999).
Euler, T., Detwiler, P.B. & Denk, W. Directionally selective calcium signals in dendrites of starburst amacrine cells. Nature 418, 845–852 (2002).
Nevian, T. & Helmchen, F. Calcium indicator loading of neurons using single-cell electroporation. Pflugers Arch. 454, 675–688 (2007).
Haas, K. et al. Single-cell electroporation for gene transfer in vivo. Neuron 29, 583–591 (2001).
Rathenberg, J., Nevian, T. & Witzemann, V. High-efficiency transfection of individual neurons using modified electrophysiology techniques. J. Neurosci. Methods 126, 91–98 (2003).
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Stuart, G.J., Dodt, H.U. & Sakmann, B. Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflugers Arch. 423, 511–518 (1993).
Koester, H.J., Baur, D., Uhl, R. & Hell, S.W. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).
Loewenstein, Y. et al. Bistability of cerebellar Purkinje cells modulated by sensory stimulation. Nat. Neurosci. 8, 202–211 (2005).
Waters, J. & Helmchen, F. Background synaptic activity is sparse in neocortex. J. Neurosci. 26, 8267–8277 (2006).
Stuart, G., Schiller, J. & Sakmann, B. Action potential initiation and propagation in rat neocortical pyramidal neurons. J. Physiol. (Lond.) 505, 617–632 (1997).
Svoboda, K., Helmchen, F., Denk, W. & Tank, D.W. Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo. Nat. Neurosci. 2, 65–73 (1999).
Larkum, M.E. & Zhu, J.J. Signaling of layer 1 and whisker-evoked Ca2+ and Na+ action potentials in distal and terminal dendrites of rat neocortical pyramidal neurons in vitro and in vivo. J. Neurosci. 22, 6991–7005 (2002).
Zhu, J.J. & Connors, B.W. Intrinsic firing patterns and whisker-evoked synaptic responses of neurons in the rat barrel cortex. J. Neurophysiol. 81, 1171–1183 (1999).
Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 micron in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).
Jung, J.C. et al. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).
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).
Ohki, K. et al. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).
Miyawaki, A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48, 189–199 (2005).
Hasan, M.T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, e163 (2004).
Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).
De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).
Zhang, F. et al. Circuit-breakers: optical technologies for probing neural signals and systems. Nat. Rev. Neurosci. 8, 577–581 (2007).
Joyner, A.L. Gene Targeting: A Practical Approach 2nd edn. (Oxford Univ. Press, Oxford, UK, 2000).
Acknowledgements
We thank T. Branco, M. Rizzi and Y. Goda (UCL) for the pEGFP-C1 plasmid; T. Tabata and K. Powell for technical help; A. Roth for advice on image processing; T. Branco, I. Duguid, M. Rizzi, S. Smith and C. Wilms for discussions and comments on the manuscript. This work was supported by the Wellcome Trust (M.H.), Gatsby Foundation (M.H.), Japan Society for the Promotion of Science (K.K.), Uehara Foundation (K.K.), MEXT (grants-in-aid for scientific research nos. 18680034, 18650086 and 18019025 to K.K., and 17023021 and 17100004 to M.K.), and the Boehringer Ingelheim Fonds (B.J.).
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Methods, Supplementary Figures 1 and 2, and Supplementary Table 1 (PDF 1004 kb)
Supplementary Movie 1
Movie of neocortical layer 2/3 neurons visualized using shadowimaging. (MOV 1570 kb)
Supplementary Movie 2
Same as Supplementary Movie 1, except image has been inverted. (MOV 3407 kb)
Supplementary Movie 3
Movie of neurons in the molecular layer of cerebellar cortex visualized using shadowimaging. Note that the dendrites of individual Purkinje cells are clearly visible, as well as the cell bodies of molecular layer interneurons. (MOV 3431 kb)
Supplementary Movie 4
Movie showing targeted patching of a Purkinje cell using the negative image. Note the appearance of the dimple just prior to GΩ seal formation. (MOV 1926 kb)
Rights and permissions
About this article
Cite this article
Kitamura, K., Judkewitz, B., Kano, M. et al. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat Methods 5, 61–67 (2008). https://doi.org/10.1038/nmeth1150
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth1150
This article is cited by
-
Shadow imaging for panoptical visualization of brain tissue in vivo
Nature Communications (2023)
-
Acute head-fixed recordings in awake mice with multiple Neuropixels probes
Nature Protocols (2023)
-
Imaging brain tissue architecture across millimeter to nanometer scales
Nature Biotechnology (2023)
-
Dense 4D nanoscale reconstruction of living brain tissue
Nature Methods (2023)
-
Fast and sensitive GCaMP calcium indicators for imaging neural populations
Nature (2023)