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Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo


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.

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Figure 1: Strategy for using two-photon microscopy to image unlabeled neurons in the intact mammalian brain.
Figure 2: Visualization and identification of unlabeled neurons in vivo.
Figure 3: Direct visualization of the process of shadowpatching.
Figure 4: Shadowpatching of neocortical and cerebellar neurons in vivo.
Figure 5: Targeted dendritic patch-clamp recordings in vivo.
Figure 6: Targeted electroporation of DNA into single neurons in vivo.

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  1. Koch, C. & Segev, I. The role of single neurons in information processing. Nat. Neurosci. 3 (suppl.), 1171–1177 (2000).

    Article  CAS  Google Scholar 

  2. Häusser, M. & Mel, B. Dendrites: bug or feature? Curr. Opin. Neurobiol. 13, 372–383 (2003).

    Article  Google Scholar 

  3. Jagadeesh, B., Gray, C.M. & Ferster, D. Visually evoked oscillations of membrane potential in cells of cat visual cortex. Science 257, 552–554 (1992).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. 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 

  6. Chadderton, P., Margrie, T.W. & Häusser, M. Integration of quanta in cerebellar granule cells during sensory processing. Nature 428, 856–860 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. Euler, T., Detwiler, P.B. & Denk, W. Directionally selective calcium signals in dendrites of starburst amacrine cells. Nature 418, 845–852 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Haas, K. et al. Single-cell electroporation for gene transfer in vivo. Neuron 29, 583–591 (2001).

    Article  CAS  Google Scholar 

  16. 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 

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

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Waters, J. & Helmchen, F. Background synaptic activity is sparse in neocortex. J. Neurosci. 26, 8267–8277 (2006).

    Article  CAS  Google Scholar 

  22. Stuart, G., Schiller, J. & Sakmann, B. Action potential initiation and propagation in rat neocortical pyramidal neurons. J. Physiol. (Lond.) 505, 617–632 (1997).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Jung, J.C. et al. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).

    Article  Google Scholar 

  28. 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 

  29. Ohki, K. et al. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    Article  CAS  Google Scholar 

  30. Miyawaki, A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48, 189–199 (2005).

    Article  CAS  Google Scholar 

  31. Hasan, M.T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, e163 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).

    Article  CAS  Google Scholar 

  34. Zhang, F. et al. Circuit-breakers: optical technologies for probing neural signals and systems. Nat. Rev. Neurosci. 8, 577–581 (2007).

    Article  CAS  Google Scholar 

  35. Joyner, A.L. Gene Targeting: A Practical Approach 2nd edn. (Oxford Univ. Press, Oxford, UK, 2000).

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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.).

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Correspondence to Michael Häusser.

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)

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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).

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