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Two-photon targeted patching (TPTP) in vivo

Nature Protocols volume 1, pages 647–652 (2006)Cite this article

Abstract

Two-photon–excited fluorescence laser-scanning microscopy (2PLSM) has provided a wealth of information about the spatiotemporal properties of biological processes at the single cell and population level. Because such nonlinear optical methods allow for imaging deep within biological tissue, 2PLSM can be combined with patch-clamp techniques to obtain electrophysiological recordings from specific fluorescently labeled cells in vivo. Here a protocol referred to as two-photon targeted patching (TPTP) describes a method that may be used to record from cells in the intact animal labeled by virtually any type of fluorophore. We target neurons that have been optically and genetically identified using green fluorescent protein (GFP) expressed under the control of a specific promoter. TPTP when combined with genetic approaches therefore permits electrophysiological recordings from specified neurons and their compartments, including dendrites. This technique may be repeated in the same preparation many times over the course of several hours and is equally applicable to non-neuronal cell types.

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Figure 1: Setup for TPTP.
Figure 2: Monitoring pipette resistance while navigating through upper layers of tissue.
Figure 3: Detecting pipette-cell interactions using visual and electrophysiological cues.
Figure 4: Verification of TPTP.

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References

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

    Article  CAS  Google Scholar 

  2. Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    Article  CAS  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

  4. Cahalan, M.D., Parker, I., Wei, S.H. & Miller, M.J. Real-time imaging of lymphocytes in vivo. Curr. Opin. Immunol. 15, 372–377 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  5. Rubart, M. Two-photon microscopy of cells and tissue. Circ. Res. 95, 1154–1166 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  6. Molitoris, B.A. & Sandoval, R.M. Intravital multiphoton microscopy of dynamic renal processes. Am. J. Physiol. Renal Physiol. 288, F1084–F1089 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  7. Denk, W. & Svoboda, K. Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357 (1997).

    Article  CAS  Google Scholar 

  8. 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  PubMed Central  Google Scholar 

  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).

    Article  CAS  PubMed Central  Google Scholar 

  10. 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  PubMed Central  Google Scholar 

  11. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  12. 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  PubMed Central  Google Scholar 

  13. 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  PubMed Central  Google Scholar 

  14. Hubener, M. & Bonhoeffer, T. Visual cortex: two-photon excitement. Curr. Biol. 15, R205–R208 (2005).

    Article  PubMed Central  Google Scholar 

  15. Holtmaat, A.J. et al. Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45, 279–291 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  16. Kerr, J.N., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  17. 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 

  18. 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  PubMed Central  Google Scholar 

  19. 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  PubMed Central  Google Scholar 

  20. Helmchen, F., Svoboda, K., Denk, W. & Tank, D.W. In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nat. Neurosci. 2, 989–996 (1999).

    Article  CAS  PubMed Central  Google Scholar 

  21. 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  PubMed Central  Google Scholar 

  22. 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  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

  25. Schrader, M., Hofmann, U.G. & Hell, S.W. Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy. J. Microsc. 191, 135–140 (1998).

    Article  CAS  PubMed Central  Google Scholar 

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Acknowledgements

The authors wish to thank P. Chadderton for his assistance in generating the figures and reading the manuscript and D. Farquarson for providing the design layout of the headplate. T.W.M. is supported by The Welcome Trust and The Human Frontiers Science Program (P.O. and T.W.M.).

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Authors and Affiliations

  1. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara, 630-0192, Japan

    Shoji Komai

  2. Department of Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg, D-16920, Germany

    Winfried Denk

  3. Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, 60611, Illinois, USA

    Pavel Osten

  4. Department of Neuroscience, Erasmus Medical Center, Dr. Molewaterplein 50, Rotterdam, 3015 GE, The Netherlands

    Michael Brecht

  5. Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK

    Troy W Margrie

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  1. Shoji Komai
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  5. Troy W Margrie
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Correspondence to Troy W Margrie.

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Komai, S., Denk, W., Osten, P. et al. Two-photon targeted patching (TPTP) in vivo. Nat Protoc 1, 647–652 (2006). https://doi.org/10.1038/nprot.2006.100

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