In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons

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Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains ('hotspots') and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5–6 h, including 1–2 h of two-photon calcium imaging in combination with stable whole-cell recordings.

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Figure 1: Experimental arrangement for in vivo dendritic imaging in combination with whole-cell recordings.
Figure 2: In vivo two-photon imaging of visually evoked dendritic calcium signals.
Figure 3: Extraction of local dendritic calcium transients from two-photon images.


  1. 1

    London, M. & Häusser, M. Dendritic computation. Annu. Rev. Neurosci. 28, 503–532 (2005).

  2. 2

    Johnston, D. & Narayanan, R. Active dendrites: colorful wings of the mysterious butterflies. Trends. Neurosci. 31, 309–316 (2008).

  3. 3

    Larkum, M.E. & Nevian, T. Synaptic clustering by dendritic signalling mechanisms. Curr. Opin. Neurobiol. 18, 321–331 (2008).

  4. 4

    Ohki, K. & Reid, R.C. Specificity and randomness in the visual cortex. Curr. Opin. Neurobiol. 17, 401–407 (2007).

  5. 5

    Bloodgood, B.L. & Sabatini, B.L. Ca2+ signaling in dendritic spines. Curr. Opin. Neurobiol. 17, 345–351 (2007).

  6. 6

    Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).

  7. 7

    Müller, W. & Connor, J.A. Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses. Nature 354, 73–76 (1991).

  8. 8

    Eilers, J., Augustine, G.J. & Konnerth, A. Subthreshold synaptic Ca2+ signalling in fine dendrites and spines of cerebellar Purkinje neurons. Nature 373, 155–158 (1995).

  9. 9

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

  10. 10

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

  11. 11

    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).

  12. 12

    Murayama, M. et al. Dendritic encoding of sensory stimuli controlled by deep cortical interneurons. Nature 457, 1137–1141 (2009).

  13. 13

    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).

  14. 14

    Waters, J. & Helmchen, F. Boosting of action potential backpropagation by neocortical network activity in vivo. J. Neurosci. 24, 11127–11136 (2004).

  15. 15

    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).

  16. 16

    Helmchen, F. & Waters, J. Ca2+ imaging in the mammalian brain in vivo. Eur. J. Pharmacol. 447, 119–129 (2002).

  17. 17

    Jia, H., Rochefort, N.L., Chen, X. & Konnerth, A. Dendritic organization of sensory input to cortical neurons in vivo. Nature 464, 1307–1312 (2010).

  18. 18

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

  19. 19

    Bollmann, J.H. & Engert, F. Subcellular topography of visually driven dendritic activity in the vertebrate visual system. Neuron 61, 895–905 (2009).

  20. 20

    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).

  21. 21

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

  22. 22

    Nagayama, S. et al. In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron 53, 789–803 (2007).

  23. 23

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

  24. 24

    Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).

  25. 25

    Mank, M. & Griesbeck, O. Genetically encoded calcium indicators. Chem. Rev. 108, 1550–1564 (2008).

  26. 26

    Hires, S.A., Tian, L. & Looger, L.L. Reporting neural activity with genetically encoded calcium indicators. Brain Cell. Biol. 36, 69–86 (2008).

  27. 27

    Miyawaki, A. Fluorescence imaging of physiological activity in complex systems using GFP-based probes. Curr. Opin. Neurobiol. 13, 591–596 (2003).

  28. 28

    Lutcke, H. et al. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice. Front. Neural. Circuits 4, 9 (2010).

  29. 29

    Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

  30. 30

    Rochefort, N.L. & Konnerth, A. Genetically encoded Ca2+ sensors come of age. Nat. Methods 5, 761–762 (2008).

  31. 31

    Niell, C.M. & Stryker, M.P. Highly selective receptive fields in mouse visual cortex. J. Neurosci. 28, 7520–7536 (2008).

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We are grateful to Y. Kovalchuk for his help in the initial experiments. This study was supported by grants from Deutsche Forschungsgemeinschaft (DFG) to A.K. and from the Friedrich Schiedel Foundation. A.K. is a Carl von Linde Senior Fellow of the Institute for Advanced Study of the Technische Universität München. H.J. and N.L.R. were supported by the DFG (IRTG 1373).

Author information

H.J., N.L.R., X.C. and A.K. performed the experiments and the analysis. H.J. developed the program for data analysis. A.K. wrote the paper together with H.J., N.L.R. and X.C.

Correspondence to Arthur Konnerth.

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Jia, H., Rochefort, N., Chen, X. et al. In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons. Nat Protoc 6, 28–35 (2011) doi:10.1038/nprot.2010.169

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