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.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
London, M. & Häusser, M. Dendritic computation. Annu. Rev. Neurosci. 28, 503–532 (2005).
Johnston, D. & Narayanan, R. Active dendrites: colorful wings of the mysterious butterflies. Trends. Neurosci. 31, 309–316 (2008).
Larkum, M.E. & Nevian, T. Synaptic clustering by dendritic signalling mechanisms. Curr. Opin. Neurobiol. 18, 321–331 (2008).
Ohki, K. & Reid, R.C. Specificity and randomness in the visual cortex. Curr. Opin. Neurobiol. 17, 401–407 (2007).
Bloodgood, B.L. & Sabatini, B.L. Ca2+ signaling in dendritic spines. Curr. Opin. Neurobiol. 17, 345–351 (2007).
Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).
Müller, W. & Connor, J.A. Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses. Nature 354, 73–76 (1991).
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).
Häusser, M. & Mel, B. Dendrites: bug or feature? Curr. Opin. Neurobiol. 13, 372–383 (2003).
Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).
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).
Murayama, M. et al. Dendritic encoding of sensory stimuli controlled by deep cortical interneurons. Nature 457, 1137–1141 (2009).
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).
Waters, J. & Helmchen, F. Boosting of action potential backpropagation by neocortical network activity in vivo. J. Neurosci. 24, 11127–11136 (2004).
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).
Helmchen, F. & Waters, J. Ca2+ imaging in the mammalian brain in vivo. Eur. J. Pharmacol. 447, 119–129 (2002).
Jia, H., Rochefort, N.L., Chen, X. & Konnerth, A. Dendritic organization of sensory input to cortical neurons in vivo. Nature 464, 1307–1312 (2010).
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).
Bollmann, J.H. & Engert, F. Subcellular topography of visually driven dendritic activity in the vertebrate visual system. Neuron 61, 895–905 (2009).
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).
Nevian, T. & Helmchen, F. Calcium indicator loading of neurons using single-cell electroporation. Pflugers Arch. 454, 675–688 (2007).
Nagayama, S. et al. In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron 53, 789–803 (2007).
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).
Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).
Mank, M. & Griesbeck, O. Genetically encoded calcium indicators. Chem. Rev. 108, 1550–1564 (2008).
Hires, S.A., Tian, L. & Looger, L.L. Reporting neural activity with genetically encoded calcium indicators. Brain Cell. Biol. 36, 69–86 (2008).
Miyawaki, A. Fluorescence imaging of physiological activity in complex systems using GFP-based probes. Curr. Opin. Neurobiol. 13, 591–596 (2003).
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).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
Rochefort, N.L. & Konnerth, A. Genetically encoded Ca2+ sensors come of age. Nat. Methods 5, 761–762 (2008).
Niell, C.M. & Stryker, M.P. Highly selective receptive fields in mouse visual cortex. J. Neurosci. 28, 7520–7536 (2008).
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).
The authors declare no competing financial interests.
About this article
Cite this article
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
Cell Reports (2019)
Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies
BMC Neuroscience (2019)
Nature Neuroscience (2019)
Vasoactive Intestinal Polypeptide-Expressing Interneurons in the Hippocampus Support Goal-Oriented Spatial Learning
Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks