Abstract
Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5–6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.
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References
Beaulieu, C. & Colonnier, M. A laminar analysis of the number of round-asymmetrical and flat-symmetrical synapses on spines, dendritic trunks, and cell bodies in area 17 of the cat. J. Comp. Neurol. 231, 180–189 (1985).
Segal, M. Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6, 277–284 (2005).
Yuste, R. Dendritic spines and distributed circuits. Neuron 71, 772–781 (2011).
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).
Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).
Bloodgood, B.L. & Sabatini, B.L. Ca(2+) signaling in dendritic spines. Curr. Opin. Neurobiol. 17, 345–351 (2007).
Tsien, R.Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 2396–2404 (1980).
Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).
Lendvai, B., Stern, E.A., Chen, B. & Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404, 876–881 (2000).
Zuo, Y., Yang, G., Kwon, E. & Gan, W.B. Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex. Nature 436, 261–265 (2005).
Nagayama, S. et al. In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron 53, 789–803 (2007).
Chen, X., Leischner, U., Rochefort, N.L., Nelken, I. & Konnerth, A. Functional mapping of single spines in cortical neurons in vivo. Nature 475, 501–505 (2011).
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).
Jia, H., Rochefort, N.L., Chen, X. & Konnerth, A. Dendritic organization of sensory input to cortical neurons in vivo. Nature 464, 1307–1312 (2010).
Jia, H., Rochefort, N.L., Chen, X. & Konnerth, A. In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons. Nat. Protoc. 6, 28–35 (2011).
Zeng, S. et al. Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism. Opt. Lett. 31, 1091–1093 (2006).
Zeng, S. et al. Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging. J. Biomed. Opt. 12, 024015 (2007).
Salome, R. et al. Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors. J. Neurosci. Methods 154, 161–174 (2006).
Varga, Z., Jia, H., Sakmann, B. & Konnerth, A. Dendritic coding of multiple sensory inputs in single cortical neurons in vivo. Proc. Natl. Acad. Sci. USA 108, 15420–15425 (2011).
Takahashi, N. et al. Locally synchronized synaptic inputs. Science 335, 353–356 (2012).
Kovalchuk, Y., Eilers, J., Lisman, J. & Konnerth, A. NMDA receptor-mediated subthreshold Ca2+ signals in spines of hippocampal neurons. J. Neurosci. 20, 1791–1799 (2000).
Schneggenburger, R., Zhou, Z., Konnerth, A. & Neher, E. Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron 11, 133–143 (1993).
Schiller, J., Major, G., Koester, H.J. & Schiller, Y. NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 404, 285–289 (2000).
Kitamura, K., Judkewitz, B., Kano, M., Denk, W. & Hausser, M. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat. Methods 5, 61–67 (2008).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
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).
Ji, N., Magee, J.C. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat. Methods 5, 197–202 (2008).
Garaschuk, O., Milos, R.I. & Konnerth, A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat. Protoc. 1, 380–386 (2006).
Xu, H.T., Pan, F., Yang, G. & Gan, W.B. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat. Neurosci. 10, 549–551 (2007).
Wickersham, I.R., Finke, S., Conzelmann, K.K. & Callaway, E.M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat. Methods 4, 47–49 (2007).
Lo, L. & Anderson, D.J. A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).
Roth, D.M., Swaney, J.S., Dalton, N.D., Gilpin, E.A. & Ross, J. Jr. Impact of anesthesia on cardiac function during echocardiography in mice. Am. J. Physiol. Heart Circ. Physiol. 282, H2134–H2140 (2002).
Acknowledgements
We thank J. Lou for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (IRTG 1373), the European Research Area (ERA)-Net Program and the Schiedel Foundation. A.K. is a Carl von Linde Senior Fellow of the Institute for Advanced Study of the Technische Universität München.
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X.C., U.L., Z.V., H.J., D.D., N.L.R. and A.K. performed the experiments and the analysis. X.C. and A.K. wrote the paper together with all authors.
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Chen, X., Leischner, U., Varga, Z. et al. LOTOS-based two-photon calcium imaging of dendritic spines in vivo. Nat Protoc 7, 1818–1829 (2012). https://doi.org/10.1038/nprot.2012.106
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DOI: https://doi.org/10.1038/nprot.2012.106
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