Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Nature Protocols volume 7, pages 1818–1829 (2012)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: AOD-based high-speed two-photon microscope.
Figure 2: Mouse surgery for the craniotomy preparation.
Figure 3: Craniotomy preparation before imaging.
Figure 4: Reduction of heartbeat-induced noise by agarose application.
Figure 5: Flowchart for the entire experimental procedure.
Figure 6: Filtering procedure for spine calcium signals.

Similar content being viewed by others

References

  1. 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).

    Article  CAS  PubMed  Google Scholar 

  2. Segal, M. Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6, 277–284 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Yuste, R. Dendritic spines and distributed circuits. Neuron 71, 772–781 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Bloodgood, B.L. & Sabatini, B.L. Ca(2+) signaling in dendritic spines. Curr. Opin. Neurobiol. 17, 345–351 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. 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).

    Article  CAS  PubMed  Google Scholar 

  11. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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).

    Article  CAS  PubMed  Google Scholar 

  14. 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  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. 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).

    Article  CAS  PubMed  Google Scholar 

  17. 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).

    Article  PubMed  Google Scholar 

  18. Zeng, S. et al. Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging. J. Biomed. Opt. 12, 024015 (2007).

    Article  PubMed  Google Scholar 

  19. Salome, R. et al. Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors. J. Neurosci. Methods 154, 161–174 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Takahashi, N. et al. Locally synchronized synaptic inputs. Science 335, 353–356 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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).

    Article  CAS  PubMed  Google Scholar 

  24. Schiller, J., Major, G., Koester, H.J. & Schiller, Y. NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 404, 285–289 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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).

    PubMed  PubMed Central  Google Scholar 

  29. Ji, N., Magee, J.C. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat. Methods 5, 197–202 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  PubMed  Google Scholar 

  31. 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).

    Article  CAS  PubMed  Google Scholar 

  32. 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).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. Ulrich Leischner

    Present address: Present address: Institute of Photonic Technology, Jena, Germany (U.L).,

Authors and Affiliations

  1. Institute of Neuroscience and Center for Integrated Protein Science, Technische Universität München, München, Germany

    Xiaowei Chen, Ulrich Leischner, Zsuzsanna Varga, Hongbo Jia, Diana Deca, Nathalie L Rochefort & Arthur Konnerth

  2. Brain Research Center, Third Military Medical University, Chongqing, China

    Xiaowei Chen

Authors
  1. Xiaowei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ulrich Leischner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zsuzsanna Varga
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongbo Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Diana Deca
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nathalie L Rochefort
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Arthur Konnerth
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Arthur Konnerth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.106

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing