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.

  • Letter
  • Published:

Functional mapping of single spines in cortical neurons in vivo

Nature volume 475, pages 501–505 (2011)Cite this article

Subjects

Abstract

The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain1,2,3,4. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex5, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored6. Here we used a new variant of high-resolution two-photon imaging7 to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.

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: Calcium signals in dendritic spines of cortical neurons in vivo.
Figure 2: Widespread dendritic distribution and sound-intensity-dependent recruitment of active spines.
Figure 3: Frequency tuning and heterogeneous distribution of individual active spines.
Figure 4: Dendritic arrangement of spines activated by the best and tail frequencies of neurons.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Cash, S. & Yuste, R. Linear summation of excitatory inputs by CA1 pyramidal neurons. Neuron 22, 383–394 (1999)

    Article  CAS  Google Scholar 

  3. Sabatini, B. L., Maravall, M. & Svoboda, K. Ca2+ signaling in dendritic spines. Curr. Opin. Neurobiol. 11, 349–356 (2001)

    Article  CAS  Google Scholar 

  4. Yuste, R., Majewska, A. & Holthoff, K. From form to function: calcium compartmentalization in dendritic spines. Nature Neurosci. 3, 653–659 (2000)

    Article  CAS  Google Scholar 

  5. 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  ADS  CAS  Google Scholar 

  6. Branco, T. & Häusser, M. The single dendritic branch as a fundamental functional unit in the nervous system. Curr. Opin. Neurobiol. 20, 494–502 (2010)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  8. Donnert, G., Eggeling, C. & Hell, S. W. Major signal increase in fluorescence microscopy through dark-state relaxation. Nature Methods 4, 81–86 (2007)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Jia, H., Rochefort, N. L., Chen, X. & Konnerth, A. In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons. Nature Protocols 6, 28–35 (2011)

    Article  CAS  Google Scholar 

  12. Chadderton, P., Agapiou, J. P., McAlpine, D. & Margrie, T. W. The synaptic representation of sound source location in auditory cortex. J. Neurosci. 29, 14127–14135 (2009)

    Article  CAS  Google Scholar 

  13. Linden, J. F., Liu, R. C., Sahani, M., Schreiner, C. E. & Merzenich, M. M. Spectrotemporal structure of receptive fields in areas AI and AAF of mouse auditory cortex. J. Neurophysiol. 90, 2660–2675 (2003)

    Article  Google Scholar 

  14. Scholl, B., Gao, X. & Wehr, M. Nonoverlapping sets of synapses drive on responses and off responses in auditory cortex. Neuron 65, 412–421 (2010)

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. Mainen, Z. F., Malinow, R. & Svoboda, K. Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151–155 (1999)

    Article  ADS  CAS  Google Scholar 

  19. Noguchi, J., Matsuzaki, M., Ellis-Davies, G. C. & Kasai, H. Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. Neuron 46, 609–622 (2005)

    Article  CAS  Google Scholar 

  20. Berretta, N. & Jones, R. S. Tonic facilitation of glutamate release by presynaptic N-methyl-D-aspartate autoreceptors in the entorhinal cortex. Neuroscience 75, 339–344 (1996)

    Article  CAS  Google Scholar 

  21. Wong, E. H. et al. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc. Natl Acad. Sci. USA 83, 7104–7108 (1986)

    Article  ADS  CAS  Google Scholar 

  22. Bloodgood, B. L., Giessel, A. J. & Sabatini, B. L. Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines. PLoS Biol. 7, e1000190 (2009)

    Article  Google Scholar 

  23. Bandyopadhyay, S., Shamma, S. A. & Kanold, P. O. Dichotomy of functional organization in the mouse auditory cortex. Nature Neurosci. 13, 361–368 (2010)

    Article  CAS  Google Scholar 

  24. Rothschild, G., Nelken, I. & Mizrahi, A. Functional organization and population dynamics in the mouse primary auditory cortex. Nature Neurosci. 13, 353–360 (2010)

    Article  CAS  Google Scholar 

  25. Wu, G. K., Li, P., Tao, H. W. & Zhang, L. I. Nonmonotonic synaptic excitation and imbalanced inhibition underlying cortical intensity tuning. Neuron 52, 705–715 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Holthoff, K., Kovalchuk, Y., Yuste, R. & Konnerth, A. Single-shock LTD by local dendritic spikes in pyramidal neurons of mouse visual cortex. J. Physiol. 560, 27–36 (2004)

    Article  CAS  Google Scholar 

  28. Polsky, A., Mel, B. W. & Schiller, J. Computational subunits in thin dendrites of pyramidal cells. Nature Neurosci. 7, 621–627 (2004)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  30. 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  ADS  CAS  Google Scholar 

  31. Franklin, K. & Paxinos, G. The Mouse Brain In Stereotaxic Coordinates. (Academic, 2001)

    Google Scholar 

  32. Busche, M. A. et al. Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer’s disease. Science 321, 1686–1689 (2008)

    Article  ADS  CAS  Google Scholar 

  33. de Kock, C. P. & Sakmann, B. High frequency action potential bursts (≥100 Hz) in L2/3 and L5B thick tufted neurons in anaesthetized and awake rat primary somatosensory cortex. J. Physiol. 586, 3353–3364 (2008)

    Article  CAS  Google Scholar 

  34. 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 . Nature Neurosci. 2, 65–73 (1999)

    Article  CAS  Google Scholar 

  35. Lechleiter, J. D., Lin, D. T. & Sieneart, I. Multi-photon laser scanning microscopy using an acoustic optical deflector. Biophys. J. 83, 2292–2299 (2002)

    Article  ADS  CAS  Google Scholar 

  36. Roorda, R. D., Hohl, T. M., Toledo-Crow, R. & Miesenbock, G. Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes. J. Neurophysiol. 92, 609–621 (2004)

    Article  Google Scholar 

  37. Kremer, Y. et al. A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view. Opt. Express 16, 10066–10076 (2008)

    Article  ADS  CAS  Google Scholar 

  38. Gerig, J. S. & Montague, H. A simple optical filter for chirp radar. Proc. IEEE 52, 1753 (1964)

    Article  Google Scholar 

  39. Kerlin, A. M., Andermann, M. L., Berezovskii, V. K. & Reid, R. C. Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex. Neuron 67, 858–871 (2010)

    Article  CAS  Google Scholar 

  40. Sohya, K., Kameyama, K., Yanagawa, Y., Obata, K. & Tsumoto, T. GABAergic neurons are less selective to stimulus orientation than excitatory neurons in layer II/III of visual cortex, as revealed by in vivo functional Ca2+ imaging in transgenic mice. J. Neurosci. 27, 2145–2149 (2007)

    Article  CAS  Google Scholar 

  41. Peters, A. & Jones, E. G. in Cerebral Cortex: Cellular Components of the Cerebral Cortex Vol. 1 (eds Peters, A. & Jones, E. G.) 107–121 (Plenum, 1984)

    Google Scholar 

  42. Kawaguchi, Y., Karube, F. & Kubota, Y. Dendritic branch typing and spine expression patterns in cortical nonpyramidal cells. Cereb. Cortex 16, 696–711 (2006)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Lou for technical assistance, D. Bayer, F. Bayer and W. Zeitz for building the scanning device, A. Fohr for software support and Y. Kovalchuk and H. Adelsberger for help during the initial experiments. This work was supported by the Schiedel Foundation, the German-Israeli Foundation (GIF grant 1002/2008 to I.N. and A.K.), the Deutsche Forschungsgemeinschaft (IRTG 1373) and the Bundesministerium für Bildung und Forschung (BMBF) in the frame of ERA-NET NEURON. 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. Xiaowei Chen and Ulrich Leischner: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Neuroscience and Center for Integrated Protein Science, Technical University Munich, Biedersteinerstrasse 29, 80802 Munich, Germany ,

    Xiaowei Chen, Ulrich Leischner, Nathalie L. Rochefort & Arthur Konnerth

  2. Department of Neurobiology, Silberman Institute of Life Sciences and the Edmond and Lily Safra Center for Brain Sciences, Hebrew University, Jerusalem 91904, Israel,

    Israel Nelken

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. Nathalie L. Rochefort
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Israel Nelken
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arthur Konnerth
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.C., U.L., I.N., N.L.R. and A.K. carried out the experiments. U.L. and A.K. designed and constructed the imaging device. X.C., U.L., N.L.R., I.N. and A.K. performed the analysis. A.K. designed the study and wrote the manuscript with the help of all authors.

Corresponding author

Correspondence to Arthur Konnerth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-11 with legends, Supplementary Table 1, the legend for Supplementary Movie 1 and additional references. (PDF 1020 kb)

Supplementary Movie 1

The movie shows three consecutive trials of spine calcium responses (S1-S4) to pure tone stimulation (0 dB attenuation; 100 ms duration) – see Supplementary Information file for full legend. (MOV 14577 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, X., Leischner, U., Rochefort, N. et al. Functional mapping of single spines in cortical neurons in vivo. Nature 475, 501–505 (2011). https://doi.org/10.1038/nature10193

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10193

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