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Spine growth precedes synapse formation in the adult neocortex in vivo

Nature Neuroscience volume 9, pages 1117–1124 (2006)Cite this article

Abstract

Dendritic spines appear and disappear in an experience-dependent manner. Although some new spines have been shown to contain synapses, little is known about the relationship between spine addition and synapse formation, the relative time course of these events, or whether they are coupled to de novo growth of axonal boutons. We imaged dendrites in barrel cortex of adult mice over 1 month, tracking gains and losses of spines. Using serial section electron microscopy, we analyzed the ultrastructure of spines and associated boutons. Spines reconstructed shortly after they appeared often lacked synapses, whereas spines that persisted for 4 d or more always had synapses. New spines had a large surface-to-volume ratio and preferentially contacted boutons with other synapses. In some instances, two new spines contacted the same axon. Our data show that spine growth precedes synapse formation and that new synapses form preferentially onto existing boutons.

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Figure 1: High-resolution in vivo imaging and retrospective serial section electron microscopy.
Figure 2: Synapses on new spines.
Figure 3: Morphometric analysis of new spines.
Figure 4: Analysis of the neuropil surrounding imaged spines.
Figure 5: Analysis of synaptic terminals.

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  Google Scholar 

  2. Harris, K.M. & Stevens, J.K. Dendritic spines of CA1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982–2997 (1989).

    Article  CAS  Google Scholar 

  3. Nusser, Z. et al. Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 21, 545–559 (1998).

    Article  CAS  Google Scholar 

  4. Kharazia, V.N. & Weinberg, R.J. Immunogold localization of AMPA and NMDA receptors in somatic sensory cortex of albino rat. J. Comp. Neurol. 412, 292–302 (1999).

    Article  CAS  Google Scholar 

  5. Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E. & Ottersen, O.P. Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nat. Neurosci. 2, 618–624 (1999).

    Article  CAS  Google Scholar 

  6. Katz, L.C. & Shatz, C.J. Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138 (1996).

    Article  CAS  Google Scholar 

  7. Chklovskii, D.B., Mel, B.W. & Svoboda, K. Cortical rewiring and information storage. Nature 431, 782–788 (2004).

    Article  CAS  Google Scholar 

  8. Stepanyants, A., Hof, P.R. & Chklovskii, D.B. Geometry and structural plasticity of synaptic connectivity. Neuron 34, 275–288 (2002).

    Article  CAS  Google Scholar 

  9. Knott, G.W., Quairiaux, C., Genoud, C. & Welker, E. Formation of dendritic spines with GABAergic synapses induced by whisker stimulation in adult mice. Neuron 34, 265–273 (2002).

    CAS  PubMed  Google Scholar 

  10. Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

    CAS  PubMed  Google Scholar 

  11. Turner, A.M. & Greenough, W.T. Differential rearing effects on rat visual cortex synapses. I. Synaptic and neuronal density and synapses per neuron. Brain Res. 329, 195–203 (1985).

    Article  CAS  Google Scholar 

  12. Yankova, M., Hart, S.A. & Woolley, C.S. Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study. Proc. Natl. Acad. Sci. USA 98, 3525–3530 (2001).

    Article  CAS  Google Scholar 

  13. Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Holtmaat, A., Wilbrecht, L., Knott, G.W., Welker, E. & Svoboda, K. Experience-dependent and cell-type-specific spine growth in the neocortex. Nature 441, 979–983 (2006).

    Article  CAS  Google Scholar 

  17. Engert, F. & Bonhoeffer, T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399, 66–70 (1999).

    Article  CAS  Google Scholar 

  18. Maletic-Savatic, M., Malinow, R. & Svoboda, K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 1923–1927 (1999).

    Article  CAS  Google Scholar 

  19. Toni, N., Buchs, P.A., Nikonenko, I., Bron, C.R. & Muller, D. LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402, 421–425 (1999).

    Article  CAS  Google Scholar 

  20. Harris, K.M. Structure, development, and plasticity of dendritic spines. Curr. Opin. Neurobiol. 9, 343–348 (1999).

    Article  CAS  Google Scholar 

  21. Miller, M. & Peters, A. Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons. J. Comp. Neurol. 203, 555–573 (1981).

    Article  CAS  Google Scholar 

  22. Yuste, R. & Bonhoeffer, T. Genesis of dendritic spines: insights from ultrastructural and imaging studies. Nat. Rev. Neurosci. 5, 24–34 (2004).

    Article  CAS  Google Scholar 

  23. Ziv, N.E. & Smith, S.J. Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17, 91–102 (1996).

    CAS  Google Scholar 

  24. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Vaughan, D.W. & Peters, A. A three dimensional study of layer I of the rat parietal cortex. J. Comp. Neurol. 149, 355–370 (1973).

    Article  CAS  Google Scholar 

  27. Adams, I. & Jones, D.G. Quantitative ultrastructural changes in rat cortical synapses during early-, mid- and late-adulthood. Brain Res. 239, 349–363 (1982).

    Article  CAS  Google Scholar 

  28. Colonnier, M. Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res. 9, 268–287 (1968).

    Article  CAS  Google Scholar 

  29. Okabe, S., Miwa, A. & Okado, H. Spine formation and correlated assembly of presynaptic and postsynaptic molecules. J. Neurosci. 21, 6105–6114 (2001).

    Article  CAS  Google Scholar 

  30. Peters, A. & Kaiserman-Abramof, I.R. The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines. Am. J. Anat. 127, 321–355 (1970).

    Article  CAS  Google Scholar 

  31. Petrak, L.J., Harris, K.M. & Kirov, S.A. Synaptogenesis on mature hippocampal dendrites occurs via filopodia and immature spines during blocked synaptic transmission. J. Comp. Neurol. 484, 183–190 (2005).

    Article  Google Scholar 

  32. Peters, A., Palay, S.L. & Webster, H.D. The Fine Structure of the Nervous System. (Oxford Univ. Press, New York, 1991).

  33. Spacek, J. & Harris, K.M. Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. J. Neurosci. 17, 190–203 (1997).

    Article  CAS  Google Scholar 

  34. Mizrahi, A. & Katz, L.C. Dendritic stability in the adult olfactory bulb. Nat. Neurosci. 6, 1201–1207 (2003).

    Article  CAS  Google Scholar 

  35. Lee, W.C. et al. Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex. PLoSBiol. 4, e29 (2006).

    Article  Google Scholar 

  36. De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).

    Article  CAS  Google Scholar 

  37. Friedman, H.V., Bresler, T., Garner, C.C. & Ziv, N.E. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69 (2000).

    Article  CAS  Google Scholar 

  38. Harris, K.M., Jensen, F.E. & Tsao, B. Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation. J. Neurosci. 12, 2685–2705 (1992).

    Article  CAS  Google Scholar 

  39. Pozzan, T., Rizzuto, R., Volpe, P. & Meldolesi, J. Molecular and cellular physiology of intracellular calcium stores. Physiol. Rev. 74, 595–636 (1994).

    Article  CAS  Google Scholar 

  40. Braitenberg, V. & Schutz, A. Anatomy of the Cortex (Springer Verlag, Berlin, 1991).

  41. Micheva, K.D. & Beaulieu, C. Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry. J. Comp. Neurol. 373, 340–354 (1996).

    Article  CAS  Google Scholar 

  42. Jones, T.A., Klintsova, A.Y., Kilman, V.L., Sirevaag, A.M. & Greenough, W.T. Induction of multiple synapses by experience in the visual cortex of adult rats. Neurobiol. Learn. Mem. 68, 13–20 (1997).

    Article  CAS  Google Scholar 

  43. Shepherd, G.M. & Harris, K.M. Three-dimensional structure and composition of CA3–CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization. J. Neurosci. 18, 8300–8310 (1998).

    Article  CAS  Google Scholar 

  44. Lu, S.M. & Lin, R.C.S. Thalamic afferents of the rat barrel cortex: a light- and electron-microscopic study using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. Somatosens. Mot. Res. 10, 1–16 (1993).

    Article  CAS  Google Scholar 

  45. Malinow, R. & Malenka, R.C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126 (2002).

    Article  CAS  Google Scholar 

  46. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy (SBFSEM) to reconstruct 3D tissue nanostructure. PLoSBiol. 2, e329 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Fox, K. Anatomical pathways and molecular mechanisms for plasticity in the barrel cortex. Neuroscience 111, 799–814 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C. Musetti, V. DePaola and B. Burbach for help with the experiments, and M. Chklovskii, R. Weinberg, R. Weimer and K. Zito for comments on the manuscript. This work was supported by the Swiss National Science Foundation (E.W., No. 310000-108246), the Howard Hughes Medical Institute and the US National Institutes of Health (A.H., K.S. and L.W.).

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Author notes

  1. Graham W Knott and Anthony Holtmaat: These authors contributed equally to this work.

Authors and Affiliations

  1. Départment de Biologie Cellulaire et de Morphologie, University of Lausanne, Lausanne, CH1005, Switzerland

    Graham W Knott & Egbert Welker

  2. Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724, USA

    Anthony Holtmaat, Linda Wilbrecht & Karel Svoboda

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  1. Graham W Knott
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Contributions

G.W.K. and A.H. contributed equally to this work. G.W.K., A.H. and K.S. planned the experiments. G.W.K. performed the ssEM. A.H. and L.W. performed the in vivo imaging experiments. K.S. built the custom two-photon microscope. K.S. and E.W. contributed reagents, materials and analysis tools, and provided financial support. G.W.K., A.H. and K.S. analyzed the data and wrote the paper. G.W.K., A.H., L.W., E.W. and K.S. discussed the results and commented on the manuscript.

Note: Supplementary information is available on the Nature Neuroscience website.

Corresponding author

Correspondence to Graham W Knott.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

The segments of dendrites imaged in vivo and reconstructed from serial electron micrographs. (PDF 834 kb)

Supplementary Fig. 2

Serial electron micrographs of spine 1 from Figure 3. (PDF 4872 kb)

Supplementary Fig. 3

Time lapse images (upper), 3d reconstruction (middle) and serial electron micrographs (lower) of 3 spines. (PDF 932 kb)

Supplementary Fig. 4

Two modes of synapse formation by spine growth. (PDF 64 kb)

Supplementary Table 1

Spine parameters. (PDF 49 kb)

Supplementary Table 2

Parameters of boutons synapsing on dendritic shafts. (PDF 50 kb)

Supplementary Methods (PDF 41 kb)

Supplementary Note (PDF 24 kb)

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Knott, G., Holtmaat, A., Wilbrecht, L. et al. Spine growth precedes synapse formation in the adult neocortex in vivo. Nat Neurosci 9, 1117–1124 (2006). https://doi.org/10.1038/nn1747

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