Spine growth precedes synapse formation in the adult neocortex in vivo

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.

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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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Authors

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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