Abstract
In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.
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References
Zito, K., Knott, G., Shepherd, G.M., Shenolikar, S. & Svoboda, K. Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton. Neuron 44, 321–334 (2004).
Zito, K., Scheuss, V., Knott, G., Hill, T. & Svoboda, K. Rapid functional maturation of nascent dendritic spines. Neuron 61, 247–258 (2009).
Holtmaat, A.J. et al. Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45, 279–291 (2005).
Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).
Knott, G.W., Holtmaat, A., Wilbrecht, L., Welker, E. & Svoboda, K. Spine growth precedes synapse formation in the adult neocortex in vivo. Nat. Neurosci. 9, 1117–1124 (2006).
De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).
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).
Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).
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).
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
Hoffpauir, B.K., Pope, B.A. & Spirou, G.A. Serial sectioning and electron microscopy of large tissue volumes for 3D analysis and reconstruction: a case study of the calyx of Held. Nat. Protoc. 2, 9–22 (2007).
Harris, K.M. et al. Uniform serial sectioning for transmission electron microscopy. J. Neurosci. 26, 12101–12103 (2006).
Acknowledgements
This work was supported by the Swiss National Science Foundation (G.W.K. no. 3100A0-112335 and E.W. no. 310000-108245). The authors would like to thank Stéphanie Rosset for technical help in the development of this protocol.
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G.W.K., J.T.T., K.S. and E.W. conceived the strategy for carrying out the electron microscopy on the imaged neurites. J.T.T., A.H. and K.S. carried out all of the in vivo imaging. K.S. and E.W. provided equipment and reagents. G.W.K. carried out the electron microscopy and wrote the manuscript.
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Knott, G., Holtmaat, A., Trachtenberg, J. et al. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat Protoc 4, 1145–1156 (2009). https://doi.org/10.1038/nprot.2009.114
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DOI: https://doi.org/10.1038/nprot.2009.114
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