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Three-Dimensional Imaging of Cerebellar Mossy Fiber Rosettes by Ion-Abrasion Scanning Electron Microscopy

Published online by Cambridge University Press:  06 August 2013

Hyun-Wook Kim
Affiliation:
Department of Anatomy, College of Medicine, Korea University, 126-1 Anam dong 5 ka SungBuk Ku, Seoul 136-705, Korea
Namkug Kim
Affiliation:
Department of Radiology, Research Institute of Radiology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea
Ki Woo Kim*
Affiliation:
School of Ecology and Environmental System, Kyungpook National University, Sangju 742-711, Korea
Im Joo Rhyu*
Affiliation:
Department of Anatomy, College of Medicine, Korea University, 126-1 Anam dong 5 ka SungBuk Ku, Seoul 136-705, Korea
*
Corresponding author. E-mail: [email protected]
Corresponding author. E-mail: [email protected]
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Abstract

The detailed knowledge of the three-dimensional (3D) organization of the nervous tissue provides essential information on its functional elucidation. We used serial block-face scanning electron microscopy with focused ion beam (FIB) milling to reveal 3D morphologies of the mossy fiber rosettes in the mice cerebellum. Three-week-old C57 black mice were perfused with a fixative of 1% paraformaldehyde/1% glutaraldehyde in phosphate buffer; the cerebellum was osmicated and embedded in the Araldite. The block containing granule cell layer was sliced with FIB and observed by field-emission scanning electron microscopy. The contrast of backscattered electron image of the block-face was similar to that of transmission electron microscopy and processed using 3D visualization software for further analysis. The mossy fiber rosettes on each image were segmented and rendered to visualize the 3D model. The complete 3D characters of the mossy fiber rosette could be browsed on the A-Works, in-house software, and some preliminary quantitative data on synapse of the rosette could be extracted from these models. Thanks to the development of two-beam imaging and optimized software, we could get 3D information on cerebellar mossy fiber rosettes with ease and speedily, which would be an additive choice to explore 3D structures of the nervous systems and their networks.

Type
Research Article
Copyright
Copyright © Microscopy Society of America 2013 

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References

Arenkiel, B.R. & Ehlers, M.D. (2009). Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature 461(7266), 900907.10.1038/nature08536Google Scholar
Bushby, A.J., P'Ng, K.M., Young, R.D., Pinali, C., Knupp, C. & Quantock, A.J. (2011). Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat Protoc 6(6), 845858.10.1038/nprot.2011.332Google Scholar
Denk, W. & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11), e329.10.1371/journal.pbio.0020329Google Scholar
Eisenstein, M. (2009). Neural circuits: Putting neurons on the map. Nature 461(7267), 11491152.10.1038/4611149aGoogle Scholar
Fiala, J.C., Allwardt, B. & Harris, K.M. (2002). Dendritic spines do not split during hippocampal LTP or maturation. Nat Neurosci 5(4), 297298.Google Scholar
Giannuzzi, L.A., Phifer, D., Giannuzzi, N.J. & Capuano, M.J. (2007). Two-dimensional and 3-dimensional analysis of bone/dental implant interfaces with the use of focused ion beam and electron microscopy. J Oral Maxillofac Surg 65(4), 737747.10.1016/j.joms.2006.10.025Google Scholar
Gray, E.G. (1959). Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex. Nature 183(4675), 15921593.10.1038/1831592a0Google Scholar
Hamori, J. & Somogyi, J. (1983). Differentiation of cerebellar mossy fiber synapses in the rat: A quantitative electron microscope study. J Comp Neurol 220(4), 365377.10.1002/cne.902200402Google Scholar
Heng, D., Tang, P., Cairney, J.M., Chan, H.K., Cutler, D.J., Salama, R. & Yun, J. (2007). Focused-ion-beam milling: A novel approach to probing the interior of particles used for inhalation aerosols. Pharm Res 24(9), 16081617.10.1007/s11095-007-9276-6Google Scholar
Hou, K. & Yao, N. (2007). Applications for Biological Materials. In Focused Ion Beam Systems. Cambridge, UK: Cambridge University Press.Google Scholar
Jakab, R.L. (1989). Three-dimensional reconstruction and synaptic architecture of cerebellar glomeruli in the rat. Acta Morphol Hung 37(1-2), 1120.Google Scholar
Jakab, R.L. & Hamori, J. (1988). Quantitative morphology and synaptology of cerebellar glomeruli in the rat. Anat Embryol (Berl) 179(1), 8188.10.1007/BF00305102Google Scholar
Kim, K., Baek, S., Park, B., Kim, H. & Rhyu, I. (2010). Applications of focused ion beam for biomedical resarch. Kor J Microsc 40(4), 177183.Google Scholar
Kim, K.W. & Jaksch, H. (2009). Compositional contrast of uncoated fungal spores and stained section-face by low-loss backscattered electron imaging. Micron 40(7), 724729.10.1016/j.micron.2009.05.001Google Scholar
Knott, G., Marchman, H., Wall, D. & Lich, B. (2008). Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28(12), 29592964.10.1523/JNEUROSCI.3189-07.2008Google Scholar
Kudo, M., Kameda, J., Saruwatari, K., Ozaki, N., Okano, K., Nagasawa, H. & Kogure, T. (2010). Microtexture of larval shell of oyster, Crassostrea nippona: A FIB-TEM study. J Struct Biol 169(1), 15.10.1016/j.jsb.2009.07.014Google Scholar
Merchan-Perez, A., Rodriguez, J.R., Alonso-Nanclares, L., Schertel, A. & Defelipe, J. (2009). Counting synapses using FIB/SEM microscopy: A true revolution for ultrastructural volume reconstruction. Front Neuroanat 3, 18.10.3389/neuro.05.018.2009Google Scholar
Muller-Reichert, T., Mancuso, J., Lich, B. & McDonald, K. (2010). Three-dimensional reconstruction methods for Caenorhabditis elegans ultrastructure. Methods Cell Biol 96, 331361.10.1016/S0091-679X(10)96015-9Google Scholar
Noske, A.B., Costin, A.J., Morgan, G.P. & Marsh, B.J. (2008). Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets. J Struct Biol 161(3), 298313.10.1016/j.jsb.2007.09.015Google Scholar
Ostroff, L.E., Fiala, J.C., Allwardt, B. & Harris, K.M. (2002). Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices. Neuron 35(3), 535545.10.1016/S0896-6273(02)00785-7Google Scholar
Palay, S. & Chan-Palay, V. (1974). Cerebellar Cortex: Cytology and Organization. New York: Springer.10.1007/978-3-642-65581-4Google Scholar
Park, I.S., Lee, K.J., Han, J.W., Lee, N.J., Lee, W.T., Park, K.A. & Rhyu, I.J. (2009). Experience-dependent plasticity of cerebellar vermis in basketball players. Cerebellum 8(3), 334339.10.1007/s12311-009-0100-1Google Scholar
Toni, N., Buchs, P.A., Nikonenko, I., Bron, C.R. & Muller, D. (1999). LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402(6760), 421425.10.1038/46574Google Scholar
White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Phil Trans R Soc Lond B 314(1165), 1340.Google Scholar
Xu-Friedman, M.A. & Regehr, W.G. (2003). Ultrastructural contributions to desensitization at cerebellar mossy fiber to granule cell synapses. J Neurosci 23(6), 21822192.10.1523/JNEUROSCI.23-06-02182.2003Google Scholar