Analysis of Synaptic Connections at the Electron Microscopic Level Using Viral Vectors

  • Takahiro Furuta
  • Keiko Okamoto-Furuta
  • Hiroyuki Hioki
Part of the Neuromethods book series (NM, volume 110)

Abstract

Anterograde tracing using Sindbis virus vector resolves several problems of conventional tracers. Usage of the Sindbis virus as a neural tracer prevents the axons passing through the injection site from being labeled because the virus does not infect from axons. Combining the methodological merit of the virus tracer with electron microscopic technique, we analyze morphological characteristics of ultrastructure of axon terminals which are provided specifically from the region of interest, and thus we can systematically extract information of synaptic organization from complicated neural networks. Here, we introduce a method where Sindbis virus labeling is combined with an immunoelectron microscopic technique. Using this technique, we observe synaptic contacts and GABA immunoreactivity in ultrastructure of axon terminals which are revealed to be projected from the virus injection site.

Keywords

Sindbis virus Anterograde tracing Electron microscopy GFP Post-embedding immunostaining 

Notes

Acknowledgments

This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan Grants (15H04266, 15H01430 and 15K14333). We thank Dr. H. Bokor for the helpful advice on post-embedding immunostaining methods.

References

  1. 1.
    Deller T, Naumann T, Frotscher M (2000) Retrograde and anterograde tracing combined with transmitter identification and electron microscopy. J Neurosci Methods 103:117–126CrossRefPubMedGoogle Scholar
  2. 2.
    McDonald AJ (1992) Neuroanatomical labeling with biocytin: a review. Neuroreport 3:821–827CrossRefPubMedGoogle Scholar
  3. 3.
    Furuta T, Timofeeva E, Nakamura K, Okamoto-Furuta K, Togo M, Kaneko T, Deschenes M (2008) Inhibitory gating of vibrissal inputs in the brainstem. J Neurosci 28:1789–1797CrossRefPubMedGoogle Scholar
  4. 4.
    Furuta T, Tomioka R, Taki K, Nakamura K, Tamamaki N, Kaneko T (2001) In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins. J Histochem Cytochem 49:1497–1508CrossRefPubMedGoogle Scholar
  5. 5.
    Somogyi P, Hodgson AJ, Chubb IW, Penke B, Erdei A (1985) Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J Histochem Cytochem 33:240–248CrossRefPubMedGoogle Scholar
  6. 6.
    Tamamaki N, Nakamura K, Furuta T, Asamoto K, Kaneko T (2000) Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo. Neurosci Res 38:231–236CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Takahiro Furuta
    • 1
  • Keiko Okamoto-Furuta
    • 2
  • Hiroyuki Hioki
    • 1
  1. 1.Department of Morphological Brain ScienceGraduate School of Medicine, Kyoto UniversityKyotoJapan
  2. 2.Division of Electron Microscopic StudyCenter for Anatomical Studies, Graduate School of Medicine, Kyoto UniversityKyotoJapan

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