The frog neuromuscular junction revisited after quick-freezing-freeze-drying: Ultrastructure, immunogold labelling and high resolution calcium mapping

R. Pezzati, F. Grohovaz

Research output: Contribution to journalArticlepeer-review

Abstract

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments - with fixatives and/or dehydration agents - that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick-freezing and freeze-drying (with or without exposure to OsO4 vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape. These preparations were also investigated by electron spectroscopic imaging and electron energy loss spectroscopy, obtaining high resolution maps of native total calcium distribution within the nerve terminal. Finally, thin sections from analogously processed, however unfixed, preparations embedded in Lowicryl, were immunogold labelled before exposure to OsO4. Nerve muscle preparations treated this way exhibited adequate preservation of ultrastructure and revealed the distribution of synaptophysin with high sensitivity and resolution. In conclusion, we provide an overview of the potential of the quick-freezing-freeze-drying approach in the study of the neuromuscular junction function.

Original languageEnglish
Pages (from-to)373-378
Number of pages6
JournalPhilosophical transactions of the Royal Society of London. Series B: Biological sciences
Volume354
Issue number1381
Publication statusPublished - Feb 28 1999

Keywords

  • Election spectroscopic imaging
  • Electron energy loss spectroscopy
  • Endoplasmic reticulum
  • Synaptic vesicles
  • Synaptophysin

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Agricultural and Biological Sciences (miscellaneous)

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