Autoradiographic measurements of protein synthesis in hippocampal slices from rats and guinea pigs

Methods. 1999 Jun;18(2):127-43. doi: 10.1006/meth.1999.0766.

Abstract

Protein synthesis is an extremely important cell function and there is now good evidence that changes in synthesis play important roles both in neuronal cell damage from ischemic insults and in neural plasticity though the mechanisms of these effects are not at all clear. The brain slice, and particularly the hippocampal slice, is an excellent preparation for studying these effects although, as with all studies on slices, caution must be exercised in that regulation in the slice may be different from regulation in vivo. Studies on neural tissue need to take into account the heterogeneity of neural tissue as well as the very different compartments within neurons. Autoradiography at both the light and electron microscope levels is a very powerful method for doing this. Successful autoradiography depends on many factors. These include correct choice of precursor amino acid, mechanisms for estimating changes in the specific activity of the precursor amino acid pool, and reliable methods for quantitation of the autoradiographs. At a more technical level these factors include attention to detail in processing tissue sections so as to avoid light contamination during exposure and developing and, also, appropriate choices of the various parameters such as exposure time and section thickness. The power of autoradiography is illustrated here by its ability to discern effects of ischemia and of plasticity-related neural input on distinct cell types and also in distinct compartments of neurons. Ischemia inhibits protein synthesis in principal neurons but activates synthesis in other cell types of the brain slice. Plasticity-related neural input immediately enhances protein synthesis in dendrites but does not affect cell bodies.

MeSH terms

  • Animals
  • Autoradiography / methods
  • Cycloheximide / pharmacology
  • Dissection
  • Guinea Pigs
  • Hippocampus / cytology
  • Hippocampus / metabolism*
  • Hippocampus / ultrastructure
  • In Vitro Techniques
  • Leucine / metabolism
  • Microscopy, Electron / methods
  • Nerve Tissue Proteins / biosynthesis*
  • Neurons / cytology
  • Neurons / metabolism*
  • Neurons / ultrastructure
  • Protein Synthesis Inhibitors / pharmacology
  • Rats
  • Tritium

Substances

  • Nerve Tissue Proteins
  • Protein Synthesis Inhibitors
  • Tritium
  • Cycloheximide
  • Leucine