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Keywords:

  • γ-aminobutyric acid;
  • boutons;
  • freeze-substitution;
  • glutamate;
  • glycine;
  • motoneuron;
  • post-embedding immunohistochemistry;
  • spinal cord

Abstract

The spinal cord motor nuclei have been the focus of a number of investigations exploring neurodegenerative mechanisms, e.g. excitotoxicity mediated by glutamate and oxidative stress. Here, high-resolution quantitative post-embedding immunocytochemistry with antibodies to oxidized and reduced glutathione (GSH), an ubiquitously expressed scavenger of free radicals, was used to examine if GSH synthesis is upregulated pre- and/or postsynaptically in the lumbar motor nuclei of aged (30 month old) rats. The purpose was, moreover, to resolve the extent of correlation between GSH expression, transmitter identity and degenerative changes. Tissue from young adult rats was co-processed for comparison. The quantitative immunogold analysis revealed an increase in GSH-immunoreactivity in both pre- and postsynaptic compartments in the lumbar motor nuclei of aged rats. Presynaptically, the enrichment of GSH-immunoreactivity was seen in axonal boutons of normal appearance, and was furthermore restricted to the extra-mitochondrial compartment. Postsynaptically, the aged rats disclosed, in comparison with young adults, higher values for GSH-immunoreactivity both over mitochondria (+49%) and cytoplasmic matrix (+130%). When analysing the transmitter identity of the bouton profiles, it turned out that close to 50% of all glutamate-immunoreactive boutons in the aged rats contained very high levels (> 40 gold particles/μm2) of GSH-immunoreactivity. Strong GSH-immunoreactivity was also a typical feature of a subset of axon terminal- and axon fibre-like profiles in the aged rat that showed signs of axon dystrophy and degeneration. When comparing with normally appearing axon fibre profiles located in close vicinity, the population of aberrant axons had higher average levels of glutamate-immunoreactivity (+93%), and lower average levels of glycine-immunoreactivity (–88%). No difference was seen regarding the levels of GABA. The results of this study lend support to the idea that aging in the spinal cord motor nuclei is associated with an increased oxidative stress and indicate that different transmitter systems are differentially affected by the degenerative process.