Improvements in an in vitro assay for excitotoxicity by measurement of early gene (c-fos) levels

A Rogers, G Schmuck, G Scholz, Roger Griffiths, C Meredith, A Schousboe, G Campiani, DC Williams

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9 Citations (Scopus)

Abstract

Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15-30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.

Original languageEnglish
Pages (from-to)129-139
Number of pages11
JournalArchives of Toxicology
Volume79
DOIs
Publication statusPublished - Mar 2005

Keywords

  • excitotoxicity
  • c-fos
  • RT-PCR
  • in-vitro testing
  • Glu receptor
  • legs
  • CEREBELLAR GRANULE CELLS
  • GLOBAL CEREBRAL-ISCHEMIA
  • PRIMARY CULTURES
  • NEURONAL DEATH
  • NMDA RECEPTOR
  • AMINO-ACIDS
  • EXPRESSION
  • GLUTAMATE
  • ACTIVATION
  • CHANNELS

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