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

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.