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

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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.

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Acknowledgements

This work was funded by an EU Biotech Demonstration Programme (Contract No. BIO4-CT98–0223), by BioResearch Ireland and Bayer HealthCare, Germany. We would like to thank Wibke Lofink for excellent technical assistance and Udo Krella and Wolfgang Hain for technical insights and discussions. We declare that all experiments complied with the current laws of Germany in which the experiments were performed.

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Author notes

  1. Gabriele Scholz

    Present address: Nestlé Research Center Lausanne, Vers-chez-les-Blanc, 1000, Lausanne 26, Switzerland

Authors and Affiliations

  1. Bayer HealthCare, Pharma Research Centre, Aprather Weg, 42096, Wuppertal, Germany

    Annamarie Rogers, Gabriele Schmuck & Gabriele Scholz

  2. School of Biology, University of St Andrews, Fife, Scotland

    Roger Griffiths

  3. Bibra International Ltd, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, UK

    Clive Meredith

  4. Department of Pharmacology, Danish University of Pharmaceutical Sciences, 2 Universitetsparken, 2100, Copenhagen, Denmark

    Arne Schousboe

  5. Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery & Development, University of Siena, via Aldo Moro 2, 53100, Siena, Italy

    Giuseppe Campiani

  6. Department of Biochemistry, Trinity College, Dublin 2, Ireland

    D. Clive Williams

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  1. Annamarie Rogers
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Correspondence to Annamarie Rogers.

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Rogers, A., Schmuck, G., Scholz, G. et al. Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels. Arch Toxicol 79, 129–139 (2005). https://doi.org/10.1007/s00204-004-0617-5

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  • DOI: https://doi.org/10.1007/s00204-004-0617-5

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