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Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels
- Source :
- Archives of Toxicology. 79:129-139
- Publication Year :
- 2004
- Publisher :
- Springer Science and Business Media LLC, 2004.
-
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.
- Subjects :
- Cell Survival
Excitatory Amino Acids
Health, Toxicology and Mutagenesis
Excitotoxicity
Mice, Inbred Strains
Kainate receptor
AMPA receptor
Biology
Toxicology
medicine.disease_cause
Receptors, N-Methyl-D-Aspartate
c-Fos
Mice
Cerebellum
medicine
Animals
Excitatory Amino Acid Agents
RNA, Messenger
Receptor
6-Cyano-7-nitroquinoxaline-2,3-dione
Reverse Transcriptase Polymerase Chain Reaction
Glutamate receptor
General Medicine
Molecular biology
Reverse transcription polymerase chain reaction
Gene Expression Regulation
NIH 3T3 Cells
biology.protein
NMDA receptor
Biological Assay
Excitatory Amino Acid Antagonists
Proto-Oncogene Proteins c-fos
Subjects
Details
- ISSN :
- 14320738 and 03405761
- Volume :
- 79
- Database :
- OpenAIRE
- Journal :
- Archives of Toxicology
- Accession number :
- edsair.doi.dedup.....4717cb35e348c3d7c5e507c7630c5e25