Biochemical and Biophysical Research Communications
Glutamate receptor-mediated regulation of c-fos expression in cultured microglia
Section snippets
Materials and methods
Microglial cultures. Primary microglial cells were prepared from cerebral cortices of 1-day-old Sprague–Dawley rats as described previously [21], [22]. Briefly, the cortices were triturated into single cells with fire-polished Pasteur pipettes. The cells were plated into 75 cm2-T-flasks (Corning) and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Life Technologies) containing 10% fetal bovine serum (FBS, Life Technologies) for 2 weeks at 37 °C in a humidified atmosphere of 5% CO2.
Glutamate-stimulated IEGs induction in microglia
Microglial cells were treated with glutamate (500 μM) for the indicated time (15, 30, and 120 min) and immediately prepared for RT-PCR analyses. We examined here gene expression of c-fos and c-jun, inducible nitric oxide synthase (iNOS), and TNF-α. Their basal mRNA expressions were barely detectable. However, following glutamate treatment, their expression levels were dramatically increased, peaked at 30 min-time point (for c-fos, 23.1 ± 5.56-fold, p < 0.05 shown in Fig. 1B), and decreased to basal
Discussion
We demonstrated here that all the subtypes of glutamate receptor agonists examined (NMDA, KA, AMPA, and DHPG) dramatically increased both c-fos mRNA and protein levels in cultured microglial cells (Fig. 2). These effects were significantly suppressed by the specific glutamate receptor antagonists MK-801, CNQX, and AIDA (Fig. 3) and also by calcium chelating agents EGTA and BAPTA-AM (Fig. 5). To our knowledge, our findings are the first to demonstrate glutamate-stimulated c-fos induction and
Acknowledgments
This work was supported by intramural Grants (347-6111-211-000-207, to S.-Y. Eun) from the Korean National Institute of Health and by Biomedical Brain Research Center Grant (0405-NS010704-0001, to S.-Y. Eun) from the Korean Ministry of Health and Welfare. We thank S.H. Go for technical support of confocal microscopy and S. Hur for preparation of manuscript.
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