Rapid, sensitive, and simple method for quantification of both neurotoxic and neurotrophic effects of NMDA on cultured cerebellar granule cells.
A simple and sensitive method adapted from the staining of living cells with fluorescein diacetate was developed to rapidly estimate the number of living cells remaining in a culture dish 24 hr after a few min of NMDA treatment of cerebellar neurons. This method consists of the measurement, after cell lysis, of the total amount of fluorescein produced from fluorescein diacetate by the living granule cells present in each culture dish. We show that this method can also be used to quantify the survival effect of chronic exposure of granule cells to either K+ or NMDA. In both cases, the fluorescence measured was found to be proportional to the number of fluorescein-labelled cells counted under a fluorescence microscope, indicating that the present method can be used to quantify both toxic and trophic effects of NMDA on cerebellar granule cells. This study confirms that these two NMDA effects occur at the same NMDA concentration, and both are inhibited by MK 801 in the same concentration range. We showed, moreover, that granule neurons developed in the presence of NMDA are much less sensitive to NMDA toxicity than neurons developed in K(+)-enriched medium.
Didier
M
M
SANOFI Recherche, Ligne de Neuropsychiatrie, Montpellier, France.
Heaulme
M
M
Soubrié
P
P
Bockaert
J
J
Pin
J P
JP
D000818:Animals; D001224:Aspartic Acid / Q000032:analysis; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D051379:Mice; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000494:pharmacology* / Q000633:toxicity; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology
D003470:Culture Media; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D011188:Potassium