NMDAR1 mRNA expression and glutamate receptor stimulated increase in cytosolic calcium concentration in rat and mouse cerebellar granule cells.
We have previously reported that, unlike their rat counterparts, the survival of mouse cerebellar granule cells is independent of chronic stimulation whether owing to elevated K(+)-induced depolarization or NMDA (N-methyl-D-aspartate) receptor activation. One explanation could be that during the critical period mouse granule cells are very sensitive to NMDA receptor stimulation by endogenous glutamate released in the cultures. If so, this might be reflected by an increased expression of NMDA receptors or an increased response to their activation. We tested this hypothesis by measuring (a) the concentration of mRNA for the obligatory NMDA receptor subunit, NMDAR1, and (b) the glutamate/NMDA stimulated increase in cytosolic Ca(2+)-ion concentration in cultures at physiological or elevated K(+)-ion concentration. The expression of NMDAR1 mRNA was measured by competitive PCR of reversely transcribed mRNA and was normalized to that of the constitutively expressed H3.3 histone mRNA. The glutamate and NMDA stimulated increase in cytosolic Ca(2+)-ion concentration was measured using the fluorescent Ca(2+)-chelator Fluo3. In contrast to the hypothesis, we found NMDAR1 mRNA expression to be lower in mouse than in rat granule cells cultured for 4 days at physiological K(+)-ion concentration. However, the NMDA stimulated increase in cytosolic Ca(2+)-ion concentration did not differ in 4-day rat and mouse cultures. Although the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration in 2-day cultures was higher in mouse granule cells than in rat granule cells, the developmental profile of the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration was the same in both cases. In conclusion, we found no obvious evidence for increased NMDA receptor activity in mouse cerebellar granule cells cultured at physiological K(+)-ion concentration.
Mogensen
H S
HS
Department of Pharmacology, University of Copenhagen, Rigshospitalet, Denmark.
Jørgensen
O S
OS
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000502:physiology; D016014:Linear Models; D051379:Mice; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017470:Receptors, Glutamate / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D014966:Xanthenes
D000814:Aniline Compounds; D002118:Calcium; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D012333:RNA, Messenger; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; C059715:Fluo-3