Temporal and depolarization-induced changes in the absolute amounts of mRNAs encoding metabotropic glutamate receptors in cerebellar granule neurons in vitro.
Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 mM than in 25 mM KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 mM KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 mM KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.
Santi
M R
MR
Fidia-Georgetown Institute for the Neurosciences, Georgetown University, Washington, D.C.
Ikonomovic
S
S
Wroblewski
J T
JT
Grayson
D R
DR
D000818:Animals; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D017931:DNA Primers; D015870:Gene Expression / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017470:Receptors, Glutamate / Q000096:biosynthesis*; D013997:Time Factors
D017931:DNA Primers; D012333:RNA, Messenger; D017470:Receptors, Glutamate; D011189:Potassium Chloride