Actions and interactions of extracellular potassium and kainate on expression of 13 gamma-aminobutyric acid type A receptor subunits in cultured mouse cerebellar granule neurons.
Cerebellar granule neurons in culture are a popular model for studying neuronal signaling and development. Depolarizing concentrations of K(+) are routinely used to enhance cell survival, and kainate is sometimes added to eliminate GABAergic neurons. We have investigated the effect of these measures on expression of mRNA for gamma-aminobutyric acid type A (GABA(A)) receptor alpha1-6, beta1-3, gamma1-3, and delta subunits in cultures of mouse cerebellar granule neurons grown for 7 or 12 days in vitro (DIV) using semiquantitative reverse transcription-PCR. We detected mRNA for the alpha1, alpha2, alpha5, alpha6, beta2, beta3, gamma2, and delta subunits in all the cell cultures, but the expression levels of the alpha5-, alpha6-, and beta2-subunit mRNAs were significantly dependent on the composition of the culture medium. Both an increase of the extracellular K(+) concentration from 5 to 25 mm and the addition of 50 microm kainate immediately depolarized the neurons but prolonged exposure (7-8 DIV)-induced compensatory hyperpolarization. 25 mm K(+) caused a shift from alpha6 to alpha5 expression measured at 7 and 12 DIV, which was mimicked by kainate in 12 DIV cultures. The expression of beta2 was decreased by 25 mm K(+) in 7 DIV cultures and by kainate in 12 DIV cultures. The effects on beta2 expression could not be ascribed to depolarization. Alterations of alpha6 mRNA expression were reflected in altered sensitivity to GABA and furosemide of the resulting receptors. Our study has shown that a depolarizing K(+) concentration as well as kainate in the culture medium significantly disturbs maturation of GABA(A) receptor subunit expression.
Engblom
A Christine
AC
Department of Pharmacology, Royal Danish School of Pharmacy, Copenhagen 2100, Denmark.
Johansen
Flemming F
FF
Kristiansen
Uffe
U
D000818:Animals; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000502:physiology*; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism / Q000494:pharmacology*; D005110:Extracellular Space / Q000502:physiology; D018757:GABA Modulators / Q000494:pharmacology; D007608:Kainic Acid / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D009474:Neurons / Q000502:physiology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects
D018690:Excitatory Amino Acid Agonists; D018757:GABA Modulators; D011963:Receptors, GABA-A; D011188:Potassium; D007608:Kainic Acid