Actions and interactions of extracellular potassium and kainate on expression of 13 γ-aminobutyric acid type A receptor subunits in cultured mouse cerebellar granule neurons

Abstract

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 γ-aminobutyric acid type A (GABAA) receptor α1-6, β1-3, γ1-3, and δ subunits in cultures of mouse cerebellar granule neurons grown for 7 or 12 days in vitro (DIV) using semi-quantitative reverse transcription-PCR. We detected mRNA for the α1, α2, α5, α6, β2, β3, γ2 and δ subunits in all the cell cultures, but the expression levels of the α5-, α6-, and β2-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 μM kainate immediately depolarized the neurons but prolonged exposure (7-8 DIV)-induced compensatory hyperpolarization. 25 mM K+ caused a shift from α6 to α5 expression measured at 7 and 12 DIV, which was mimicked by kainate in 12 DIV cultures. The expression of β2 was decreased by 25 mM K+ in 7 DIV cultures and by kainate in 12 DIV cultures. The effects on β2 expression could not be ascribed to depolarization. Alterations of α6 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 GABAA receptor subunit expression.