Gonadal steroids regulate GABA A receptor subunit mRNA expression in NT2-N neurons

Abstract

Changes in gonadal steroid hormone levels during the menstrual cycle affect seizure frequency in women with catamenial epilepsy. Since GABA A receptors (GABARs) contribute to the prevention and termination of seizures by reducing neuronal excitability, we hypothesized that fluctuating gonadal steroid levels might affect GABAR subunit expression, which could alter inhibitory tone leading to increased seizure activity. To address this question in a simplified environment in vitro, we examined the effects of gonadal steroids on NT2-N neuronal cells. We have previously shown that NT2-N cells express functional GABARs, and that the expression pattern of GABAR subunits is regulated by chronic benzodiazepine exposure and hypoxia. NT2-N neurons were exposed to progesterone (0.1 μM), β-estradiol (3 nM), or vehicle (DMSO) for 2 days or 7 days prior to RNA harvesting. GABAR subunit mRNA levels were assessed by semiquantitative RT-PCR normalized to actin levels. Progesterone exposure for 7 days increased α2 and γ3 and decreased α5 subunit mRNAs, while β-estradiol caused significant increases in α3, β3 and ε expression. Further analysis revealed differential regulation of α4, α5, ε and π subunit expression. Plots of relative PCR density in progesterone-treated cells for α2 vs. α5, α5 vs. γ3 and α2 vs. γ3 showed correlation between samples, suggesting coordinate regulation. Both progesterone and estrogen nuclear receptor mRNAs were detected by RT-PCR, and 2 days but not 7 days estrogen exposure upregulated progesterone receptor mRNA. Gonadal steroid fluctuations regulate GABA A receptor subunit expression in NT2-N cells. Such changes, if observed in vivo, could affect seizure frequency.