PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

OBJECTIVE

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABAA receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg2+).

METHODS

A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg2+ solutions to generate a low Mg2+ model of epilepsy. The low Mg2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABAA receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABAA receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method.

RESULTS

After the neurons were exposed to a low-Mg2+ solution for 3 h, GABAA receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABAA receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABAA subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA.

CONCLUSIONS

The PKC/CREB pathway may negatively regulate the expressions of GABAA receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg2+ model of epilepsy.