Gamma-aminobutyric acid A receptor gamma 2 subunit following Mg-free-induced seizures in cultured developing neurons

BACKGROUND: Studies have demonstrated that in vitro cultured cortical neurons from embryonic rats can produce spontaneous recurrent epileptiform discharges following transient Mg2+-free extracellular solution culture. OBJECTIVE: To explore gamma-aminobutyric acid A receptor (GABA(A)R) gamma 2 subunit expression following Mg2+ free-induced seizures in cultured developing neurons. DESIGN, TIME AND SETTING: Cellular and molecular biology. The in vitro experiment was performed at the Department of Pediatrics, Second Xiangya Hospital of Central Southern University between January 2007 and February 2008. MATERIALS: Cortical neurons of Wistar rats on gestational days 16-17 were used. Normal extracellular solution (pH 7.3) consisted of NaCl 145 mmol/L, KCI 2.5 mmol/L, HEPES 10 mmol/L, MgCl2 1 mmol/L, CaCl2 2 mmol/L, glucose 10 mmol/L, and glycine 0.01 mmol/L. In addition, there was no MgCl2 in the Mg2+-free extracellular solution. METHODS: Cortical neurons cultured for 6 days were exposed to normal extracellular solution (control group) and Mg2+-free media (Mg2+-free group) respectively for 3 hours, followed by continuous culture in DMEM solution. MAIN OUTCOME MEASURES: On days 1, 7 and 12 after Mg2+-free treatment, real-time RT-PCR, immunochenustry, and flow cytometry were used to detect GABAAR 72 subunit expression. RESULTS: Compared with the control group, GABAAR gamma 2-positive cells decreased significantly on days 1 and 7 after Mg2+-free treatment (P < 0.01), but significantly increased on day 12 (P < 0.01). GABA(A)R gamma 2 subunit mRNA expression decreased significantly at 7 days Mg2+-free treatment when measured by real-time RT-PCR compared with the control group (P < 0.05). CONCLUSION: GABAAR Y 2 subunit expression is modified following Mg-free-induced seizures in cultured developing neurons. This indicates the possibility that abnormal GABAA receptor expression might play an important role in development of neuronal injury.