Effects Of γ-aminobutyric Acid Receptor A Agonists On Hippocampal Neurons Epileptiform Discharge Model In Neonate Rat

Objective:To establish an ex-vivo epileptic model and test the effects of GABAa agonists(Clonazepam, Muscimol and Allopregnanolone) on hippocampal neurons epileptiform discharge. To assess the effect of GABA-mediated during early central nervous system(CNS) development.Methods: The hippocampal tissues of neonatal S-D rats(<24h)were taken out and the neurons were isolated for primary culture in cell incubator(37℃、5%CO2、95%O2、saturated humidity).On day 9,seizure like events(SLEs) were induced by culturing in lowering magnesium concentration.We chose active neurons with smooth surface and clear contours for electrophysiological recording. In current mode of EPC-10 amplifier, the discharge activities were recorded.The effects of three GABAa agonists were analyzed.Results:1)Neurons epileptiform discharge model: The hippocampal neurons of neonatal rats are plump in shape with intensive axon and dendrites after cultured for 9 days. After treated with lowering magnesium concentration for 3 hours, neurons excitability elevated obviously and appeared spontaneous “epileptiform discharge”(Discharge frequency 5-20 Hz,amplitude 45-60Mv). This “epileptiform discharge” continued to exist until the culture medium returnd to be normal.2) GABAa receptor agonist(1)Clonazepam: “epileptiform discharge” frequency and amplitude showed no significant change when neurons were treated with Clonazepam(P>O.05).(2)Muscimol:SLEs were significantly suppressed, frequency and amplitude of epileptiform discharge decreased significantly after treated with muscimol(p<0.001).(3)Allopregnanolone: SLEs were signficantly suppressed and even blocked. After treated with allopregnanolone,the frequency and amplitude of epileptiform discharge decreased significantly(p<0.001).Conclusions:(1)Spontaneous epileptiform discharge was induced by lowering magnesium concentration, so the ex-vivo epileptic model was established successfully.(2)GABAa receptor agonist did not aggravate epileptiform discharge of early CNS development, whereas, remains mediated inhibition effect.(3) Clonazepam could not obviously suppress newborn rat hippocampal neurons epileptiform discharge,this maybe associated with immature brain relevant receptorin adequate expression, and that the GABA as exhausted because of neurons persistent spontaneous discharge,and lowering magnesium concentration Extracellular fluid reduced GABAergic neurons conduction.(4)Muscimol and Allopregnanolone could signficantly suppress SLEs in neonate rat. It indicated that neurosteroids and other GABA analogues play a part in treatment of epilepsy in neonate.