The Expressions Of GABA_AR Subunits Were Effected In Developing Cortical Neurons With Magnesium-free And Phenobarbital Treatment

Objective:To study the expressions of GABAARα1 subunit andγ2 subunit and the effect of ion currents of the cultured developing cortical neurons injured with Magnesium-free treatment in vitro.Methods:Rat embryo cortical neurons cultured in vitro on 6d were exposed transient to Mg2+-free media for 3h to induce injure, then they were returned to regular media containing normal level magnesium as INJ groups. Rat embryo cortical neurons were cultured in regular media containing normal level magnesium in vitro as control (CTL) groups. At different time points of 1d,7d,12d with Mg2+-free treatment, the expression of GABAARα1 subunit andγ2 subunit protein and mRNA were measured by real-time PCR, immunochemistry and flow cytometry methods; at the same time points, the change of K+and Ca2+on cultured cortical neurons were detected by whole-cell patch-clamp method.Results:Compared with CTL, MTT metabolic rates had no difference at 1d,7d,12d after Mg2+-free treatment in INJ. Compared with CTL, GABAAR ai subunit mRNA expression decreased significantly at Id after Mg2+-free treatment in INJ by real-time PCR (P<0.05); GABAARα1-positive cell ratio decreased significantly at 12d after Mg2+-free treatment in INJ by flow cytometry method (P<0.05); and the accumulated optical density (AOD) of GABAARα1 subunit protein immunoreactivity (IR) decreased significantly at 7d and 12d after Mg2+-free treatment in INJ by immunochemistry method (P<0.05). And compared with control, GABAARγ2 subunit mRNA expression increased significantly at 7d after Mg2+-free treatment by real-time PCR(P<0.05); GABAARγ2-positive cell ratio decreased significantly at 7d, whereas increased significantly at 12d after Mg2+-free treatment by flow cytometry method(P<0.05); the accumulated optical density (AOD) of GABAARγ2 subunit immunoreactivity(IR) decreased significangtly at 1d and 7d, but increased significantly at 12d after Mg2+-free treatment by immunochemistry method(P<0.05). Whole-cell patch-clamp recordings were applied in cultured rat cortical neurons. The inward currents of K+, Ca2+ were decreasing slowly in normal neurons. Compared with control, the currents of K+ and Ca2+ increased significantly at 7d and 12d after Mg2+-free treatment (P<0.05).Conclusion:The expressions of GABAARα1 subunit andγ2 subunit were modified following Mg2+-free-induced injure in developing neurons cultured in vitro. The change of GABAARγ2 subunit is earlier thanα1 subunit. The currents of K+ and Ca2+ on the developing neurons were effected with Mg2+-free-induced injure. Objective:To study the effect of the expressions of GABAARα1 subunit andγ2 subunit and membrane ion currents were induced by GABAAR agonist, phenobarbital, in developing normal cortical neurons and the cortical neurons injured with Magnesium-free treatment in vitro.Methods:Rat embryo cortical neurons cultured in vitro on 6d were exposed transient to Mg2+-free media for 3h to induce injure, then they were returned to regular media containing normal level magnesium as INJ groups. Or they were returned to normal media containing phenobarbital for 3d, then were returned to cultur in normal media as INJ+PB groups. Rat embryo cortical neurons were cultured in regular media containing normal level magnesium in vitro as negative (CTL) groups. Rat embryo cortical neurons cultured in vitro on 6d were exposed to regular media containing phenobarbital for 3d as positive (CTL+PB) groups. At different time points of 1d,7d,12d after Mg2+-free treatment, the expression of GABAARα1 subunit and 72 subunit protain and mRNA were measured by real-time PCR, immunochemistry and flow cytometry methods; at the same time points, the change of K+and Ca2+on cultured cortical neurons were deteced by whole-cell pate h-clamp method.Results:With phnobarbital treatment, compared with CTL and INJ, MTT metabolic rate decreased significantly in CTL+PB and INJ+PB (P<0.05); compared with CTL, MTT metabolic rate decreased significantly in CTL+PB and INJ+PB (P<0.05). Compared with CTL, the expression of GABAARα1 subunit mRNA decreased significantly at Id after Mg2+-free treatment in INJ+PB and CTL+PB by real-time PCR (P<0.05), and compared with CTL and INJ+PB, the expression of GABAARα1 subunit mRNA decreased significantly at 7d after Mg2+-free treatment in CTL+PB (P<0.05); Compared with CTL and INJ+PB, the expression of GABAAγ2 subunit mRNA increased significantly in CTL+PB at 7d,12d with Mg2+-free treatment by real-time PCR (P＜0.05). Compared INJ+PB with CTL+PB and CTL, GABAARα1-positive cells ratio decreased significantly at 12d after Mg2+-free treatment (P＜0.05), and the accumulated optical density (AOD) of GABAARα1 subunit immunoreactivity (IR) decreased significantly at 7d and 12d after Mg2+-free treatment by immunochemistry method (P<0.05); Compared INJ+PB with CTL+PB and CTL, GABAARγ2-positive cells ratio decreased significantly at 7d after Mg2+-free treatment, whereas increased significantly at 12d with Mg2+-free treatment by flow cytometry method(P<0.05). Compared INJ+PB with CTL+PB and CTL, the accumulated optical density (AOD) of GABAAγ2 subunit immunoreactivity (IR) decreased significangtly at 1d and 7d after Mg2+-free treatment, but increased significantly at 12d after Mg2+-free treatment by immunochemistry method (P<0.05).Whole-cell patch-clamp recordings were applied in cultured rat cortical neurons. Compared with CTL, the current of K+decreased significantly at 1d after Mg2+-free treatment in CTL+PB and INJ+PB (P＜0.05); compared with the other three groups, the current of K+ increased significantly at 7d and 12d after Mg2+-free treatment in INJ; compared with CTL, the current of K+decreased significantly at 12d after Mg2+-free treatment (P<0.05). As well as compared with CTL the current of Ca2+was higer significantly at 7d and 12d after Mg2+-free treatment in INJ and INJ+PB (P＜O.05).Conclusion:The normal expressions of GABAARα1 subunit andγ2 subunit and ion channel currents in developing cortical neurons in vitro were affected directly by GABA receptor agonist, phenobarbital. These abnormal changes induced by Mg2+-free injure were not recov-ered by phenobarbital. Objectiv:To study neurons livability and the change of ion currents on neuron membrane after the expression of GABAARα1 subunit was inhibited in vitro.Methods:Three pairs of small interfering RNA (siRNA) for GABAARα1 subunit were designed using OligoEngine RNAi software and transfected to rat embryo cortical neurons by using Trans Messenger reagent (Lipofectamne TM2000). One pair of irrelevant fluorescence labeling RNA sequence was transfected to rat embryo cortical neurons as the positive control. The cortical neurons were transfected after 24h in fresh DMEM+10%FBS culture media. The expression of GABAARα1 subunit was tested after 72h posttranfection by real-time PCR and cell immun-oreactivity (IR) methods. K+ and Ca2+ currents on neuron membranes and GABAA receptor-induced currents were detected by whole-cell patch-clamp recordings after the expression of GABAARα1 subunit was inhibited.Results:There was no difference for the expressions of GABAARα1 mRNA and GABAARα1 protain between negative control and positive control after 72h posttranfection by real-time PCR and cell immunoreactivity (IR) (p>0.05). But there was significant difference for the expressions of GABAARα1 mRNA and GABAARα1 protain in RNAi group, compared with negative group. When the expression of GABAARα1 subunit in the neurons was inhibited significantly by the RNA interference (RNAi), the livability of neurons was no significant difference in three groups (p>0.05) after 72h posttranfection by trypan blue staining (x2=5.444; p>0.05). After 72h posttransfection by Trans messeger reagent, the membrane currents of K+and Ca2+decreased significantly in posttransfected neurons(p<0.001), compared with negtive control and positive control.Conclusion:Neurons livability were not effected signifantly after GABAARα1 subunit was inhibited. The currents of K+and Ca2+on neuron membranes decreased inderectly following GABAARα1 subunit was inhibited.