Neurosteriod Regulation of Seizures

描述（由申请人提供）：癫痫的特点是反复发作。生物节律和外部刺激可通过循环类固醇激素部分调节癫痫发作频率。像异孕酮这样的神经类固醇是由循环中的类固醇在大脑中合成的，具有强大的抗惊厥作用。aiopregnanolone通过作用于GABAA受体在中枢神经系统发挥作用。异孕酮生理浓度（10 ~ 30 nM）可增强急性分离大鼠海马齿状颗粒细胞全细胞gaba诱发电流。相反，在癫痫大鼠急性分离的齿状颗粒细胞中，这些生理浓度的异孕酮不能增强全细胞GABAA受体电流。全细胞GABA-A受体电流是通过激活突触和突触外GABAA受体产生的。在幼稚大鼠齿状颗粒细胞中，a1和g2亚基在突触上表达，a4和d亚基在突触外表达。突触外受体介导强直性抑制，突触受体介导阶段性（突触）抑制。强直性抑制对异孕酮、地西泮不敏感，对Zn2+敏感；突触抑制对异孕酮、地西泮敏感，对Zn2+不敏感。这些初步发现支持了癫痫大鼠海马齿状颗粒细胞中异孕酮对gaba能抑制的调节减弱的假设。异孕酮调节的减少是由于α 4和δ亚单位受体的表达增加，它们在癫痫大鼠中存在于突触外和突触下膜。我们将通过实现以下具体目标来验证我们假设的预测：1)表征异孕酮、地西泮、锌和速尿对GABA介导的微型抑制性突触电流（mlPSCs）的调节，以及GABAA受体介导的海马齿状颗粒细胞的背景强直抑制。2)应用免疫组织化学和免疫印迹联合激光共聚焦显微镜观察癫痫大鼠和对照组齿状颗粒细胞特异性GABAA受体亚基表达的数量和部位（突触与突触外）。

DESCRIPTION (provided by applicant): Epilepsy is characterized by recurrent unprovoked seizures. Biological rhythms and external stimuli can modulate seizure frequency partly through circulating steroid hormones. Neurosteroids like allopregnanolone are synthesized in the brain from circulating steroids and have powerful anticonvulsant effects. AIIopregnanolone exerts its actions in the central nervous system by acting on GABAA receptors. Physiological concentrations of allopregnanolone (10 -30 nM) potently enhanced whole cell GABA-evoked currents in hippocampal dentate granule cells acutely isolated from naive rats. In contrast, in dentate granule cells acutely isolated from epileptic rats these physiological concentrations of allopregnanolone failed to enhance whole cell GABAA receptor currents. Whole cell GABA-A receptor currents are generated by activation synaptic and extrasynaptic GABAA receptors. In dentate granule cells of naive rats, a1 and g2 subunits are expressed at synapses while a4 and d subunits are extrasynaptic. The extrasynaptic receptors mediate tonic inhibition while synaptic receptors mediate phasic (synaptic) inhibition. The tonic inhibition is insensitive to allopregnanolone, diazepam, and sensitive to Zn2+ while synaptic inhibition is sensitive to allopregnanolone, diazepam, and insensitive to Zn2+. These preliminary findings support the hypothesis that in the hippocampal dentate granule cells of epileptic rats, there is diminished allopregnanolone modulation of GABAergic inhibition. Decreased allopregnanolone modulation is due to increased expression of alpha4 and delta subunit-containing receptors, which in epileptic rats are present extrasynaptically and in subsynaptic membrane. We will test the predictions of our hypotheses by accomplishing the following specific aims: 1) To characterize allopregnanolone, diazepam, zinc, and furosemide modulation of GABA mediated miniature inhibitory synaptic currents (mlPSCs), and of GABAA receptor mediated background tonic inhibition of hippocampal dentate granule cells of epileptic and control rats. 2) To characterize the amount and site (synaptic versus extrasynaptic) of expression of specific GABAA receptor subunits in dentate granule cells in epileptic and control rats by immunohistochemistry and immunoblot studies combined with confocal laser microscopy. Significance: These experiments are of particular relevance to understanding epilepsy-induced alterations in GABA-A receptors and their modulation by neurosteroids.