Neurosteriod Regulation of Seizures

描述（由申请人提供）：癫痫的特征是反复的无端癫痫发作。生物节律和外部刺激可以部分地通过循环类固醇激素来调节癫痫发作频率。神经类固醇如别孕烯醇酮是在大脑中从循环类固醇合成的，具有强大的抗惊厥作用。Allopregnanolone通过作用于GABAA受体在中枢神经系统中发挥作用。生理浓度的allopregnanolone（10 - 30 nM）有效地增强从幼稚大鼠急性分离的海马齿状颗粒细胞中的全细胞GABA诱发电流。与此相反，在急性分离的癫痫大鼠齿状颗粒细胞中，这些生理浓度的别孕烯醇酮未能增强全细胞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。在幼稚大鼠的齿状颗粒细胞中，a1和g2亚基在突触处表达，而a4和d亚基在突触外表达。突触外受体介导紧张性抑制，而突触受体介导相位（突触）抑制。紧张性抑制对别孕烯醇酮、地西泮不敏感，对Zn ~（2+）敏感;突触性抑制对别孕烯醇酮、地西泮敏感，对Zn ~（2+）不敏感。这些初步研究结果支持了这样的假设，即在癫痫大鼠海马齿状颗粒细胞中，GABA能抑制的别孕烯醇酮调节减少。别孕烷醇酮调节作用的降低是由于含有α 4和δ亚基的受体表达增加所致，这些受体在癫痫大鼠中存在于突触外和突触下膜中。 我们将通过实现以下具体目标来测试我们的假设的预测：1）表征别孕烯醇酮、地西泮、锌和呋塞米对GABA介导的微型抑制性突触电流（mlPSC）和GABAA受体介导的癫痫和对照大鼠海马齿状颗粒细胞的背景紧张性抑制的调制。2)通过免疫组织化学和免疫印迹研究结合共聚焦激光显微镜，表征癫痫和对照大鼠齿状回颗粒细胞中特异性GABAA受体亚单位表达的量和部位（突触与突触外）。 重要性：这些实验是特别相关的理解癫痫引起的GABA-A受体的变化和它们的调制神经类固醇。

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.