Neurosteriod Regulation of Seizures

描述(申请人提供)：癫痫的特征是反复无端发作。生物节律和外部刺激可以部分通过循环类固醇激素来调节癫痫发作的频率。像别孕酮这样的神经类固醇是在大脑中由循环类固醇合成的，具有强大的抗惊厥作用。AIIopregnanolone通过作用于GABAA受体在中枢神经系统发挥作用。生理浓度的别孕酮(10-30 nM)可显著增强幼年大鼠海马齿状颗粒细胞的全细胞GABA诱发电流。相反，在从癫痫大鼠急性分离的齿状颗粒细胞中，这些生理浓度的别孕酮未能增强整个细胞的GABAA受体电流。整个细胞的GABA-A受体电流是由突触和突触外的GABAA受体激活产生的。幼年大鼠齿状回颗粒细胞中，A1和G2亚基在突触内表达，而A4和d亚基在突触外表达。突触外受体介导紧张性抑制，而突触受体介导时相(突触)抑制。紧张性抑制对别孕酮、安定不敏感，对锌离子敏感；突触抑制对别孕酮、安定敏感，对锌离子不敏感。这些初步发现支持癫痫大鼠海马齿状回颗粒细胞对GABA能抑制的别孕酮调节作用减弱的假说。癫痫大鼠突触外和突触下膜上α4和delta亚单位受体的表达增加，导致别孕酮调节减弱。我们将通过实现以下特定目标来验证我们的假设：1)表征别孕酮、安定、锌和速尿对GABA介导的微小抑制性突触电流(MlPSCs)以及GABAA受体介导的背景紧张性抑制癫痫和对照组大鼠海马齿状回颗粒细胞的调节作用。2)用免疫组织化学和免疫印迹结合激光共聚焦显微镜观察癫痫大鼠和正常大鼠齿状回颗粒细胞GABAA受体亚基表达的数量和部位。 意义：这些实验对于理解癫痫引起的GABA-A受体的改变以及神经类固醇对它们的调节特别相关。

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.