Early Life Seizures and Development of Gabaergic Inhibition in the Human Brain

描述（由申请人提供）：婴儿和新生儿癫痫发作是常见的，通常对目前使用的治疗有抗药性，并可能产生毁灭性的后果。这一年龄组最常用的抗惊厥药物是巴比妥类药物和苯二氮卓类药物，它们通过增强神经元GABAA受体的电流起作用。当前研究的重点是描述从妊娠晚期到儿童早期人类皮质gaba能抑制的发展，并进一步评估这种发展是如何被引起该年龄组癫痫发作的条件所破坏的。这一提议的基本假设是，GABAA受体亚基和阳离子-氯共转运体在人类皮质中的不成熟表达模式导致的药理学特性预测了在早产儿、新生儿和幼儿中使用GABAA能药物的低疗效甚至有害后果。本课题的具体目的包括：1)明确人类皮质GABAA受体亚基和阳离子-氯共转运体表达的发育成熟时间，评估皮质发育畸形导致的早期癫痫发作相关的表达异常。这一目标将通过使用定量Western blot分析和红外荧光检测来实现。冷冻的死后和手术后的皮质标本将从妊娠20周至3岁的对照婴儿，以及因皮质发育的胶质神经元畸形或迁移障碍而癫痫发作的年龄相近的婴儿中收集。此外，将建立一个临床数据库，并与我们的实验数据相关联，以评估在实验性癫痫模型中显示的调节受体或转运蛋白表达的变量是否也可能在我们的人类癫痫人群中发挥重要作用。2)确定人类皮质GABAA受体亚基和阳离子-氯共转运蛋白表达的发育和癫痫相关变化的功能和药理学后果。将利用一种创新的实验范式，允许对冷冻人脑组织中的GABAA受体进行电生理分析，从而将天然人类受体“微移植”到非洲爪蟾卵母细胞质膜中。3)分析与早期癫痫发作相关的人类皮层GABAA受体亚基和氯离子转运体的细胞分布变化。该职业发展奖的资金将允许PI获得荧光免疫组织化学和原位杂交技术的专业知识，以确定在正常人类发育和由于皮质畸形引起的癫痫发作中表现出gaba能特性变化的皮质细胞群。从这项研究中获得的信息可以直接应用于临床，从而改善婴儿和新生儿的医疗和神经系统护理。

DESCRIPTION (provided by applicant): Seizures in infants and neonates are common, are often resistant to currently used treatments, and can have devastating outcomes. The most frequently used anticonvulsant medications in this age group, the barbiturates and the benzodiazepines, act by enhancement of current through neuronal GABAA receptors. The focus of the current study is to characterize the development of human cortical GABAergic inhibition from late gestation through early childhood, and further to assess how this development is disrupted by conditions causing seizures in this age group. The underlying hypothesis of this proposal is that immature patterns of GABAA receptor subunit and cation-chloride cotransporter expression in human cortex result in pharmacologic properties predictive of low efficacy or even harmful consequences of the use of GABAergic agents in the treatment of premature infants, neonates, and young children. The Specific Aims of this proposal include: 1) Define the time course of the developmental maturation of human cortical GABAA receptor subunit and cation-chloride cotransporter expression, and assess abnormalities in expression related to early-life seizures due to malformations of cortical development. This aim will be achieved through the use of quantitative Western blot analysis with infrared fluorescence detection. Frozen postmortem and surgical cortical specimens will be collected from control infants at 20 weeks gestation through 3 years of age, as well as similarly-aged infants with seizures due to glioneuronal malformations of cortical development or migrational disorders. In addition, a clinical database will be constructed and correlated with our experimental data to assess whether variables that have been shown to modulate receptor or transporter expression in experimental epilepsy models might also play a significant role in our human epilepsy population. 2) Determine the functional and pharmacological consequences of developmental and seizure-associated changes in GABAA receptor subunit and cation-chloride cotransporter expression in human cortex. An innovative experimental paradigm will be utilized which allows electrophysiological analysis of GABAA receptors from frozen human brain tissue resulting in "microtransplantation" of native human receptors into the Xenopus oocyte plasma membrane. 3) Analyze alterations in the cellular distribution of GABAA receptor subunits and chloride transporters in human cortex related to early-life seizures. Funding from this Career Development Award will permit the PI to acquire expertise in the techniques of fluorescence immunohistochemistry and in situ hybridization to determine the cortical cell populations exhibiting changes in GABAergic properties during both normal human development and its disruption by seizures due to cortical malformations. Information gained as a result of this study could have direct clinical applications that produce improved medical and neurologic care of infants and neonates.