Early-Life Seizures and Development of GABAergic Inhibition in the Human Brain

描述(由申请人提供)：婴儿和新生儿的癫痫发作很常见，通常对当前使用的治疗方法具有抵抗力，并可能产生毁灭性的后果。这个年龄段最常用的抗惊厥药物是巴比妥类和苯二氮卓类，它们通过增强神经元GABAA受体的电流发挥作用。本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，并进一步评估这一发展是如何被导致这一年龄组癫痫发作的条件所干扰的。这一建议的基本假设是，GABAA受体亚单位和阳离子-氯离子共转运体在人脑皮质中表达的不成熟模式导致了预测使用GABA能药物治疗早产儿、新生儿和幼儿的低效甚至有害后果的药理学特性。这一建议的具体目的包括：1)确定人皮质GABAA受体亚单位和阳离子-氯共转运体表达的发育成熟的时间进程，并评估与皮质发育畸形引起的早期癫痫相关的表达异常。这一目标将通过使用带有红外荧光检测的定量Western印迹分析来实现。冰冻的死后和手术皮质标本将从妊娠20周到3岁的对照组婴儿以及因皮质发育的胶质神经畸形或迁移障碍而癫痫发作的相似年龄段的婴儿中收集。此外，还将建立一个临床数据库，并将其与我们的实验数据相关联，以评估在实验性癫痫模型中被证明调节受体或转运体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)确定发育和癫痫相关的人类皮质GABAA受体亚单位和阳离子氯化物共转运体表达变化的功能和药理学后果。将利用一种创新的实验范式，对冷冻的人脑组织中的GABAA受体进行电生理学分析，从而将本地人受体“微移植”到非洲爪哇卵母细胞质膜上。3)分析GABAA受体亚单位和氯离子转运体在人类大脑皮质中与早期癫痫发作相关的细胞分布变化。这项职业发展奖的资助将允许PI获得荧光免疫组织化学和原位杂交技术方面的专业知识，以确定在人类正常发育期间和因皮质畸形癫痫发作而表现出GABA能特性变化的皮质细胞群。这项研究获得的信息可能会直接应用于临床，从而改善对婴儿和新生儿的医疗和神经护理。 与公共卫生相关：目前使用的针对神经元GABAA受体的新生儿癫痫治疗方法往往无效，根据动物模型的数据，实际上可能是有害的。这项建议的目标是描述从妊娠晚期到儿童早期人类皮质GABA能抑制的发展特点，并进一步评估这一发展是如何被导致这一年龄组癫痫发作的条件所干扰的。这项研究的结果可能会鼓励重新考虑使用GABA能药物来治疗婴儿和新生儿的癫痫发作或镇静，并提出更好的治疗方案。

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. PUBLIC HEALTH RELEVANCE: Currently used treatments for neonatal seizures that target neuronal GABAA receptors are often ineffective and, based on data from animal models, may actually be detrimental. The goal of this proposal 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. Results from this study may encourage the reconsideration of the use of GABAergic medications in the treatment of seizures or sedation in infants and neonates, and also suggest better therapeutic alternatives.