GABAergic excitation in human hypothalamic hamartoma

项目描述（由申请人提供）：该项目的长期目标是了解人类弹性癫痫的发病机制。人类下丘脑错构瘤（HH）是一种罕见的发育畸形，通常以弹性癫痫为特征，药物治疗难以治愈。根据HH的脑电图记录，弹性癫痫发作是由HH损伤本身引起的。这一观点得到了新的神经外科方法对癫痫发作控制显著改善的证据的支持，该方法允许安全切除HH。然而，这种皮层下病变的癫痫发生机制尚不清楚。我们最近首次描述了从手术标本中分离的单个HH神经元的电生理特性。本研究的目的是利用手术切除的HH组织探索人类弹性癫痫发作的新机制。核心假设是，在HH神经元中表达的GABAA受体表现出兴奋性表型，这可能是人类弹性癫痫发作的一个来源。该项目的基本原理是，HH神经元可能由于其异位和异常的细胞结构以及持续的癫痫活动而表现出不成熟的特征或与其相结合。这种不成熟可能是gaba能激发的基础。中心假设将通过追求两个具体目标来检验。目的1是表征GABAA受体介导的兴奋。工作假设是GABA通过功能性GABAA受体发挥兴奋作用。这一观点将通过定义功能性GABAA受体的显性表达和功能性嗜离子性谷氨酸受体的有限表达，以及在急性解离的HH神经元中显示GABAA受体介导电流的逆转电位的正转移和使用革兰西丁穿孔补丁记录的gaba诱导的去极化和兴奋来验证。目的2是确定HH组织中Na+-K+- cl -共转运蛋白（NKCC1）和K+- cl -共转运蛋白（KCC2）的表达水平。工作假设是HH组织表达异常高比例的NKCC1和KCC2基因，导致细胞内CI水平升高和兴奋性而非抑制性GABA反应。定量RT-PCR技术将用于测量NKCC1和KCC2 mRNA水平。计划中的研究将建立和测试一种新的假设，以增强对人类弹性癫痫发作机制的理解。了解HH神经元的兴奋性GABAA受体功能也将有助于选择更好的药物开发策略，以预防和控制人类弹性癫痫发作。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the etiopathogenesis of human gelastic epilepsy. Human hypothalamic hamartoma (HH) is a rare developmental malformation often characterized by gelastic seizures, which are refractory to medical therapy. Based on ictal EEG recordings from HH, the gelastic seizures are postulated to arise from the HH lesion itself. This idea is well supported by evidence of dramatic improvements in seizure control using a novel neurosurgical approach allowing for safe resection of HH. However, the mechanisms of epileptogenesis operative in this subcortical lesion are unknown. We have recently characterized, for the first time, the electrophysiological properties of single HH neurons acutely dissociated from surgical specimens. The objective of the proposed study is to explore a novel mechanism of epileptogenesis of human gelastic seizures using surgically resected HH tissues. The central hypothesis is that GABAA receptors expressed in HH neurons exhibit an excitatory phenotype, which may serve as a source of human gelastic seizures. The rationale for the project is that HH neurons may exhibit immature features due to or combined with their ectopic location and abnormal cytoarchitecture, as well as their persistent epileptic activity. This immaturity may underlie GABAergic excitation. The central hypothesis will be tested by pursuing two specific aims. Aim 1 is to characterize GABAA receptor-mediated excitation. The working hypothesis is that GABA exerts an excitatory role mediated through functional GABAA receptors. This idea will be tested by defining a dominant expression of functional GABAA receptors with a limited expression of functional ionotropic glutamate receptors, as well as showing a positive shift of the reversal potential of the GABAA receptor-mediated currents and a GABA-induced depolarization and excitation using gramicidin-perforated patch recordings in acutely dissociated HH neurons. Aim 2 is to determine levels of expression of the Na+-K+-Cl- cotransporter (NKCC1) and the K+-Cl- cotransporter (KCC2) in HH tissues. The working hypothesis is that HH tissues express an abnormally high ratio of NKCC1 to KCC2 genes, contributing to elevated intracellular CI- levels and excitatory, rather than inhibitory, GABA responses. Quantitative RT-PCR techniques will be used to measure NKCC1 and KCC2 mRNA levels. The planned studies will establish and test a novel hypothesis that enhances understanding of mechanisms involved in human gelastic seizures. Knowledge about excitatory GABAA receptor function of HH neurons will also aid selection of superior strategies for pharmaceutical development to prevent and control human gelastic seizures.