GABAergic excitation in human hypothalamic hamartoma

描述（由申请人提供）：本项目的长期目标是了解人类痉挛性癫痫的发病机制。人类下丘脑错构瘤是一种罕见的发育畸形，常以痉挛性发作为特征，药物治疗无效。根据HH的发作性EEG记录，假设痉挛性癫痫发作是由HH病变本身引起的。这一想法得到了证据的充分支持，证据表明，使用一种新的神经外科方法可以安全切除HH，从而显著改善癫痫控制。然而，在这种皮层下病变的癫痫发生机制尚不清楚。我们最近的特点，第一次，单HH神经元的电生理特性急性分离手术标本。本研究的目的是利用手术切除的HH组织来探索人类痉挛性癫痫发作的新机制。中心假设是HH神经元中表达的GABAA受体表现出兴奋性表型，这可能是人类痉挛发作的来源。该项目的基本原理是，HH神经元可能表现出不成熟的功能，由于或结合其异位位置和异常的细胞结构，以及其持续的癫痫活动。这种不成熟可能是GABA能兴奋的基础。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受体介导发挥兴奋作用。将通过定义功能性GABAA受体的显性表达与功能性离子型谷氨酸受体的有限表达，以及在急性分离的HH神经元中使用短杆菌肽穿孔贴片记录显示GABAA受体介导的电流的逆转电位的正向偏移和GABA诱导的去极化和兴奋来测试该想法。目的2是确定HH组织中Na+-K+-Cl-协同转运体（NKCC 1）和K+-Cl-协同转运体（KCC 2）的表达水平。工作假设是HH组织表达异常高比率的NKCC 1与KCC 2基因，导致细胞内Cl-水平升高和兴奋性而非抑制性GABA反应。将使用定量RT-PCR技术测量NKCC 1和KCC 2 mRNA水平。计划中的研究将建立和测试一个新的假设，提高对人类gelastic癫痫发作机制的理解。关于HH神经元的兴奋性GABAA受体功能的知识也将有助于选择用于药物开发的上级策略，以预防和控制人的痉挛性癫痫发作。

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.