PATHOPHYSIOLOGY OF DEVELOPING DYSPLASTIC HUMAN CORTEX

根据神经成像(MRI和PET)和手术切除的组织检查，皮质发育不良(CD)已被公认为癫痫的主要病理基础。加州大学洛杉矶分校的儿科癫痫手术计划治疗了许多患有难治性癫痫的儿童，根据我们的经验，一半的病例患有CD，其余的病例有中风和脑炎(非CD)等病因。关于发育不良皮质细胞的电生理特性、使Cd区和非Cd区致痫的潜在机制(S)，以及出生后正常的皮质发育如何影响致痫新皮质，人们知之甚少。本研究项目旨在通过对这些儿童的手术组织进行协调的形态和电生理学研究来解决这些基本的病理生理学问题。我们的工作假设是，由于兴奋性和抑制性过程之间的失衡，异常的新皮质是致痫的，皮层轴突回路的异常组织是病理过程的结果。拟议的研究将有两个目标：1)检验CD和非CD组织中兴奋和抑制过程发生变化的假设；2)评估人类出生后发育过程中新皮质神经元兴奋和抑制过程的发展。每个特定的目的都将利用类似的实验设计，结合术前临床和术中ECoG来确定哪些区域需要进行细胞内电生理学和形态评估。利用最先进的形态学和电生理学技术，实验将比较CD和非CD新皮质中出现“异常”神经元的情况，以确定：1)N-甲基-D-天冬氨酸(NMDA)和非NMDAα-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)和海人酸(KA)]离子亲性谷氨酸受体是否存在差异和/或变化；2)抑制性神经元数量和/或抑制性GABAA受体增加是否存在差异；以及3)组织中癫痫发作的可能原因之一是否来自异常轴突电路。这些目标将通过检测肉眼可识别的发育异常和外观正常的细胞来实现：1)激活NMDA、AMPA、KA和GABAA受体引起的电生理膜电流的变化；2)使用原位杂交和免疫组织化学技术检测表达NMDA、非NMDA和GABAA亚单位的神经元的数量和位置；以及3)通过向记录的发育不良神经元和外观正常的细胞填充生物细胞素或荧光黄来鉴定树突和轴突连接。这些发现将为理解发育不良的新皮质的病理生理学提供必要的重要基础信息，为儿童难治性癫痫的发病机制提供建议，并为控制CD和非CD引起的儿童癫痫的可能方法提供见解。

Based on neuroimaging (MRI and PET) and examination of surgically-resected tissue, cortical dysplasia (CD) has become recognized as a major pathological substrate in epilepsy. UCLA's Pediatric Epilepsy Surgery Program treats populations of children with intractable seizures, and in our experience one-half of the cases have CD while the remaining have etiologies such as strokes and encephalitis (non-CD). Very little is known about the electrophysiological properties of cells in dysplastic cortex, the underlying mechanism(s) that make CD and non-CD areas epileptogenic, and how normal postnatal cortical development affects epileptogenic neocortex. This research project is designed to address these fundamental pathophysiologic questions by performing coordinated morphologic and electrophysiologic studies on surgical tissue from these children. Our working hypothesis is that abnormal neocortex is epileptogenic because of an imbalance between excitatory and inhibitory processes and that cortical axon circuits are abnormally organized as a consequence of the pathologic process. The proposed studies will have two goals: 1) To examine the hypothesis that excitatory and inhibitory processes are altered in CD and non-CD tissue; and 2) to assess development of excitatory and inhibitory processes in neocortical neurons during human postnatal development. Each specific aim will utilize a similar experimental design incorporating pre-surgical clinical and intraoperative ECoG to determine which regions are to be studied for intracellular electrophysiology and morphologic assessments. Using state-of-the-art morphologic and electrophysiologic techniques, experiments will compare "abnormal"-appearing neurons in CD and non-CD neocortex to determine: 1) If there are differences and/or alterations in N-methyl-D-aspartate (NMDA) and non-NMDA alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA)] ionotropic glutamate receptors; 2) if there are differences in the number of inhibitory neurons and/or an increase in inhibitory GABAA receptors; and 3) if one possible etiology of seizures in the tissue is from abnormal axon circuitry. These goals will be accomplished by examining in visually-identified dysplastic and normal-appearing cells: 1) The alterations in electrophysiologic membrane currents induced by activation of NMDA, AMPA, KA, and GABAA receptors; 2) the number and location of neurons expressing NMDA, non-NMDA, and GABAA subunits using in situ hybridization and immunohistochemical techniques; and 3) the dendritic and axonal connections as identified by filling recorded dysplastic neurons and normal-appearing cells with biocytin or Lucifer Yellow. The findings will provide important fundamental information necessary for the understanding of the pathophysiology of dysplastic neocortex, suggest pathologic mechanisms of intractable childhood epilepsy, and provide insights into possible ways of controlling childhood seizures resulting from CD and non-CD.