GLUTAMATE NMDA RECEPTORS IN EPILEPTIC CORTEX

描述：(逐字摘自申请者摘要)本项目设计 鉴定和定量NMDA受体蛋白的分子机制 它们的亚基组合物是必需的和/或足够的 经生理(EEG)证实的癫痫人大脑皮层的超兴奋性 癫痫发作。最常见的耐药新皮质癫痫发生在人类 癫痫伴皮质发育不良。这种严重的癫痫发作障碍发生在 约占所有癫痫患者的20%，与最严重的 与其他重点或全面性的社会和教育方面的障碍相比 癫痫。这些皮质发育不良中的大多数可以通过手术切除并在 在某些情况下，癫痫发作减少或消除。然而，外科手术的成功不能 从切除皮质的常规组织病理学分析可以预测。 约50%的病例会持续发作，需要药物治疗。通过 相比之下，NMDA受体上复杂的免疫细胞化学现在已经揭示 NR2亚基的上调及其与NR1亚基的共表达 癫痫，但不是非癫痫皮质。这项提案旨在揭示 N-甲基-D-天冬氨酸受体通过检测 新切除的癫痫皮质(术前皮层记录 脑电发作的记录)。将在以下方面进行量化比较 每个患者的“癫痫”和“非癫痫”皮质(无脑电发作)。 这些平行研究将揭示NMDA受体组成的差异。 其功能包括：1)NMDA受体蛋白亚单位基因产物NR2A、B和 它们与NR1 A-H)剪接变异体的共组件；2)双标记NR1-NR2 单个发育不良神经元的免疫荧光；3)免疫共沉淀印迹 用于NR1-NR2抗体；4)Northern blotts(通过mRNA杂交测试 NR2A、B和NR1剪接变异体A-H；5)定量原位杂交 确认Northern印迹mRNAs；6)体外切片和分离的神经元 场电位、膜片钳记录和选择性电生理学 NMDA受体亚单位的药物阻断。这些多学科 蛋白质、分子和药物生理分析将提供新的 关于癫痫机制的信息，并可能提出新的方法 设计新药。这些药物应该选择性地作用于过度兴奋 具有独特的NR2和NR1异构体组合的发育不良神经元 在“非癫痫”皮质神经元上没有发现亚单位。特定的 受体靶向药物将避免全身神经系统抑郁和 应提供更有效的治疗癫痫的皮质发育不良。

DESCRIPTION: (Verbatim from the Applicant's Abstract) This project is designed to identify and quantify the molecular mechanisms of NMDA receptor proteins and their subunit coassemblies that are necessary and/or sufficient for hyperexcitability of physiologically-verified (EEG) epileptic human cortical seizures. The most frequent drug-resistant neocortical seizures occur in human epileptics with cortical dysplasia. This serious seizure disorder occurs in approximately 20% OF all epileptics and is associated with the most severe social and educational retardations compared to the other focal or generalized epilepsies. Most of these cortical dysplasias can be surgically removed and in some cases seizures are reduced or eliminated. Surgical success however cannot be predicted from routine histopathologic analysis of the resected cortex. Continued seizures requiring medication occur in about 50% of cases. By contrast, sophisticated immunocytochemistry on NMDA receptors has now revealed upregulation of the NR2 subunits, and their coexpression with NR1 subunits in epileptic but not non-epileptic cortex. This proposal is designed to uncover the mechanisms by which NMDA receptors generate hyperexcitability by examining freshly resected epileptic cortex (documented by preoperative cortical recordings of the EEG seizure onset). Quantitative comparisons will be made in each patient's "epileptic" and "non-epileptic" cortex (no EEG seizure onsets). These parallel studies will uncover differences in NMDA receptor composition and function in: 1) NMDA receptor protein subunit gene products NR2A, B and their coassemblies with NR1 A-H )splice variants); 2) double-labeled NR1-NR2 immunofluorescence on single dysplastic neurons; 3) coimmunoprecipitation blots for NR1-NR2 antibodies; 4) Northern blots (with mRNA hybridization tests for NR2A, B, and NR1 splice variants A-H; 5) quantitative in situ hybridization to confirm Northern blot mRNAs; and 6) in vitro slice and dissociated neuron electrophysiology with field potential, patch clamp recordings, and selective pharmacologic blockade of NMDA receptor subunits. These multidisciplinary protein, molecular, and pharmaco-physiologic analyses will provide new information about the mechanisms of epilepsy and may suggest novel approaches to designing new drugs. These drugs should selectively act on hyperexcitable dysplastic neurons that have unique heteromeric coassemblies of NR2 and NR1 subunits not found on "non-epileptic" cortical neurons. Specific receptor-targeted drugs would avoid general nervous system depression and should provide more effective management of epilepsy in cortical dysplasia.