Origin of Extracellular Glutamate in Human Temporal Lobe Epilepsy

人颞叶癫痫细胞外谷氨酸的起源

• 批准号: 7282011
• 负责人: DENNIS D SPENCER
• 金额: $ 50.44万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282011
• 关键词: 4-Aminopyridine; Acetates; Address; Amino Acids; Anions; Appearance; Area; Bathing; Biochemical; Brain; Cell Count; Characteristics; Condition; Convulsants; Count; Data; Depth; Drug Metabolic Detoxication; Electric Stimulation; Electrodes; Electrophysiology (science); Elevation; Enzymes; Epilepsy; Epileptogenesis; Excision; Focal Seizure; Functional disorder; Glial Fibrillary Acidic Protein; Glutamate Metabolism Pathway; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Goals; High Pressure Liquid Chromatography; Hippocampus (Brain); Homeostasis; Human; In Vitro; Incubated; Infusion procedures; Intractable Epilepsy; Invasive; Label; Liquid Chromatography; Mass Spectrum Analysis; Measurement; Measures; Medial; Metabolic; Metabolism; Microdialysis; Molecular Target; Monitor; Neocortex; Neuroglia; Neurons; Operative Surgical Procedures; Patients; Physiological; Preparation; Pyramidal Cells; Rate; Recruitment Activity; Research Personnel; Resected; Role; Sampling; Sclerosis; Seizures; Simulate; Site; Slice; Source; Techniques; Temporal Lobe; Temporal Lobe Epilepsy; Testing; Tissues; Western Blotting; Work; extracellular; granule cell; human tissue; in vivo; neuronal excitability; novel; programs; protein expression; tandem mass spectrometry; uptake

DESCRIPTION (provided by applicant): Extracellular glutamate is known to be excessively elevated both ictally and interictally in the epileptic human hippocampus. However, the source and significance to epileptogenesis of these high levels of glutamate remain a matter of debate. The overall goal of this proposal is to better understand the cellular and metabolic mechanisms that underlie the elevations of glutamate seen in the hippocampus of patients with mesial temporal sclerosis (MTS). There is compelling evidence for both neuronal and glial dysfunction n MTS and much of the work to date has focused on changes in either neuronal or glial function in isolated preparations. We postulate that the "neuronal-glial unit" is impaired in MTS such that the dysfunctional glia Droduce the bulk of the elevated interictal glutamate. Moreover, while both neuronal and glial release may contribute to ictal elevation, we hypothesize that the glial component is very substantial. We propose to test this hypothesis using a wide array of techniques aimed at understanding the disposition of glutamate in the epileptogenic human temporal lobe. We will couple these studies to the intracranial monitoring used to dentify epileptogenic regions in patients with medically intractable epilepsy. In Specific Aim 1, we will use HPLC to measure extracellular glutamate and glutamine in microdialysate both interictally and following stimulation using microdialysis probes attached to the depth electrodes. Stimulation will be done to simulate focal seizure activity. In Specific Aim 2, we will infuse 13C acetate, which is exclusively taken up by glia, and will use mass spectrometry to measure the fractional enrichment of 13C in glutamate and glutamine in the microdialysate at baseline and following stimulation to determine the source(s) of extracellular glutamate under these different conditions. For both these studies, we will perform the analyses in probes placed in the hippocampus and in a non-epileptogenic cortical region. Glutamate uptake and efflux will be measured in Specific Aim 3 using resected cortical and hippocampal tissue. In addition physiological studies on the effects of elevated glial glutamate will be done as part of this aim. Possible mechanisms for glial release will be addressed as well. Finally, in Specific Aim 4, the levels of the enzymes and transporters most relevant to glutamate metabolism in glia and neurons will be measured by Western blots using resected tissue. The data obtained in each Specific Aim will be correlated with quantitative neuronal and glial counts. Relevance: Taken together these data will allow us to develop a better understanding of the origins of the elevated glutamate in MTS, and thus to devise more targeted molecular and cellular therapies to restore glutamate homeostasis and thus decrease excitability and seizures.

描述（由申请人提供）：已知在癫痫人海马中，细胞外谷氨酸在急性期和间歇期均过度升高。然而，这些高水平谷氨酸的来源和癫痫发生的意义仍然是一个有争议的问题。本研究的总体目标是更好地理解内侧颞叶硬化（MTS）患者海马中谷氨酸水平升高的细胞和代谢机制。有令人信服的证据表明MTS中存在神经元和神经胶质功能障碍，迄今为止的大部分工作都集中在分离制剂中神经元或神经胶质功能的变化上。我们假设MTS中的“神经元-神经胶质单元”受损，使得功能失调的神经胶质产生大量升高的间期谷氨酸。此外，虽然神经元和胶质细胞的释放都可能导致垂直升高，但我们假设胶质细胞的成分是非常重要的。我们建议使用一系列旨在了解谷氨酸在致癫痫人类颞叶中的配置的技术来测试这一假设。我们将把这些研究与颅内监测相结合，用于识别医学上难治性癫痫患者的癫痫发生区域。在具体目标1中，我们将使用HPLC测量细胞外谷氨酸和谷氨酰胺在微透析期间和刺激后使用微透析探针连接到深度电极。将进行刺激以模拟局灶性癫痫发作活动。在具体目标2中，我们将注入仅由胶质细胞吸收的13C醋酸盐，并将使用质谱法测量在基线和刺激后微透析液中谷氨酸和谷氨酰胺中13C的分数富集，以确定这些不同条件下细胞外谷氨酸的来源。对于这两项研究，我们将在放置在海马体和非癫痫性皮质区域的探针中进行分析。谷氨酸摄取和外排将在特异性Aim 3中使用切除的皮质和海马组织测量。此外，将对神经胶质谷氨酸升高的影响进行生理学研究，作为这一目标的一部分。神经胶质释放的可能机制也将被讨论。最后，在Specific Aim 4中，与胶质细胞和神经元中谷氨酸代谢最相关的酶和转运蛋白的水平将通过Western blots使用切除的组织来测量。在每个特定目标中获得的数据将与定量神经元和胶质细胞计数相关联。相关性：综合这些数据将使我们能够更好地理解MTS中谷氨酸升高的起源，从而设计出更有针对性的分子和细胞疗法来恢复谷氨酸稳态，从而减少兴奋性和癫痫发作。