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: 7392762
• 负责人: DENNIS D SPENCER
• 金额: $ 49.94万
• 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/7392762
• 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(申请人提供)：已知细胞外谷氨酸在癫痫患者的海马区发作和发作间歇期均过度升高。然而，这些高水平谷氨酸的来源和对癫痫发生的意义仍然存在争议。这项建议的总体目标是更好地了解内侧颞叶硬化症(MTS)患者海马区谷氨酸升高的细胞和代谢机制。有令人信服的证据表明MTS同时存在神经元和神经胶质功能障碍，到目前为止，大部分工作都集中在孤立制剂中神经元或神经胶质功能的变化上。我们推测MTS患者的“神经元-神经胶质单位”受损，因此功能障碍的胶质细胞减少了发作间期升高的大部分谷氨酸。此外，虽然神经元和神经胶质细胞的释放都可能有助于发作的抬高，但我们假设神经胶质成分是非常重要的。我们建议使用一系列广泛的技术来检验这一假设，这些技术旨在了解谷氨酸在致痫人类颞叶中的分布。我们将把这些研究与用于识别难治性癫痫患者致痫区域的颅内监测结合起来。在具体目标1中，我们将使用连接在深度电极上的微透析探针，在间歇期和刺激后使用高效液相色谱法来检测微透析液中的细胞外谷氨酸和谷氨酰胺。将进行刺激以模拟局灶性癫痫发作活动。在特定目标2中，我们将注入胶质细胞独有的13C醋酸酯，并将使用质谱仪测量在基线和随后的刺激下微透析液中13C在谷氨酸和谷氨酰胺中的浓缩程度，以确定这些不同条件下细胞外谷氨酸的来源(S)。对于这两项研究，我们将在放置在海马区和非癫痫致癫痫皮质区的探针中进行分析。谷氨酸的摄取和外流将在特定的目标3中使用切除的皮质和海马区组织来测量。此外，作为这一目标的一部分，还将对升高的胶质谷氨酸的影响进行生理学研究。神经胶质细胞释放的可能机制也将被讨论。最后，在特定的目标4中，将使用切除的组织通过Western blotts测量与胶质细胞和神经元中谷氨酸代谢最相关的酶和转运体的水平。在每个特定目标中获得的数据将与神经元和神经胶质细胞的定量计数相关联。相关性：综合这些数据，我们将能够更好地了解MTS中谷氨酸升高的来源，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。