Consequences of impaired metabolism studied in vitro

体外研究代谢受损的后果

• 批准号: 6616369
• 负责人: ANNE WILLIAMSON
• 金额: $ 22.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616369
• 关键词: bioenergetics; brain metabolism; electrophysiology; epilepsy; glutamates; glutamine; human tissue; laboratory rat; microelectrodes; mitochondria; neural plasticity; neurotransmitter metabolism; neurotransmitter transport; partial seizure; single cell analysis; temporal lobe /cortex disorder; tissue /cell culture

The central hypothesis for this grant is that interictal hypometabolism seen in epileptic human brain reflects impaired mitochondrial metabolism, on consequence of which is an alteration in glial glutamate metabolism,, especially glutamate uptake. The goal of this subsection is to test specific hypotheses regarding the functional consequences of these changes. using brain slices and cultured astrocytes. Tissue from patients as well as from chronically epileptic kainate-treated rats obtained from human tissue will be correlated with the data obtained in the other subprojects. In the first Specific Aim we will test the hypothesis that extracellular homeostasis is impaired in epileptic tissue. The regulation of [K+]o, the size of the extracellular space and extracellular pH will be assayed using ion-selective microelectrodes in human and rodent tissue. The effect of specific metabolic inhibitors on these variables will be examined. In the second Specific Aim we will examine if glutamate uptake impaired in epileptic tissues. The changes in glutamate and glutamine levels will be measured during synaptic and spontaneous activity and control tissue using HPLC of the extracellular fluid. This links directly with the microdialysis studies in the Core. The effects of glutamate uptake inhibitors on the amino acid concentrations and on seizure activity in both epileptic and control tissue will also be investigated. The third Specific Aim tests the hypothesis that an increase in extracellular glutamate associated with defective glutamate uptake acts to limit glutamate release via presynaptic mechanisms. Finally, confocal microscopy of glutamate- induced Ca2+ transients in cultured glia will be examined to test the hypothesis that elevated extracellular glutamate alters glial plasticity. Cultures from control and epileptic tissue will be used to assess whether epileptic tissue has comparable responses to that chronically exposed to glutamate. We hypothesize that this will be the condition found in tissue from the patients with impaired mitochondrial metabolism. These experiments will complement the other three projects by examining several possible functional consequences of impaired mitochondrial metabolism at the level of single neurons or glial cells.

这项赠款的中心假设是，癫痫性人脑中看到的发型疗程低代谢反映了线粒体代谢受损，结果是神经胶质谷氨酸代谢的改变，尤其是谷氨酸的摄取。该小节的目的是检验有关这些变化功能后果的特定假设。使用脑切片和培养的星形胶质细胞。来自患者以及从人体组织获得的经慢性癫痫病的大鼠的组织与其他次要注射中获得的数据相关。在第一个特定目的中，我们将检验以下假设：癫痫组织中细胞外稳态受损。 [K+] O的调节，将使用人和啮齿动物组织中的离子选择微电极分析细胞外空间和细胞外pH的大小。将检查特定代谢抑制剂对这些变量的影响。在第二个特定目的中，我们将检查是否在癫痫组织中谷氨酸摄取受损。使用细胞外液的HPLC，将在突触和自发活性和控制组织期间测量谷氨酸和谷氨酰胺水平的变化。这直接与核心中的微透析研究联系在一起。还将研究谷氨酸摄取抑制剂对氨基酸浓度以及癫痫组织和对照组织中癫痫发作活性的影响。第三个特定目的检验了以下假设：与缺陷的谷氨酸摄取作用相关的细胞外谷氨酸的增加，以通过突触前机制限制谷氨酸释放。最后，将检查谷氨酸诱导的Ca2+瞬时的共聚焦显微镜检查，以测试升高细胞外谷氨酸的假设，以改变神经胶质的可塑性。对照和癫痫组织的培养物将用于评估癫痫组织是否对长期暴露于谷氨酸的培养物具有可比的反应。我们假设这将是线粒体代谢受损患者的组织中发现的状况。这些实验将通过检查单个神经元或神经胶质细胞水平的线粒体代谢受损的几种可能功能后果来补充其他三个项目。