Consequences of impaired metabolism studied in vitro

体外研究代谢受损的后果

基本信息

批准号：
6233749
负责人：
ANNE WILLIAMSON
金额：
$ 22.6万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-13 至 2004-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6233749
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

项目摘要

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.

这项资助的核心假设是，癫痫人脑中观察到的发作间期代谢低下反映了线粒体代谢受损，其结果是神经胶质谷氨酸代谢的改变，特别是谷氨酸的摄取。本小节的目标是测试有关这些变化的功能后果的具体假设。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中获得的慢性癫痫红藻氨酸治疗的大鼠的组织将与其他子项目中获得的数据相关联。在第一个具体目标中，我们将检验癫痫组织中细胞外稳态受损的假设。 [K+]o 的调节、细胞外空间的大小和细胞外 pH 值将使用离子选择性微电极在人类和啮齿动物组织中进行测定。将检查特定代谢抑制剂对这些变量的影响。在第二个具体目标中，我们将检查癫痫组织中谷氨酸的摄取是否受损。使用细胞外液的 HPLC 测量突触和自发活动期间谷氨酸和谷氨酰胺水平的变化以及对照组织。这与核心中的微透析研究直接相关。还将研究谷氨酸摄取抑制剂对癫痫组织和对照组织中氨基酸浓度和癫痫发作活动的影响。第三个具体目标测试了以下假设：与谷氨酸摄取缺陷相关的细胞外谷氨酸的增加通过突触前机制限制谷氨酸的释放。最后，将检查培养神经胶质细胞中谷氨酸诱导的 Ca2+ 瞬变的共聚焦显微镜，以检验细胞外谷氨酸升高会改变神经胶质可塑性的假设。来自对照和癫痫组织的培养物将用于评估癫痫组织是否具有与长期暴露于谷氨酸的组织相当的反应。我们假设这将是在线粒体代谢受损患者的组织中发现的情况。这些实验将通过检查单个神经元或神经胶质细胞水平上线粒体代谢受损的几种可能的功能后果来补充其他三个项目。