Consequences of impaired metabolism studied in vitro

本研究的中心假设是，癫痫患者大脑的间期代谢降低反映了线粒体代谢受损，其后果是神经胶质谷氨酸代谢，特别是谷氨酸摄取的改变。本小节的目的是测试关于这些变化的功能后果的特定假设。使用脑切片和培养的星形胶质细胞。来自患者的组织以及从人体组织中获得的慢性癫痫盐处理大鼠的组织将与其他子项目中获得的数据相关联。在第一个特定的目的，我们将测试的假设，细胞外稳态是受损的癫痫组织。[K+]o的调节、细胞外空间的大小和细胞外pH值将在人类和啮齿动物组织中使用离子选择性微电极进行检测。我们将研究特定代谢抑制剂对这些变量的影响。在第二个特定目的，我们将检查是否谷氨酸摄取受损癫痫组织。谷氨酸和谷氨酰胺水平的变化将在突触和自发活动和控制组织使用细胞外液的高效液相色谱测量。这与核心的微透析研究直接相关。谷氨酸摄取抑制剂对癫痫和对照组织中氨基酸浓度和癫痫发作活动的影响也将被研究。第三个特异性目的是验证细胞外谷氨酸增加与谷氨酸摄取缺陷相关的假设，通过突触前机制限制谷氨酸释放。最后，将在共聚焦显微镜下检测谷氨酸诱导的钙离子在培养的胶质细胞中的瞬态，以检验细胞外谷氨酸升高改变胶质可塑性的假设。对照和癫痫组织的培养将用于评估癫痫组织是否与长期暴露于谷氨酸的组织具有可比性反应。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。这些实验将通过在单个神经元或神经胶质细胞水平上检查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。

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.