Origin of Extracellular Glutamate in Human Temporal Lobe Epilepsy

描述（由申请人提供）：已知在癫痫人类海马中，细胞外谷氨酸在发作期和发作间期均过度升高。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了解内侧颞叶硬化症（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，神经元和胶质细胞功能障碍的MTS和迄今为止的大部分工作都集中在神经元或胶质细胞功能的变化，在孤立的准备工作。我们推测MTS中的“神经元-胶质细胞单位”受损，使得功能障碍的胶质细胞产生大量升高的发作间期谷氨酸。此外，虽然神经元和神经胶质细胞的释放可能有助于发作海拔，我们假设，神经胶质成分是非常可观的。我们建议测试这一假设，使用广泛的技术，旨在了解在致癫痫的人类颞叶谷氨酸的处置。我们将把这些研究与颅内监测结合起来，颅内监测用于识别药物难治性癫痫患者的致痫区域。在具体目标1中，我们将使用HPLC测量微透析液中的细胞外谷氨酸盐和谷氨酰胺，包括使用连接到深度电极的微透析探头在发作间期和刺激后。将进行刺激以模拟局灶性癫痫发作活动。在特定目标2中，我们将输注13 C醋酸盐（仅由神经胶质吸收），并将使用质谱法测量基线和刺激后微透析液中谷氨酸盐和谷氨酰胺中13 C的富集分数，以确定这些不同条件下细胞外谷氨酸盐的来源。对于这两项研究，我们将在海马和非致癫痫皮质区放置的探针中进行分析。将在特定目标3中使用切除的皮质和海马组织测量谷氨酸摄取和外排。此外，作为这一目标的一部分，将对神经胶质谷氨酸盐升高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得到解决。最后，在具体目标4中，将使用切除的组织通过蛋白质印迹法测量与神经胶质和神经元中谷氨酸代谢最相关的酶和转运蛋白的水平。每个特定目标中获得的数据将与定量神经元和神经胶质细胞计数相关。相关性：这些数据将使我们能够更好地了解MTS中谷氨酸升高的起源，从而设计出更具针对性的分子和细胞疗法来恢复谷氨酸稳态，从而降低兴奋性和癫痫发作。

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.