GLUTAMATE RELEASE AND UPTAKE AT CEREBELLAR SYNAPSES

描述：（申请人摘要） 细胞外谷氨酸水平的控制非常精确， 在时间和空间上，实现有效和选择性的突触兴奋 并防止兴奋性毒性引起的神经元死亡。一方面，谷氨酸 突触间隙中的浓度必须迅速上升到毫摩尔浓度 浓度，以确保激活突触后离子型受体。对 另一方面，亚微摩尔水平的谷氨酸，平均而言，必须是 维持在细胞外空间以防止细胞死亡。这些要求 谷氨酸的爆炸性胞吐释放和高容量 和高亲和性谷氨酸摄取系统，由 钠依赖性谷氨酸转运蛋白。当这些传送器被渲染后 无效的，无论是在转基因小鼠中，还是在自发 人类中的突变（例如，ALS），谷氨酸盐的紧张水平升高， 突触释放部位周围的间隙可导致癫痫发作，增强 对缺血性损伤的易感性以及神经元和生物体死亡。 这项提议的目的是确定有多少谷氨酸从 突触间隙释放后，距离释放位点多远谷氨酸 达到足以激活受体的浓度， 系统螯合谷氨酸盐，以及这些过程如何受到 谷氨酸释放量的生理变化，包括 多泡释放和泡填充的可变性。我们将使用整个 细胞和外向膜片钳方法在急性大鼠小脑脑片， 解决离子型和代谢型谷氨酸受体 突触间隙内外的激活，包括 邻近的神经胶质和神经元。

DESCRIPTION: (Applicant's Abstract) Extracellular levels of glutamate are controlled with great precision both temporally and spatially, achieving efficient and selective synaptic excitation and preventing neuronal death by excitotoxicity. On one hand, glutamate concentrations in the synaptic cleft must rise rapidly to millimolar concentrations to ensure activation of postsynaptic ionotropic receptors. On the other hand, sub-micromolar levels of glutamate, on average, must be maintained in the extracellular space to prevent cell death. These requirements are met by the explosive exocytotic release of glutamate and the high capacity and high affinity glutamate uptake system provided by the family of Na-dependent glutamate transporters. When these transporters are rendered ineffective, either pharmacologically, in transgenic mice, or by spontaneous mutation in humans (e.g., ALS), elevated tonic levels of glutamate and slowed clearance around synaptic release sites can result in seizures, enhanced susceptibility to ischemic insults, and neuronal and organismal death. The aim of this proposal is to determine how much glutamate escapes from the synaptic cleft following release, how far from the release site glutamate attains concentrations sufficient to activate receptors, how rapidly the uptake system sequesters glutamate, and how these processes are affected by physiological alterations in the amount of glutamate released including multivesicular release and variability in vesicular filling. We will use whole cell and outside-out patch clamp methods in acute rat cerebellar slices to address the consequences of ionotropic and metabotropic glutamate receptor activation inside and outside of the synaptic cleft, including receptors on neighboring glia and neurons.