Glutamate release and uptake at central synapses

描述（由申请人提供）：细胞外谷氨酸水平在时间和空间上都有非常精确的控制，实现有效和选择性的突触兴奋，防止兴奋性毒性神经元死亡。一方面，突触间隙中的谷氨酸浓度必须迅速上升到毫摩尔浓度，以确保突触后嗜离子受体的激活。另一方面，细胞外谷氨酸的平均浓度必须维持在亚微摩尔水平，以防止细胞死亡。这些需要通过谷氨酸的爆炸性胞外释放和na依赖性谷氨酸转运蛋白家族提供的高容量和高亲和力的谷氨酸摄取系统来满足。当转运体功能在实验中或由于缺血等代谢危机而受损时，谷氨酸的强直性水平升高和突触释放位点周围谷氨酸清除的减慢可导致癫痫发作、缺血性损伤的扩散性损伤增强以及神经元和机体死亡。本研究的目的是确定释放后有多少谷氨酸从突触间隙中逃逸，谷氨酸在离释放位点多远的地方达到足以激活受体的浓度，摄取系统将谷氨酸隔离的速度有多快，以及这些过程如何受到谷氨酸释放量（包括多泡释放）的生理变化的影响。我们将在三种不同的突触中研究这些问题，比较它们独特的形态和受体和转运体的表达模式如何影响谷氨酸的突触释放和外源光解谷氨酸的作用。在这些研究中，我们将使用膜片钳记录结合双光子激光扫描显微镜和双光子激光谷氨酸释放在大鼠小脑和海马急性切片。通过对电和光记录的配对，我们实现了高的时间和空间分辨率。双光子激光谷氨酸释放特别适合于研究谷氨酸从单个突触释放的扩散，因为可以在小体积（~ 1¿m3）中实现高浓度（mM）的短时间应用（0.5 ms）。这种应用接近于囊泡性胞吐，但在实验上更容易控制。利用这一技术以及突触释放，我们将讨论突触间隙内外嗜离子性和代谢性谷氨酸受体激活的后果。

DESCRIPTION (provided by applicant): Extracellular levels of glutamate are controlled with great precision both temporally and spatially, achieving efficient and selective synaptic excitation and preventing excitotoxic neuronal death. 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, the average extracellular concentration of glutamate must be maintained at sub-micromolar levels 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 transporter function is compromised experimentally or by metabolic crises such as ischemia, elevated tonic levels of glutamate and slowing of glutamate clearance around synaptic release sites can result in seizures, enhanced spreading damage from ischemic insults and neuronal and organismal death. The objective of this proposal is to determine how much glutamate escapes from the synaptic cleft following release, how far from the release site glutamate reaches 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. We will investigate these issues at three dissimilar synapses to compare how their unique morphologies and expression patterns of receptors and transporters affect the actions of glutamate released synaptically and applied exogenously by the photolysis of caged glutamate. For these studies, we will use patch clamp recordings in conjunction with two photon laser scanning microscopy and two photon laser glutamate uncaging in acute slices of rat cerebellum and hippocampus. By pairing electrical and optical recording we achieve both high temporal and spatial resolution. Two photon laser glutamate uncaging is particular well suited to studying diffusion of glutamate released from individual synapses because short applications (0.5 ms) of high concentrations (mM) in small volumes (~ 1 ¿m3) can be achieved. Such applications approach those of vesicular exocytosis but are much more easily controlled experimentally. Using this technique as well as synaptic release, we will address the consequences of ionotropic and metabotropic glutamate receptor activation inside and outside of the synaptic cleft. PUBLIC HEALTH RELEVANCE: Understanding how the brain functions depends on knowledge of the fundamental unit of information processing within the brain called the synapse. To understand the synapse, we need detailed information about its physical structure, molecular constituents, biochemical and physiological mechanisms and how these properties change with age and experience. Rational design of therapies for neurological deficits is not possible without a fundamental understanding of synaptic function.