Glutamate release and uptake at central synapses

描述（由申请人提供）：谷氨酸的细胞外水平在时间和空间上都得到了非常精确的控制，实现了有效和选择性的突触兴奋并防止了兴奋性神经元死亡。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，以确保突触后离子型受体的激活。另一方面，谷氨酸的平均细胞外浓度必须保持在亚微摩尔水平，以防止细胞死亡。这些要求通过谷氨酸的爆炸性胞吐释放和由Na依赖性谷氨酸转运蛋白家族提供的高容量和高亲和力谷氨酸摄取系统来满足。当转运蛋白功能在实验上或因代谢危机（如缺血）而受损时，谷氨酸盐的紧张性水平升高和突触释放部位周围谷氨酸盐清除率降低可导致癫痫发作、缺血性损伤引起的扩散性损伤增强以及神经元和生物体死亡。 这个建议的目的是确定有多少谷氨酸逃逸突触间隙释放后，有多远的释放位点谷氨酸达到浓度足以激活受体，如何迅速的摄取系统螯合谷氨酸，以及这些过程是如何影响的生理变化的谷氨酸释放量，包括多泡释放。我们将调查这些问题在三个不同的突触，比较其独特的形态和受体和转运蛋白的表达模式如何影响突触释放的谷氨酸和应用外源性的笼状谷氨酸的光解的行动。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在大鼠小脑和海马的急性切片中打开。通过电子和光学记录的配对，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从单个突触释放的谷氨酸的扩散，因为可以实现小体积（~ 1 <$m3）的高浓度（mM）的短应用（0.5 ms）。这种应用接近囊泡胞吐，但更容易控制实验。使用这种技术以及突触释放，我们将解决的后果，亲离子型和亲代谢型谷氨酸受体激活的突触间隙内外。 公共卫生相关性：了解大脑如何运作取决于对大脑内信息处理的基本单位突触的了解。为了了解突触，我们需要详细的信息，包括它的物理结构、分子组成、生化和生理机制，以及这些特性如何随着年龄和经验而变化。如果没有对突触功能的基本理解，就不可能合理设计神经功能缺损的治疗方法。

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.