Glutamate release and uptake at central synapses

描述(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极高的精确控制，实现了有效和选择性的突触兴奋，并防止了兴奋性毒性神经元的死亡。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，以确保突触后离子亲电受体的激活。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。谷氨酸的爆炸性胞吐释放和钠依赖的谷氨酸转运体家族提供的高容量和高亲和力的谷氨酸摄取系统满足了这些要求。当转运蛋白功能受到实验或代谢危机(如缺血)的影响时，谷氨酸的紧张性水平升高和突触释放部位谷氨酸清除的放缓可导致癫痫发作，缺血性损伤造成的扩散损害加剧，以及神经元和器官的死亡。这个方案的目的是确定释放后有多少谷氨酸从突触间隙逃逸，离释放部位多远的谷氨酸达到足以激活受体的浓度，摄取系统隔离谷氨酸的速度有多快，以及这些过程如何受到谷氨酸释放量的生理变化的影响，包括多囊释放。我们将在三个不同的突触上研究这些问题，以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通过突触释放的谷氨酸和通过笼养的谷氨酸的光解而外源应用的谷氨酸的活动。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浸泡在大鼠小脑和海马区的急性切片上。通过将电记录和光记录配对，我们获得了高的时间和空间分辨率。双光子激光谷氨酸去化特别适合于研究从单个突触释放的谷氨酸的扩散，因为可以在小体积(~1？m~3)内实现短时间(0.5ms)的高浓度(Mm)应用。这种应用接近于囊泡胞吐，但在实验上更容易控制。利用这项技术和突触释放，我们将研究突触裂隙内外离子型和代谢型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.