AXON GLIAL SIGNALING IN MAMMALIAN WHITE MATTER

哺乳动物的白色物质是大脑的主要部分， 多年来，钾离子一直被认为是唯一的介质， 轴突和神经胶质细胞之间的信号传导，主要导致被动神经胶质细胞 去极化没有已知的囊泡方式释放递质 在哺乳动物的白色物质中。然而，最近的研究表明， 白色物质中的细胞表达谷氨酸受体。该提案审查了 轴突是如何动态激活这部分神经胶质受体的？ 个脑袋待研究的白色物质是大鼠视神经。 在目标1中，我们将研究谷氨酸在轴突-神经胶质细胞中是否重要。 通过使用HPLC检测谷氨酸的活性依赖性释放的信号传导 从视神经，并通过药理学解剖胶质谷氨酸 受体亚型在神经活动期间被激活。然后，我们专注于 谷氨酸释放的机制以及它们是否是载体介导的。 在目标2中，我们将为可能介导 谷氨酸盐释放。我们将用分子生物学来鉴定三种主要的 克隆的视神经谷氨酸转运蛋白（GLT、GLAST和EAAC 1）。 然后产生肽特异性抗体来检查细胞内的 这些转运蛋白在神经胶质细胞和轴突上的定位。 在目标3中，我们将观察轴突和神经胶质转运蛋白是否介导 生理性谷氨酸释放以及它们的贡献是否可以 整理出来.为了区分神经胶质和轴突释放，我们将使用许多 技术.这些包括选择性操纵神经胶质转运蛋白， 通过膜片钳进行神经切片，通过检测器检测轴突释放 补丁，以及高空间分辨率的神经胶质和轴突释放， D-天冬氨酸在电镜水平的免疫金分析。 这些结果将大大扩展我们目前对神经元的理解- 神经胶质相互作用，并可能导致了解潜在的新的 谷氨酸转运体和谷氨酸受体在调节 哺乳动物白色物质的发育。

The mammalian white matter is a major part of the brain where for many years potassium ions have been thought to be the only mediator of signaling between axons and glial cells, resulting mostly in passive glial depolarization. There are no known vesicular means of transmitter release in mammalian white matter. Yet recent studies have revealed that glial cells in white matter express glutamate receptors. This proposal examines how axons can dynamically activate glial receptors in this part of the brain. The white matter to be studied is the rat optic nerve. In Aim 1, we will examine whether glutamate is important in axon-glial signaling by using HPLC to detect activity-dependent release of glutamate from optic nerves, and by pharmacological dissection of glial glutamate receptor subtypes activated during nerve activity. We then focus on mechanisms of glutamate release and whether they are carrier-mediated. In Aim 2, we will provide evidence for carriers that might mediate glutamate release. We will use molecular biology to identify three major cloned transporters for glutamate (GLT, GLAST and EAAC1) in optic nerves. Then peptide-specific antibodies will be generated to examine the cellular localization of these transporters on glial cells and axons. In Aim 3, we will see if axonal and glial transporters mediate physiological glutamate release and whether their contributions can be sorted out. To distinguish glial and axonal release, we will use many techniques. These include selective manipulation of glial transporters in a nerve slice by patch-clamping, detection of axonal release by a detector patch, and high spatial resolution of glial and axonal release by immunogold analysis of D-aspartate at the electronmicroscopic level. The results will considerably extend our current understanding of neuron- glial interactions, and may lead to an understanding of potential new roles for glutamate transporters and glutamate receptors in regulating the development of mammalian white matter.