AXON GLIAL SIGNALING IN MAMMALIAN WHITE MATTER

哺乳动物的脑白质是大脑的主要部分，对许多人来说 多年来，钾离子一直被认为是唯一的媒介 轴突和神经胶质细胞之间的信号传递，主要导致被动神经胶质细胞 去极化。目前还没有已知的释放递质的囊泡方式。 在哺乳动物的脑白质中。然而，最近的研究表明，神经胶质 白质中的细胞表达谷氨酸受体。这份提案审查了 轴突如何动态激活这一部分的神经胶质受体 大脑。被研究的白质是大鼠的视神经。 在目标1中，我们将研究谷氨酸是否在轴突胶质细胞中起重要作用。 用高效液相色谱法检测谷氨酸活性依赖性释放的信号 从视神经和神经胶质谷氨酸的药理解剖 受体亚型在神经活动过程中被激活。然后我们专注于 谷氨酸的释放机制以及它们是否由载体介导。 在目标2中，我们将为可能进行调解的运营商提供证据 谷氨酸的释放。我们将利用分子生物学来鉴定三个主要的 视神经谷氨酸转运蛋白(GLT、GLAST和EAAC1)的克隆。 然后将产生多肽特异性抗体来检查细胞 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，我们将看到轴突和神经胶质转运蛋白是否起调节作用 生理性谷氨酸释放及其贡献是否可以 已经解决了。为了区分神经胶质释放和轴突释放，我们将使用许多 技巧。这些包括选择性操纵胶质细胞转运蛋白。 膜片钳神经切片，检测仪检测轴突释放 以及神经胶质和轴突释放的高空间分辨率 D-天冬氨酸的免疫金免疫金分析。 这一结果将极大地扩展我们目前对神经元-- 神经胶质相互作用，并可能导致对潜在的新的 谷氨酸转运体和谷氨酸受体在调节细胞周期中的作用 哺乳动物脑白质的发育。

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.