PH REGULATION IN NEURONS AND ASTROCYTES IN HYPOXIA

缺氧时神经元和星形胶质细胞的 PH 调节

• 批准号: 6410492
• 负责人: Walter F. Boron
• 金额: $ 18.52万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410492
• 关键词: acid base balance; acidity /alkalinity; age difference; astrocytes; bicarbonates; cell cell interaction; cerebral ischemia /hypoxia; electrophysiology; fluorescent dye /probe; ion transport; membrane transport proteins; neurons; pyramidal cells; tissue /cell culture; voltage /patch clamp

A well documented feature of brain hypoxia/ischemia is a change in pH, both intracellular pH )pHi) and extracellular pH (pHo). Recent evidence indicates that pH, especially pHo modulates hypoxic/ischemic injury in the mammalian central nervous system (CNS). It is clear that pHo disturbances accompanying hypoxicaanoxia are initiated by primary changes in neuron and/or astrocyte pHi. Disturbances in neuron and astrocyte pHi are critical in their own right because of the pH sensitivity of ion conductances and responses to neurotransmitters. Thus, it is critical to understand how pHi homeostasis is affected by hypoxica, as well as by the ancillary disturbances that accompany hypoxic/ischemia. The proposed research would investigate the mechanisms underlying pHi regulation in pyramidal neurons and astrocytes freshly isolated from CA1 region of the rat hippocampus, with the goal of understanding how the pHi physiologies of neurons and astrocytes interact with one another, via the extracellular space, during hypoxia/ischemia. We will load cells with a pH-sensitive dye, and compute pHi from fluorescence signals. We also will use electrophysiological approaches for monitoring membrane voltage (Vm) and assessing neuronal function. Our general approach will be to study pyramidal neurons and astrocytes freshly isolater from the CA1 region of the hippocampus, both from immature (3-10 day ole) and mature (22030 day old) rats. The proposal has three major aims. First, to understand how acute, graded hypoxia and chronic hypoxia affect steady- state pHi, as well as individual acid-base transporters responsible for pHi homeostasis in neurons and astrocytes. Of particular interest are the observations that the neurons can exist in both a low- and high-pHi state, that the distribution between low- and high pHi neurons is age dependent, and that the neurons sometimes spontaneously shift from the low-to the high-pHi state. Second, to understand how hypoxia/ischemia related disturbances such as [K+]o glutamate, GABA, (glu)o, and deltas in pHo affect pHi and individual transporters. Third, to determine how neuronal function is affected by pHi and pHo changes. We will use electrophysiological techniques to assess excitability both in freshly dissociated CA1 neurons, and in CA1 neurons examined in situ in hippocampal slices. We will corroborate the electrophysiological data in slices with confocal measurements of pHi. We will use conventional fluorescent microscopy and dyes to monitor pHi (and also [Ca++]i [Na+]i and voltage in single, freshly dissociated cells attached to cover slips. In addition, we will use the patch-clamp technique to monitor Vm. The proposed work would lead to the first comprehensive description of how hypoxia/ischemia and hypoxia/ischemia-related disturbances in extracellular parameters affect pHi regulation in either a neuron or an astrocyte from a mammalian brain. Its focus developmental changes could lead to a better understanding of how hypoxia/ischemia in the neonatal period affect brain function.

脑缺氧/缺血的一个有充分证据的特征是pH的变化， 胞内pH(Phi)和胞外pH(Pho)。最近的证据 提示pH，尤其是pho对缺氧/缺血损伤有调节作用。 哺乳动物的中枢神经系统。很明显，Pho 伴随缺氧性缺氧的紊乱是由原发改变引起的 在神经元和/或星形胶质细胞中。神经元和星形胶质细胞PHI的紊乱 由于离子的pH敏感性，它们本身就是关键的 电导和对神经递质的反应。因此，它是至关重要的 为了了解低氧对phi动态平衡的影响，以及 伴随着缺氧/缺血而来的辅助紊乱。建议数 研究将调查PHI调控的潜在机制 新分离的大鼠脑CA1区锥体神经元和星形胶质细胞 大鼠海马区，目的是了解PHI生理学 神经元和星形胶质细胞相互作用，通过 细胞外空间，在缺氧/缺血期间。我们将在单元格中加载 一种对pH敏感的染料，并根据荧光信号计算phi。我们也 将使用电生理方法来监测膜电压 (Vm)和评估神经功能。我们的总体方法将是 新分离的CA1区锥体神经元和星形胶质细胞的研究 海马区，包括未成熟(3-10天)和成熟 (22030日龄)大鼠。该提案有三个主要目标。第一，要 了解急性、分级低氧和慢性低氧如何影响稳定- 州PHI，以及负责 神经细胞和星形胶质细胞的PHI动态平衡。特别值得关注的是 神经元可同时存在于低PH值和高PH值的观察 低phi神经元和高phi神经元之间的分布是年龄 依赖的，神经元有时自发地从 低-到高-phi状态。第二，了解缺氧/缺血是如何 相关干扰，如[K+]o谷氨酸、GABA、(Glu)o和 Pho中的Deltas会影响Phi和单个转运蛋白。第三，到 确定phi和pho变化对神经元功能的影响。我们 将使用电生理技术来评估兴奋性 新分离的CA1神经元，并在原位观察 海马片。我们将证实电生理数据 在共焦测量PHI的切片中。我们将使用传统的 荧光显微镜和染料用于监测Phi(以及[Ca++]i[Na+]i 以及贴在盖片上的单个、新分离的细胞中的电压。 此外，我们还将使用膜片钳技术来监测VM。这个 拟议的工作将导致第一次全面描述 缺氧/缺血与缺氧/缺血相关的紊乱 细胞外参数影响神经元或神经细胞内的phi调节 来自哺乳动物大脑的星形细胞。它的重点发展变化可能 更好地了解新生儿缺氧/缺血是如何发生的 经期影响大脑功能。