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.

大脑缺氧/缺血的一个有据可查的特征是 pH 值的变化， 细胞内 pH (pHi) 和细胞外 pH (pHo)。 最近的证据 表明 pH，尤其是 pHo 调节缺氧/缺血性损伤 哺乳动物的中枢神经系统（CNS）。 很明显，pHo 伴随缺氧缺氧的紊乱是由原发性变化引发的 在神经元和/或星形胶质细胞 pHi 中。 神经元和星形胶质细胞 pHi 紊乱 由于离子的 pH 敏感性，它们本身就至关重要 电导和对神经递质的反应。 因此，至关重要的是 了解 pHi 稳态如何受到缺氧以及 伴随缺氧/缺血的辅助紊乱。 拟议的 研究将调查 pHi 调节的潜在机制 新鲜分离自 CA1 区的锥体神经元和星形胶质细胞 大鼠海马体，目的是了解 pHi 生理学原理 神经元和星形胶质细胞通过 缺氧/缺血期间的细胞外空间。 我们将用 pH 敏感染料，并根据荧光信号计算 pHi。 我们也 将使用电生理学方法监测膜电压 (Vm) 并评估神经元功能。 我们的一般方法是 研究从 CA1 中新鲜分离的锥体神经元和星形胶质细胞 海马体区域，包括未成熟（3-10 天 ole）和成熟 （22030 天龄）大鼠。 该提案有三个主要目标。 首先，要 了解急性、分级缺氧和慢性缺氧如何影响稳态 状态 pHi，以及负责的各个酸碱转运蛋白 神经元和星形胶质细胞中的 pHi 稳态。 特别感兴趣的是 观察到神经元可以在低 pHi 和高 pHi 中存在 状态，低 pHi 神经元和高 pHi 神经元之间的分布是年龄 依赖性，并且神经元有时会自发地从 低至高 pHi 状态。 二、了解缺氧/缺血是如何发生的 相关干扰，例如 [K+]o 谷氨酸、GABA、(glu)o 和 pHo 的变化会影响 pHi 和单个转运蛋白。 三、至 确定 pHi 和 pHo 变化如何影响神经元功能。 我们 将使用电生理学技术来评估兴奋性 新鲜分离的 CA1 神经元，以及在原位检查的 CA1 神经元中 海马切片。 我们将验证电生理数据 在切片中进行 pHi 共聚焦测量。 我们将使用常规的 荧光显微镜和染料监测 pHi（以及 [Ca++]i [Na+]i 和附着在盖玻片上的单个、新分离的细胞的电压。 此外，我们将使用膜片钳技术来监测Vm。 这 拟议的工作将首次全面描述如何 缺氧/缺血和缺氧/缺血相关的紊乱 细胞外参数影响神经元或神经元的 pHi 调节 来自哺乳动物大脑的星形胶质细胞。 其重点发展变化可能 有助于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。