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: 6272332
• 负责人: Walter F. Boron
• 金额: $ 20.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272332
• 关键词: acid base balance; age difference; astrocytes; cell cell interaction; cerebral ischemia /hypoxia; confocal scanning microscopy; electrophysiology; extracellular; fluorescence microscopy; fluorescent dye /probe; hippocampus; immature animal; laboratory rat; mature animal; membrane transport proteins; neurons; single cell analysis; 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/缺血的有充分记录的特征是pH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会调节低氧/缺血性损伤 哺乳动物中枢神经系统（CNS）。 很明显pho 伴随性降压毒素毒素的干扰是由主要变化引发的 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的干扰 由于离子的pH敏感性，它们本身至关重要 电导和对神经递质的反应。 因此，这很关键 了解PHI稳态如何受到phoxica的影响，以及 缺氧/缺血的辅助干扰。 提议 研究将研究PHI调节的基础机制 锥体神经元和星形胶质细胞从新鲜分离的 大鼠海马，目的是了解PHI生理方式 神经元和星形胶质细胞通过 在缺氧/缺血期间，细胞外空间。 我们将加载电池 pH敏感的染料，并根据荧光信号计算PHI。 我们也是 将使用电生理方法监测膜电压 （VM）和评估神经元功能。 我们的一般方法将是 从CA1研究锥体神经元和星形胶质细胞 海马的区域，包括未成熟（3-10天的OLE）和成熟 （22030天大）大鼠。 该提案具有三个主要目标。 首先，到 了解急性，分级缺氧和慢性缺氧如何影响稳定 状态PHI以及负责的单个酸碱转运蛋白 神经元和星形胶质细胞中的PHI体内平衡。 特别感兴趣的是 观察到神经元可以存在于低phi和高phi中 指出，低PHI神经元之间的分布是年龄 依赖，神经元有时会自发地从 低到高态状态。 第二，了解低氧/缺血如何 相关干扰，例如[k+] o谷氨酸，gaba，（glu）o和 pho中的三角洲会影响phi和单个转运蛋白。 第三，到 确定神经元功能如何受PHI和PHO变化的影响。 我们 将使用电生理技术来评估兴奋性 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。 我们将证实电生理数据 在具有PHI共聚焦测量的切片中。 我们将使用常规 荧光显微镜和染料以监测PHI（以及[Ca ++] I [Na+] I 和单个新鲜分离的细胞中的电压，附着在覆盖滑板上。 此外，我们将使用贴片钳技术监视VM。 这 拟议的工作将导致有关如何的第一个全面描述 缺氧/缺血和低氧/缺血相关的疾病 细胞外参数会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。 它的重点发展变化可能 可以更好地了解新生儿中低氧/缺血的方式 周期会影响大脑功能。