PH REGULATION IN NEURONS AND ASTROCYTES IN HYPOXIA

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值，尤其是pHo调节缺氧/缺血性损伤， 哺乳动物中枢神经系统（CNS）。 很明显，pH 伴随缺氧的紊乱是由原发性改变引起的， 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， 传导和对神经递质的反应。 因此， 了解缺氧如何影响pHi稳态，以及 伴随缺氧/缺血的辅助紊乱。 拟议 研究将调查pHi调节的潜在机制， 锥体神经元和星形胶质细胞新鲜分离的CA 1区的 大鼠海马，目的是了解pHi生理学 的神经元和星形胶质细胞相互作用，通过 细胞外间隙，在缺氧/缺血期间。 我们将在传感器上加载 pH敏感染料，并从荧光信号计算pHi。 我们也 将使用电生理学方法监测膜电压 (Vm)和评估神经元功能。 我们的总体方针是 研究从CA 1新鲜分离的锥体神经元和星形胶质细胞 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。 一是 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 神经元和星形胶质细胞中的pHi稳态。 特别感兴趣的是 观察到神经元可以存在于低和高pHi 国家，低和高pHi神经元之间的分布是年龄 依赖性，神经元有时会自发地从 从低pH值到高pH值状态。 第二，了解缺氧/缺血是如何 相关干扰，如[K+]o 谷氨酸盐， GABA、（glu）o和 pHo中的Δ影响pHi和单个转运蛋白。 三是 确定pHi和pHo变化如何影响神经元功能。 我们 将使用电生理技术来评估兴奋性， 新鲜分离的CA 1神经元，并在CA 1神经元原位检查， 海马切片。 我们会证实电生理数据 用共焦测量的pHi切片。 我们将使用传统的 荧光显微镜和染料监测pHi（以及[Ca++]i [Na+]i 和电压。 此外，我们将使用膜片钳技术来监测Vm。 的 拟议的工作将导致第一次全面描述如何 缺氧/缺血和缺氧/缺血相关紊乱 细胞外参数影响神经元或神经元中的pHi调节。 哺乳动物大脑中的星形胶质细胞。 其重点发展变化可能 从而更好地了解新生儿缺氧/缺血 经期影响大脑功能。

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.