Dynamics of pH Regulation in the Brain

描述（由申请人提供）：在大脑中，正常和病理的电活动引起细胞外和细胞内pH的快速变化。这种pH的调节可以反馈影响神经活动，并影响对脑缺氧和缺血的反应。众所周知，由于钠驱动、电致、碳酸氢盐的进入，神经胶质细胞的细胞内pH值随着膜去极化而增加。这种流入同时使细胞外液酸化。相反，神经元的去极化会缓慢地使细胞质酸化，这是一种与钙离子进入有关的反应。对海马神经元的初步研究表明，这种神经反应代表了钙依赖性酸化和去极化触发的几乎等效的碱化机制之间的平衡。这些结果表明，神经元利用一种或多种响应膜电位的转运蛋白来预先调节其pH值。与胶质细胞的转运机制不同，初步数据表明，神经元碱化是通过一种需要氯离子的机制来响应去极化的，另一种是不依赖氯离子的单独过程。阐明这两种机制是本建议的头两个目的。第三个目的是澄清它们在酸中毒、重复放电和缺氧缺血性损伤反应中的功能相关性。最后两个目标关注神经元在快速调节细胞外pH中的作用。第四个目标关注海马神经元的细胞外碳酸酐酶（14型），并假设这种酶调节突触周围液体的pH。最后一个目标再次集中在氯依赖的碳酸氢盐运输的作用，但从细胞外的角度来看。该项目将使用具有特定碳酸氢盐转运体基因缺失的小鼠组织。研究将通过细胞内pH成像和全细胞记录技术进行，辅以定量聚合酶链反应方案来量化转运蛋白的表达。这些实验的结果提供了潜在的突破性见解，了解pH是如何在中枢神经系统中调节的，现在这种调节影响了正常的生理机能，以及对缺氧、心脏骤停、中风和创伤性脑损伤等情况的反应。公共卫生相关性：该项目将重点研究神经细胞在正常和异常电活动期间调节内部（细胞质）酸碱平衡的机制。阐明这些机制对于理解大脑如何维持有利于正常功能的内部微环境至关重要。此外，这一信息应该为神经细胞如何对心脏骤停、中风和创伤性脑损伤等情况作出反应提供重要的新见解。

DESCRIPTION (provided by applicant): In the brain, normal and pathological electrical activity gives rise to rapid changes in extracellular and intracellular pH. This modulation of pH can feedback to influence neural activity and can impact the response to brain hypoxia and ischemia. It is known that the intracellular pH of glial cells increases in response to membrane depolarization, due to sodium driven, electrogenic, entry of bicarbonate. This influx simultaneously acts to acidify the extracellular fluid. By contrast, depolarization of neurons slowly acidifies the cytosol, a response associated with entry of calcium. Preliminary studies on hippocampal neurons indicate that such neural responses represent a balance between a calcium dependent acidification, and a nearly equivalent alkalinizing mechanism that is triggered by depolarization. These results suggest that neurons regulate their pH preemptively, utilizing one or more transporters responsive to membrane potential. Unlike the transport mechanism of glia, preliminary data indicate that neurons alkalinize in response to depolarization by one mechanism that requires chloride ions, and a separate process that is chloride-independent. Elucidation of these two mechanisms occupies the first two aims of this proposal. The third aim seeks to clarify their functional relevance in response to acidosis, repetitive firing, and hypoxic-ischemic insults. The last two aims concern the role of neurons in rapid regulation of extracellular pH. The fourth aim focuses on an extracellular carbonic anhydrase (type 14) localized to neurons in the hippocampus, and posits that this enzyme regulates pH in the fluid around synapses. The last aim focuses again on role of chloride dependent bicarbonate transport, but from the extracellular perspective. The project will employ tissue from mice with gene deletions of specific bicarbonate transporters. Studies will be conducted with intracellular pH imaging and whole cell recording techniques, complemented by quantitative polymerase chain reaction protocols to quantify transporter expression. Results of these experiments offer potentially groundbreaking insights into how pH is regulated in the CNS, and now that regulation impacts normal physiology, and the response to conditions such as hypoxia, cardiac arrest, stroke and traumatic brain injury. PUBLIC HEALTH RELEVANCE: This project will focus on the mechanisms used by nerve cells to regulate internal (cytoplasmic) acid base balance, during both normal and abnormal electrical activity. Elucidation of these mechanisms is critical to understanding how the brain maintains an internal microenvironment conducive to proper function. Moreover, this information should provide important new insights into how nerve cells respond to conditions such as cardiac arrest, stroke and traumatic brain injury.