Comparison between pH regulation in chemosensitive vs nonchemosensitive neurons

化学敏感与非化学敏感神经元 pH 调节的比较

• 批准号: 7662264
• 负责人: Vernon A. Ruffin
• 金额: $ 5.17万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-08 至 2010-10-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662264
• 关键词: Acids; Acute; Affect; Alkalosis; Bicarbonates; Brain; Buffers; Carbon Dioxide; Cells; Development; Electrodes; Environmental air flow; Exhibits; Family; Fellowship; Goals; Hand; Hippocampus (Brain); Individual; Kidney; Knock-out; Ligand Binding Domain; Lung diseases; Metabolic; Metabolic acidosis; Molecular; Monitor; Names; Neurons; Neurotransmitters; Patch-Clamp Techniques; Physiology; Population; Proteins; Recording of previous events; Regulation; Resistance; Respiratory Acidosis; Respiratory Alkalosis; Respiratory Failure; Role; Signal Transduction; Sleep Apnea Syndromes; Solutions; Sudden infant death syndrome; Testing; Time; Training; Tyrosine; Western Blotting; Work; base; experience; extracellular; falls; fluorescence imaging; hippocampal pyramidal neuron; immunocytochemistry; insight; interest; molecular phenotype; prevent; receptor; respiratory; response

DESCRIPTION (provided by applicant): Two earlier studies-using different criteria to characterize intracellular-pH (pHi) physiology-each identified two functional classes of neurons. In the first study, the initial pHi in a CO2/HCO3-free (i.e., HEPES- buffered) solution defines two populations of freshly dissociated pyramidal hippocampal (HC) neurons, a relatively low-pHi neuronal population (80%) and a relatively high-pHi neuronal population (20%). The second study comparing the response to acid/ base disturbances in HC and MR neurons saw similar results during respiratory acidosis, respiratory alkalosis, and metabolic alkalosis . The second criterion is the sensitivity to metabolic acidosis (MAc), a decrease in intracellular pH (pHo) caused by a decrease in [HCO3-] at fixed [CO2]. About 20% of cultured HC neurons are MAc-sensitive, exhibiting a large pHi decrease in response to MAc, whereas ~80% are MAc-resistant. In the medullary raphe (MR),15% of neurons were MAc-resistant and 85% were MAc-sensitive. An exciting preliminary result is that a second episode of MAc causes MAc-sensitive HC neurons to become MAc-resistant-perhaps the first example of history-dependent modulation of acid-base transport. Differences in CO2/HCO3-dependent transporter expression or regulation may explain differences in pHi regulation and MAc sensitivity. MR neurons are chemosensitive and altering the acid- base regulation through MAc exposure may affect their respiratory chemosensitivity and/or firing rate. Aim 1. To define the molecular basis of MAc sensitivity vs. MAc resistance using pH sensitive electrodes, immunocytochemistry, single cell PCR, KOs, and Western Blot. (A) Do MR neurons (like HC neurons) exist in high-pHi vs. low-pHi states, and are the high-pHi neurons identical to the MAc-sensitive ones? (B) Does MAc sensitivity vs. resistance correlate with the presence of specific SLC4 transporters, RPTP gamma or beta, or a specific neurotransmitter? (C) Does the knockout of specific SLC4 transporters, RPTP gamma, or RPTP beta shift the ratio of MAc-sensitive vs. resistant (or high- vs. low-pHi) neurons? Aim 2. To determine if a second MAc exposure (as in HC neurons) shifts MAc- sensitive MR neurons toward MAc resistance. Aim 3. To determine if pH-chemosensitivity in MR neurons correlates with pHi physiology. I will use patch clamp techniques. (A) Does the ability of MR neurons to alter firing rate in response to MAc correlate with MAc sensitivity vs. resistance, SLC4 profile, RPTP expression, or transmitter content. (B) Must pHi change in order for an MR neuron to alter firing rate? The proposed work will provide a valuable training experience as well as important new insights into respiratory control and could have important implications for several respiratory diseases, including sleep apnea, SIDS.

描述（由申请人提供）：两个早期的研究-使用不同的标准来表征细胞内pH（pHi）生理学-每个确定两个功能类的神经元。在第一项研究中，初始pHi在无CO2/HCO 3（即，HEPES缓冲的）溶液限定了两个新鲜解离的锥体海马（HC）神经元群体，相对低pHi神经元群体（80%）和相对高pHi神经元群体（20%）。第二项研究比较了HC和MR神经元对酸/碱紊乱的反应，在呼吸性酸中毒、呼吸性酸中毒和代谢性酸中毒期间观察到了类似的结果。第二个标准是对代谢性酸中毒（MAc）的敏感性，即固定[CO2]时[HCO 3-]减少引起的细胞内pH值（pHo）降低。约20%的培养HC神经元是MAc敏感的，表现出响应于MAc的大的pHi降低，而约80%是MAc抗性的。在延髓中缝核（MR）中，15%的神经元对MAc呈抗性，85%的神经元对MAc呈敏感性。一个令人兴奋的初步结果是，第二次发作的MAc导致MAc敏感的HC神经元成为MAc抗性-也许是第一个例子的历史依赖性调制的酸碱转运。CO2/HCO 3依赖性转运蛋白表达或调节的差异可以解释pHi调节和MAc敏感性的差异。MR神经元是化学敏感的，并且通过MAc暴露改变酸碱调节可能影响它们的呼吸化学敏感性和/或放电率。目标1.使用pH敏感电极、免疫细胞化学、单细胞PCR、科斯和蛋白质印迹法确定MAc敏感性与MAc耐药性的分子基础。(A)MR神经元（如HC神经元）是否存在于高pHi与低pHi状态，高pHi神经元与MAc敏感神经元是否相同？(B)MAc敏感性与耐药性是否与特定SLC 4转运蛋白、RPTP γ或β或特定神经递质的存在相关？(C)特异性SLC 4转运蛋白、RPTP γ或RPTP β的敲除是否改变了MAc敏感神经元与耐药神经元（或高pHi神经元与低pHi神经元）的比例？目标2.确定第二次MAc暴露（如HC神经元中）是否使MAc敏感的MR神经元向MAc抗性转变。目标3.确定MR神经元的pH化学敏感性是否与pHi生理学相关。我将使用膜片钳技术。(A)MR神经元响应MAc改变放电频率的能力是否与MAc敏感性与电阻、SLC 4谱、RPTP表达或递质含量相关。(B)MR神经元要改变放电频率，pHi必须改变吗？拟议的工作将提供宝贵的培训经验以及对呼吸控制的重要新见解，并可能对几种呼吸系统疾病，包括睡眠呼吸暂停，SIDS产生重要影响。