Comparison between pH regulation in chemosensitive vs nonchemosensitive neurons

描述(由申请人提供)：早期的两项研究-使用不同的标准来表征细胞内pH(Phi)生理学-每项研究都识别了两种功能类别的神经元。在第一项研究中，在无CO2/HCO3(即HEPES缓冲)的溶液中，初始PHI定义了两组新分离的锥体(HC)神经元，相对低PHI神经元群(80%)和相对高PHI神经元群(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、呼吸性碱中毒和代谢性碱中毒中发现了类似的结果。第二个标准是对代谢性酸中毒(Mac)的敏感性，即由于固定[CO2]时[HCO3-]减少而导致的细胞内pH(Pho)下降。约20%的培养的HC神经元对Mac敏感，表现出对Mac的反应显著降低，而约80%的HC神经元对Mac不敏感。在延髓中缝(MR)，15%的神经元对Mac不敏感，85%的神经元对Mac敏感。一个令人兴奋的初步结果是，Mac的第二次发作导致对Mac敏感的HC神经元对Mac产生抵抗力--这可能是酸碱运输依赖于历史的第一个例子。依赖CO2/HCO3的转运蛋白表达或调控的差异可能解释了phi调控和Mac敏感性的差异。MR神经元是化学敏感的，通过暴露Mac改变酸碱调节可能会影响它们的呼吸化学敏感性和/或放电频率。目的1.应用pH敏感电极、免疫细胞化学、单细胞聚合酶链式反应、KOS和Western Blot等方法，研究Mac敏感性与Mac耐药性的分子基础。(A)MR神经元(如HC神经元)是否存在高Phi与低Phi状态，以及高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SLC4转运体、RPTPγ或β转运体或特定神经递质的存在相关？(C)特定的SLC4转运体、RPTPγ或RPTPβ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)的比率？目的2.确定第二次Mac暴露(如在HC神经元中)是否使对Mac敏感的MR神经元向Mac抵抗方向移动。目的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神经元对Mac反应而改变放电频率的能力是否与Mac敏感性与阻力、SLC4谱、RPTP表达或递质含量相关。(B)为了让MR神经元改变放电频率，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，并可能对包括睡眠呼吸暂停、小岛屿发展中国家在内的几种呼吸系统疾病产生重要影响。

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.