Comparison between pH regulation in chemosensitive vs nonchemosensitive neurons

描述（由申请人提供）：两项早期研究-使用不同的标准来表征细胞内ph （pHi）生理学-分别确定了两种功能类别的神经元。在第一项研究中，无CO2/ hco3（即HEPES缓冲）溶液中的初始pHi定义了两种新解离的锥体海马（HC）神经元群体，相对低pHi的神经元群体（80%）和相对高pHi的神经元群体（20%）。第二项研究比较了HC和MR神经元对酸碱干扰的反应，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，即在固定[CO2]下[HCO3-]的减少导致细胞内pH （pHo）的降低。培养的HC神经元中约有20%对MAc敏感，对MAc的反应显着降低pHi，而约80%对MAc有抗性。在中脑髓（MR）中，15%的神经元为mac抵抗性神经元，85%为mac敏感性神经元。一个令人兴奋的初步结果是，MAc的第二次发作会导致MAc敏感的HC神经元变得耐MAc——这可能是酸碱转运历史依赖调节的第一个例子。CO2/ hco3依赖性转运体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神经元具有化学敏感性，通过MAc暴露改变酸碱调节可能会影响其呼吸化学敏感性和/或放电速率。目的1。使用pH敏感电极、免疫细胞化学、单细胞PCR、KOs和Western Blot来定义MAc敏感性和MAc耐药性的分子基础。(A) MR神经元（像HC神经元一样）是否存在于高phi和低phi状态，高phi神经元是否与mac敏感神经元相同？(B) MAc敏感性与耐药是否与特异性SLC4转运体、RPTP γ或β或特异性神经递质的存在相关？(C)敲除特定的SLC4转运体、RPTP γ或RPTP β是否会改变mac敏感与耐药（或高phi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）是否使MAc敏感的MR神经元转向MAc抗性。目标3。目的：确定MR神经元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神经元在MAc反应中改变放电速率的能力是否与MAc敏感性与阻抗、SLC4谱、RPTP表达或递质含量相关？(B)为了使一个MR神经元改变放电速率，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具有重要意义。

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.