Proton as co-transmitter of neuronal signaling

描述（由申请人提供）：本提案的总体目标是研究质子是否可以作为胆碱能神经元突触信号传导的共递质。这种可能性的基本原理来自我们最近的发现，酸化独特地调节重组神经元烟碱乙酰胆碱受体（nAChRs），部分基于它们的脱敏动力学和β亚基组成。与酸化通常抑制配体和电压门控阳离子通道的报道形成鲜明对比的是，我们发现快速（约20 ms）酸化增强了alpha3/beta4神经元的nAChR电流，并加速了其激活和衰减动力学。由于众所周知：a)大脑中的重复放电导致短暂的酸性转移，b) ACh储存在酸性囊泡中，以及c)囊泡质子的释放抑制突触前Ca2+通道，我们假设突触间隙的短暂间歇酸化调节突触后nachr，为突触信号传递提供局灶可塑性。本文提出的实验旨在：1)在近似突触传递的时间尺度上检验酸化如何调节原代培养的嗜铬细胞、皮质和小脑神经元中的天然神经元nachr；2)在PC12和染色质细胞培养中，成像酸化微域，探测“质子火花”的存在，并释放递质；3)直接评估释放质子在皮质和小脑神经元突触信号调节中的作用。为了在可行的实验条件下验证这一假设，我们将使用两种神经分泌模型：1)PC12和染色质细胞；2)皮质和小脑神经元培养，其中激动剂和质子可以快速（大约2毫秒）和短暂地应用，同时测量膜电流。在染色质细胞和皮质神经元中，我们将量化质子对nachr的调节作用，并使用定制的ph敏感染料，结合快速（240-480 f/s）共聚焦显微镜和我们实验室开发的一种新型高分辨率光学技术（全反射荧光显微镜，TIRF），对分泌囊泡释放到细胞旁空间所产生的酸化谱进行成像。我们相信，我们的发现将为突触信号传导提供新的见解，这可能对阿尔茨海默氏症等神经退行性疾病的发病机制至关重要，在这些疾病中，突触囊泡的质子含量可能被改变，导致胆碱能信号传导受损。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to examine whether protons can serve as co-transmitters in cholinergic neuronal synaptic signaling. The rationale for this possibility carne from our recent findings that acidification uniquely modulated the recombinant neuronal nicotinic acetylcholine receptors (nAChRs) in part based on their desensitization kinetics and their beta subunit composition. In sharp contrast to reports that acidification generally inhibits ligand- and voltage-gated cation channels, we found that rapid (approximately 20 ms) acidification enhanced the alpha3/beta4 neuronal nAChR current and accelerated its activation and decay kinetics. Since it is well known that: a) repetitive firing in the brain causes brief acidic shifts, b) ACh is stored in acidic vesicles, and c) release of vesicular protons inhibits presynaptic Ca2+ channels, we hypothesize that brief intermittent acidification of synaptic cleft modulates the postsynaptic nAChRs providing focal plasticity to synaptic signaling. The experiments proposed here are aimed at: 1) examining, on a time scale approximating synaptic transmission, how acidification modulates the native neuronal nAChRs in primary culture of chromaffin cells, cortical, and cerebellar neurons; 2) imaging the microdomains of acidification and probing for presence of "proton sparks" with transmitter release in PC12 and chromaffin cell cultures; 3) evaluating directly the roles of released protons in modulation of synaptic signaling in cortical and cerebellar neurons. To test this hypothesis under feasible experimental conditions, we will use two models of neurosecretion: 1) PC12 and chromaffin cells and 2) cultures of cortical and cerebellar neurons, where agonist and protons may be applied rapidly (approximately 2 ms) and briefly, and membrane currents measured simultaneously. In chromaffin cells and cortical neurons, we shall quantify the effect of protons in modulation of nAChRs, and image the acidification profiles resulting from the release of secretory vesicles into paracellular space, using custom-made pH-sensitive dyes, in combination with rapid (240-480 f/s) confocal microscopy and a novel high-resolution optical technique (Total Internal Reflection Fluorescence Microscopy, TIRF), developed in our laboratory. Our finding, we believe, will provide novel insights into synaptic signaling that may be critical in the pathogenesis of neurodegenerative diseases such as Alzheimer's, where proton content of synaptic vesicle may be altered leading to impaired cholinergic signaling.