High-fidelity synaptic transmission from hair cells to auditory afferent fibers

项目摘要 听觉毛细胞通过带状突触与传入神经纤维相连，其中听觉 信号从毛细胞的分级膜电位转化为传入的尖峰电位。 神经纤维。在成年动物身上进行的活体研究表明，对声音的反应 传入纤维以惊人的时间精确度发射尖峰，但潜在的突触 机制仍然鲜为人知。一个主要的障碍是缺乏一个完全成熟的模式 体外双膜片钳分析系统。成人的传入纤维终末 哺乳动物的耳蜗很小，大部分无法进行膜片钳分析，其结果来自 未成熟的耳蜗不足以解释活体内的发现。在这里，我们建议研究一下 成年牛蛙两栖类乳头的听觉传递是一种独特的模式系统 活体传入纤维的尖峰模式已用成对膜片钳重现 体外实验。利用这个模型系统，我们将结合双膜片钳记录， 谷氨酸去化和计算机建模，以解决 听觉：听觉毛细胞中微小而快速变化的信号是如何传递到传入的 纤维精准，不知疲倦。首先，我们将研究AMPA的减敏特性 传入纤维终末中的受体，并决定这些特性如何允许带状 在毛细胞持续释放谷氨酸的情况下维持突触强度的突触。 其次，我们将演示由预除极引起的钙离子内流导致快速启动 使突触小泡可在短暂的电压变化时释放。我们会 检验这种突触小泡启动是由第二个钙离子感受器介导的假设 具有不同于突触囊泡融合的生物物理特性。最后，我们将 阐明毛细胞带的标志--多泡释放的细胞机制 突触。我们将测试三种主要的候选机制：融合孔闪烁，纳米域 突触小泡和钙通道的偶联，以及突触带的协调。这个 通过这个项目获得的结果将回答内耳的几个长期存在的问题 生理学，并扩大我们对支配突触的基本规则的了解 听觉毛细胞突触的传递。

Project Summary Auditory hair cells connect to afferent nerve fibers through ribbon synapses, where auditory signals are converted from graded membrane potentials in hair cells into spikes in afferent nerve fibers. In vivo studies conducted in adult animals have shown that in response to sound afferent fibers fire spikes with remarkable temporal precision, but the underlying synaptic mechanisms are still poorly understood. A major hurdle is the lack of a fully mature model system for dual patch-clamp analysis in vitro. The afferent fiber terminals in the adult mammalian cochlea are small and mostly inaccessible to patch-clamp analysis, and results from the immature cochlea are insufficient to explain in vivo findings. Here we propose to examine auditory transmission in the amphibian papilla of adult bullfrogs, which is a unique model system where spiking patterns of afferent fibers in vivo have been recapitulated with paired patch-clamp experiments in vitro. Using this model system, we will combine dual patch-clamp recording, glutamate uncaging and computer modeling to address a central and fundamental question in hearing: how small and fast-changing signals in auditory hair cells are transmitted to afferent fibers precisely and tirelessly. First, we will examine the desensitization properties of AMPA receptors in afferent fiber terminals, and determine how these properties allow the ribbon synapses to maintain synaptic strength under continuous glutamate release from hair cells. Second, we will demonstrate that Ca2+ influx caused by pre-depolarization leads to rapid priming of synaptic vesicles and makes them releasable in response to brief voltage changes. We will test the hypothesis that this priming of synaptic vesicles is mediated by a second Ca2+ sensor with biophysical properties distinct from the one for synaptic vesicle fusion. Lastly, we will elucidate the cellular mechanisms for multivesicular release, a hallmark of hair cell ribbon synapses. We will test three major candidate mechanisms: fusion pore flickering, nanodomain coupling of synaptic vesicles and Ca2+ channels, and coordination of synaptic ribbons. The results obtained through this project will answer several long-standing questions of inner ear physiology, and expand our knowledge of the fundamental rules governing synaptic transmission at auditory hair cell synapses.