Auditory coding at the hair cell ribbon synapse

描述（申请者提供）：耳蜗中的毛细胞通过带状突触连接听觉传入纤维。突触前的梯度电位在这里转化为突触后的尖峰电位。本研究的长期目标是探讨该突触的听觉信号编码策略。有研究表明，毛细胞倾向于一次释放多个突触囊泡（多囊泡释放：MVR）。然而，MVR的细胞机制尚不清楚，其功能优势尚不清楚。我们的第一个假设是，当毛细胞中的[Ca2+]j越过一个阈值时，MVR就会发生，这个阈值会触发条带上相邻的囊泡相互预融合，并同时将其所有内容物释放到突触间隙中。我们将确定量子响应大小（即单个囊泡融合引起的兴奋性突触后电流（EPSC）振幅），并以此来量化EPSC的量子内容。我们将发现MVR中的囊泡是否来自单个带，并研究MVR的Ca2+依赖性（即确定其Ca2+阈值）。第二个假设涉及MRV的功能，分为两个部分。一方面，MVR可以更快地对传入纤维膜进行充电和放电，帮助它们以更高的时间精度发射锁相尖峰。第二部分是MRV为毛细胞反复正弦去极化诱发的EPSC振幅提供了必要的变化因子。这允许传入纤维避免在每个正弦周期发射尖峰，并且尖峰的时序不会因尖峰不应期而恶化。我们将测量EPSC对正弦突触前去极化的响应，然后模拟这些EPSC，在一个时间窗口（例如0.1 ms）内用均匀分布的单个囊泡释放代替MVR，或者在量子响应的变化中限制EPSC振幅的变化（从而消除其量子内容的变化）。然后，这些模拟的EPSCs将被实验性地注入电流箝位下的传入纤维中，以确定当MVR缺失时，尖峰锁相的恶化程度。第三个有待验证的假设是融合的突触囊泡可以通过MVR后的快速内吞作用进行循环。我们将使用双光子显微镜观察FM1-43染料负载情况以监测囊泡循环，我们还将在毛细胞上进行细胞附着电容测量，通过监测电容变化来研究囊泡循环，时间分辨率为50 ps或更高。

DESCRIPTION (provided by applicant): Hair cells in the cochlea connect to auditory afferent fibers via ribbon synapses. Presynaptic graded potentials are converted here into postsynaptic spikes. The long-term objective of this study is to investigate the strategies for auditory signal encoding at this synapse. It has been suggested that hair cells tend to release more than one synaptic vesicle at a time (multivesicular release: MVR). However, the cellular mechanisms underlying MVR are poorly understood and its functional advantages are not known. Our first hypothesis is that MVR occurs when [Ca2+]j in hair cells crosses a threshold that triggers neighboring vesicles on a ribbon to pre-fuse with each other and release all their contents into the synaptic cleft simultaneously. We will determine the quantal response size (i.e., excitatory postsynaptic current (EPSC) amplitude evoked by a single vesicle fusion) and use this to quantify EPSC quantal content. We will find out if vesicles in MVR are from a single ribbon and investigate the Ca2+-dependence of MVR (i.e. determine its Ca2+ threshold). The second hypothesis concerns the function of MRV and has two parts. One part is that MVR can charge and discharge the membrane of afferent fibers more rapidly, helping them to fire spikes with higher temporal precision for phase-locking. The second part is that MRV provides a necessary varying factor on EPSC amplitudes evoked by repeated sinusoidal depolarizations of hair cells. This allows afferent fibers to avoid firing spikes at every sinusoidal cycle and the timing of spikes will not deteriorate due to spike refractory periods. We will measure EPSCs in response to a sinusoidal presynaptic depolarization and then simulate these EPSCs to either substitute MVR with evenly distributed single vesicle releases within a time window (e.g. 0.1 ms), or limit the variation of EPSC amplitudes within the variation of quantal responses (removing thus the variation in their quantal content). These simulated EPSCs will then be experimentally injected into afferent fibers under current-clamp to determine to what extent the phase-locking of spikes becomes deteriorated when MVR is absent. The third hypothesis to be tested is that fused synaptic vesicles can be recycled through fast endocytosis following MVR. We will use a 2-photon microscope to visualize FM1-43 dye loading to monitor vesicle recycling, and we will also make cell-attached capacitance measurements on hair cells to study vesicle recycling by monitoring capacitance changes with a time resolution of 50 ps or higher. RELEVANCE: In the United States, roughly 23,000 adults and 15,500 children have received cochlear implants, which restore part of their hearing by directly stimulating auditory nerve fibers with electrodes. However, the algorithms to stimulate the fibers according to the sound signal have been determined only empirically. The fundamental studies of afferent fiber spiking proposed here will provide guidance for significantly improving these algorithms, especially for adult patients whose auditory systems are fully developed and may thus have lost some of their plasticity and adaptability to different stimulus protocols.