Synaptic Specializations in Auditory Hair Cells

 描述(申请人提供)：中枢神经系统接收到的所有听觉信息都通过内毛细胞-传入纤维突触传递。由于衰老、噪音或遗传疾病导致的突触功能障碍会极大地降低听力。这种非凡的突触必须在长时间内忠实地传递强度和计时信息。带状突触，就像毛细胞中的突触一样，通常存在于使用分级受体潜力的细胞中，需要有持续的释放。毛细胞带状突触比大多数突触更强壮，释放速度更快，持续释放的时间更长。为了让突触以这种方式运作，它们必须有一种强大的方法来回收和补充囊泡到突触释放部位。我们已经确认了一种非线性的钙依赖的电容变化，我们假设这是囊泡补充和补充的反映。我们的第一个主要目标(目标1)是确定这种非线性变化是否实际上是基于突触的，我们将使用成对的记录来识别突触后反应，使用新的谷氨酸传感器来确定谷氨酸释放是否模仿电容反应，并通过光学灭活带来看看我们是否可以消除这种非线性成分。我们还发现了与非线性电容变化相关的钙诱导钙释放过程(CICR)。我们假设囊泡运输是钙依赖的，CICR被用作确保囊泡持续供应的一种手段，而突触处的钙受到严格调控。我们将使用光学、电生理和免疫细胞化学手段在AIM 2中研究这种可能性。Otoferlin是一种参与调控囊泡融合、运输和重摄取的独特蛋白质。我们推测，通过CaM Kinase II的磷酸化或泛素化对该蛋白进行的生化修饰负责靶向这些不同的功能。我们将使用电生理、光学、分子和免疫细胞化学技术来研究这一假说。

 DESCRIPTION (provided by applicant): All auditory information received by the central nervous system is transferred across the inner hair cell - afferent fiber synapse. Dysfunction of this synapse due to aging, noise or genetic disorders reduces hearing dramatically. This remarkable synapse must transfer intensity and timing information faithfully for long periods of time. Ribbon synapses, like those in hair cells, are typically found in cells that use graded receptor potentials and need to have sustained release. The hair cell ribbon synapse is more robust than most, releasing at higher rates and sustaining release for longer periods of time. In order for synapses to operate in this manner they must have a robust means of recycling and replenishing vesicles to synaptic release sites. We have identified a nonlinear calcium dependent capacitance change that we hypothesize is a reflection of vesicle recruitment and replenishment. Our first major goal (Aim 1) is to determine whether this nonlinear change is in fact synaptically based and we will do that using paired recordings to identify a postsynaptic response, using a new glutamate sensor to determine if glutamate release mimics the capacitance response and by inactivating ribbons optically to see if we can ablate this nonlinear component. We have also identified a calcium induced calcium release process (CICR) associated with the nonlinear capacitance change. We are hypothesizing that vesicle trafficking is calcium dependent and that CICR is used as a means of ensuring continual vesicle supply while calcium at the synapse is tightly regulated. We will use optical, electrophysiological and immunocytochemical tools to investigate this possibility in aim 2. Otoferlin is a unique protein implicated to regulate vesicle fusion, trafficking and reuptake. We are postulating that biochemical modification of this protein via phosphorylation by CaM Kinase II or ubiquitination is responsible for targeting these different functions. We will investigate this hypothesis using electrophysiological, optical, molecular and immunocytochemical technologies.