Dynamics of calcium signals control neurotransmitter release in retinal ribbon synapses

视网膜双极细胞是脊椎动物视觉系统的第一个“投射神经元”，它传递所有的 视觉所需的信息。双极电池可以发出信号变化的对比信号，同时提供模拟读出 通过改变神经递质释放速率(NTR)来调节亮度。为了保持这种能力，双极细胞 必须动态控制释放速率和有效地招募可释放的囊泡以进行融合 网站。然而，控制NTR的钙信号的时空特性，以及控制NTR的分子实体 在维持动态不同的NTR中控制钙信号和囊泡动力学之间的相互作用 人们对其组成部分仍知之甚少。长期目标是揭示视网膜中钙信号的调节。 发育、正常成年期和疾病期间的带状突触。在这一目标范围内， 这一提议是为了确定控制动态不同池的钙信号的时空特性 以及局部钙信号在维持神经传递的囊泡动力学中的作用 双极细胞带突触。中心假设是，钙离子结构域在动力学上是截然不同的 NTR的组件不同，因为色带本身增加了一个额外的隔间，负责 运动学上不同的突触小泡的空间分离及其潜在的分子靶点 钙离子浓度和/或改变钙离子信号。这一假设是基于在 申请人的实验室使用新技术评估单个突触小泡的流量 同时测量潜在的[Ca~(2+)]变化的同时，都以毫秒级的时间 精确度。这一假设将通过追求两个具体目标来检验，使用最新技术的汇合 荧光成像，电压钳电生理学，计算建模，电子显微镜 单个生理识别的细胞和药理工具：1)揭示决定 控制神经递质动态释放的钙信号的时空特性 以及2)确定局部钙信号和囊泡补充之间的相互作用，即 以视杆双极细胞带突触为模型维持NTR运动学不同成分所需 系统。钙信号转导失调是老年性神经退行性变的一个关键早期过程 视网膜变性、青光眼、糖尿病和视神经病变。从研究钙离子中获得的知识 双极细胞突触传递的动力学将使我们能够确定局部钙稳态缺陷 是未来疾病的前奏。此提案产生的数据将产生广泛的影响 超越我们对视杆双极细胞的具体研究，并将适用于类似的带状突触定位 在视觉系统内部和外部，并编码感觉信息的不同方面。更广泛地说，我们的 数据将与整个中枢神经系统的突触相关，因为带状突触的CAZ 与传统的突触有许多相同的分子成分。

Retinal bipolar cells are the first 'projection neurons' of the vertebrate visual system and transmit all of the information needed for vision. Bipolar cells can signal change in contrast while providing an analog read-out of luminance via changing the rate of neurotransmitter release (NTR). To maintain this ability, the bipolar cells must have dynamic control over release rate and the efficient recruitment of release-ready vesicles to fusion sites. However, the spatiotemporal properties of Ca2+ signals that control NTR, and the molecular entities that control the interplay between Ca2+ signal and vesicle dynamics in sustaining kinetically distinct NTR components remain poorly understood. The long-term goal is to unveil the regulation of Ca2+ signaling in retinal ribbon synapses during development, normal adulthood, and disease. Within this goal, the overall objective of this proposal is to determine the spatiotemporal properties of Ca2+ signals that control kinetically distinct pools of NTR and the role of local Ca2+ signals in governing vesicle dynamics that sustain neurotransmission in bipolar cell ribbon synapses. The central hypothesis is that Ca2+ domains governing kinetically distinct components of NTR are different because the ribbon itself adds an additional compartment responsible for spatial segregation of kinetically different synaptic vesicles and the underlying molecular targets that sense Ca2+ concentration and/or alter Ca2+ signals. This hypothesis is based on preliminary data, acquired in applicant’s laboratory using novel techniques developed for evaluating the traffic of single synaptic vesicles at ribbons while simultaneously measuring the underlying changes in [Ca2+], all with millisecond temporal precision. This hypothesis will be tested by pursuing two specific aims using a confluence of state-of-the-art fluorescence imaging, voltage-clamp electrophysiology, computational modeling, electron microscopy of individual physiologically identified cells, and pharmacological tools: 1) Reveal the mechanisms that determine the spatiotemporal properties of calcium signals which control kinetically distinct neurotransmitter release pools; and 2) Determine the interplay between local calcium signaling and vesicle replenishment that is required for sustaining kinetically distinct components of NTR in rod bipolar cell ribbon synapses as a model system. Dysregulation of Ca2+ signaling is a key early–stage process of neurodegeneration in age-related retinal degenerations, glaucoma, diabetic, and optic neuropathies. The knowledge gained from studying Ca2+ dynamics in bipolar cell synaptic transmission will allow us to determine if defects with local Ca2+ homeostasis are a prelude to disease in the future. Data generated from this proposal will have a broad impact that extends beyond our specific investigation of rod bipolar cells and will be applicable to similar ribbon synapses located within and outside the visual system and encoding distinct aspects of sensory information. More widely, our data will be relevant to synapses throughout the central nervous system because the CAZ of ribbon synapses shares many molecular components with conventional synapses.