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，双极电池必须 动态控制释放速率，并有效地将可释放的囊泡招募到融合部位。 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成分方面，钙信号和囊泡动力学之间的相互作用仍然存在 人们对此知之甚少。长期目标是揭示视网膜带状突触中钙信号的调节。 在发育期间，正常的成年期，和疾病。在这样的目标范围内，本提案的总体目标是 确定控制动态不同的NTR池的钙信号的时空特性及其作用 局部钙信号在调控双极细胞带内维持神经传递的囊泡动力学中的作用 突触。中心假设是，控制NTR动态不同组成部分的钙结构域是 不同，因为功能区本身添加了一个额外的隔间，负责 动力学上不同的突触小泡，以及感知钙浓度和/或 改变钙信号。这一假设是基于申请者的实验室使用新奇技术获得的初步数据 用于同时评估带上单个突触小泡的交通的技术 测量[钙]的潜在变化，都是以毫秒的时间精度进行的。这一假设将得到检验。 通过使用最先进的荧光成像技术的融合来实现两个特定目标，电压钳 电生理学，计算模型，单个生理鉴定细胞的电子显微镜，以及 药理学工具：1)揭示决定钙的时空特性的机制 控制动态不同神经递质释放池的信号；以及2)决定 维持不同运动成分所需的局部钙信号和囊泡补充 视杆双极细胞突触中NTR的表达作为模型系统。钙信号的失调是一个关键的早期- 老年性视网膜变性、青光眼、糖尿病和视神经变性的分期过程 神经病。从研究双极细胞突触传递的钙动力学中获得的知识将使 以确定局部钙稳态缺陷是否是未来疾病的前奏。生成的数据 这项提议将产生广泛的影响，不仅限于我们对杆状双极细胞的具体调查，但 将适用于位于视觉系统内外的类似的带状突触，并编码不同 感觉信息的各个方面，更广泛地说，与整个中枢神经系统的突触有关，因为 带状突触的CAZ与传统突触有许多共同的分子成分。

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 such 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 but will be applicable to similar ribbon synapses located within and outside the visual system and encoding distinct aspects of sensory information and, more widely, to synapses throughout the central nervous system because the CAZ of ribbon synapses shares many molecular components with conventional synapses.