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”， 视觉所需的信息。双极单元可以用信号通知对比度的变化，同时提供对比度的模拟读出。 通过改变神经递质释放速率（NTR）来调节亮度。为了维持这种能力，双极细胞必须 具有对释放速率的动态控制和释放准备好的囊泡到融合位点的有效募集。 然而，控制NTR的Ca 2+信号的时空特性，以及控制NTR的分子实体， 在维持动力学上不同的NTR组分中，Ca 2+信号和囊泡动力学之间的相互作用仍然存在 不太了解。长期目标是揭示视网膜带状突触中Ca 2+信号的调节 在发育、正常成年和疾病期间。在这一目标范围内，本提案的总体目标是 确定控制NTR的动力学不同池的Ca 2+信号的时空特性， 在双极细胞带中，局部Ca 2+信号在控制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的动力学不同组分的Ca 2+结构域是 不同之处在于，带状物本身增加了一个额外的隔间，负责空间隔离， 动力学上不同的突触囊泡，以及感知Ca 2+浓度和/或 改变Ca 2+信号。该假设是基于在申请人的实验室中使用新的方法获得的初步数据。 技术开发用于评估交通的单一突触囊泡在丝带，同时 测量[Ca 2 +]的潜在变化，所有这些都具有毫秒级的时间精度。这一假设将得到检验 通过使用最先进的荧光成像、电压钳 电生理学、计算建模、单个生理学鉴定的细胞的电子显微镜，以及 药理学工具：1）揭示决定钙离子时空特性的机制 控制动力学上不同的神经递质释放池的信号;和2）确定 维持动力学上不同的组分所需的局部钙信号和囊泡补充 作为模型系统的视杆双极细胞带状突触的NTR。Ca 2+信号传导的失调是一个关键的早期- 年龄相关性视网膜变性、青光眼、糖尿病和视神经变性分期过程 神经病从研究双极细胞突触传递中的Ca 2+动力学中获得的知识将允许 我们来确定局部Ca 2+稳态的缺陷是否是未来疾病的前奏。生成的数据 从这个建议将有一个广泛的影响，超出了我们的具体调查杆双极细胞， 将适用于位于视觉系统内外的类似带状突触， 感觉信息的各个方面，更广泛地说，是整个中枢神经系统的突触， 带状突触的CAZ与常规突触共享许多分子成分。

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.