Effect of Nanoscale Active Zone Morphology on Synaptic Vesicle Release Probability

纳米级活性区形态对突触小泡释放概率的影响

• 批准号: 10251901
• 负责人: Andres Crane
• 金额: $ 4.6万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251901
• 关键词: Affect; Age; Biological Models; Buffers; Calcium; Calcium Channel; Cells; Communication; Confocal Microscopy; Coupling; Data; Drosophila genus; Electrophysiology (science); Equipment; Gene Expression; Genes; Genetic; Genetic Transcription; Glutamates; Heterogeneity; Image; Individual; Institutes; Knock-out; Knowledge; Learning; Massachusetts; Membrane; Memory; Microscopy; Microtubules; Modeling; Molecular; Morphology; Motor Neurons; Muscle; Neuromuscular Junction; Neurons; Optics; Output; Play; Positioning Attribute; Probability; Process; Property; Proteins; RNA; Regulation; Reporting; Research; Resolution; Resource Development; Role; Shapes; Signal Transduction; Site; Source; Structural Protein; Structure; Synapses; Synaptic Vesicles; Synaptic plasticity; System; Technology; Training; Variant; differential expression; experimental study; genetic manipulation; imaging approach; insight; member; muscle form; mutant; nanoscale; neurotransmitter release; novel; organizational structure; postsynaptic; presynaptic; programs; quantum; receptor; sensor; single-cell RNA sequencing; tool; vesicular release; voltage

Regulation of the release probability (Pr) of single synaptic vesicles (SVs) from active zones (AZs) determines the presynaptic contribution to synaptic strength. Dynamic regulation of synaptic strength is thought to underlie learning and memory, so fundamental knowledge of the molecular mechanisms governing Pr is critical to understanding these vital neuronal processes. The Drosophila neuromuscular junction (NMJ) is a widely-used model synapse for studying Pr due to its accessibility and genetic toolkit. In this system, two glutamatergic motor neuron classes (termed 1b and 1s) with distinct excitability and synaptic properties each form hundreds of AZs onto individual body wall muscles. At each AZ, structural proteins position SVs near voltage-gated calcium channels. Calcium influx is detected by calcium-sensing proteins on SV membranes, triggering SV release. However, calcium is also quickly buffered once inside the cell, making the distance between SVs and calcium channels extremely important for determining Pr. Recent studies have shown that Pr is heterogeneous across AZs formed by 1b and 1s and can vary as much as 50-fold between neighboring AZs. However, the sources of this heterogeneity have not been fully identified. Accumulating evidence suggests that structural AZ proteins play a large role in regulating Pr by regulating position of SVs relative to calcium channels. Super-resolution stimulated emission depletion (STED) imaging reveals that AZ structural proteins vary widely in their nanoscale morphology across individual AZs, suggesting that heterogeneity in AZ morphology could explain heterogeneity in Pr across AZs. Additionally, recent data indicates that average AZ morphology and Pr are significantly different between AZs formed by 1b and 1s, suggesting that a difference in gene expression between these neurons is likely to control cell-wide AZ morphology. Single-cell RNA sequencing indicates that Toll-6 is one of the most differentially expressed genes between 1b and 1s neurons, and preliminary data indicates that it is capable of affecting AZ morphology and electrophysiology making it a likely candidate underlying difference in AZ properties between these neuronal classes. To determine how AZ morphology correlates with Pr across 1b and 1s neurons, a novel genetically encoded fluorescent release sensor and STED microscopy will be used on Drosophila NMJs to compare nanoscale AZ morphology and Pr at single AZs. An exogenous calcium buffer will be used to investigate importance of morphology in determining nanodomain distance coupling between SVs and calcium channels. To determine how Toll-6 affects cell-wide AZ morphology and Pr, Toll-6 expression levels in 1b and 1s neurons will be individually genetically manipulated. This project will be carried out in the lab of Dr. Troy Littleton in the Picower Institute for Learning and Memory (PILM) at the Massachusetts Institute of Technology (MIT). All necessary equipment is available through Dr. Littleton’s lab and appropriate training for STED microscopy and electrophysiology will be carried out by Dr. Littleton and senior lab members. MIT and PILM additionally provide excellent scientific and professional development resources and opportunities.

单个突触囊泡（SV）从活动区（AZ）释放概率（Pr）的调节决定了 突触前对突触强度的贡献。突触强度的动态调节被认为是 学习和记忆，所以对Pr的分子机制的基础知识是至关重要的 了解这些重要的神经元过程。果蝇神经肌肉接头（NMJ）是一种广泛使用的 模型突触研究Pr由于其可访问性和遗传工具包。在该系统中，两个电磁能电机 具有不同兴奋性和突触特性的神经元类（称为1b和1 s）各自形成数百个AZ 贴到个别的体壁肌肉上。在每个AZ，结构蛋白将SV定位在电压门控钙附近 渠道SV膜上的钙敏感蛋白检测到钙内流，触发SV释放。 然而，一旦进入细胞，钙也会迅速缓冲，使SV和钙之间的距离 最近的研究表明，Pr是异质的， 由1b和1 s形成的AZ，相邻AZ之间的变化高达50倍。然而， 这种异质性还没有被完全确定。越来越多的证据表明，结构AZ蛋白发挥作用， 通过调节SV相对于钙通道的位置在调节Pr中起重要作用。超分辨率受激 发射耗尽（STED）成像显示AZ结构蛋白在其纳米级形态上差异很大 在个体AZ中，表明AZ形态的异质性可以解释Pr的异质性， 阿兹此外，最近的数据表明，平均AZ形态和Pr在 AZs由1b和1 s形成，这表明这些神经元之间基因表达的差异可能会导致神经元的死亡。 对照全细胞AZ形态。单细胞RNA测序表明，Toll-6是一个最具差异的 在1b和1 s神经元之间表达基因，初步数据表明它能够影响AZ 形态学和电生理学使其成为可能的候选者，潜在的AZ性质差异， 这些神经元类。为了确定AZ形态如何与1b和1 s神经元的Pr相关，一种新的 基因编码荧光释放传感器和STED显微镜将用于果蝇NMJ， 比较纳米级AZ形态和单个AZ处的Pr。外源性钙缓冲液将用于研究 形态学在确定SV和钙通道之间的纳米畴距离偶联中的重要性。 确定Toll-6如何影响1b和1 s神经元中全细胞AZ形态以及Pr、Toll-6表达水平 都将被单独基因操控该项目将在特洛伊利特尔顿博士的实验室进行， 马萨诸塞州理工学院（MIT）的皮考尔学习与记忆研究所（PILM）。所有 利特尔顿博士的实验室提供了必要的设备，并进行了适当的STED显微镜培训， 电生理学将由利特尔顿博士和高级实验室成员进行。MIT和PILM还提供 优秀的科学和专业发展资源和机会。