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

基本信息

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
项目类别：
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

项目摘要

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.

调节活性区 (AZ) 中单个突触小泡 (SV) 的释放概率 (Pr) 决定突触前对突触强度的贡献被认为是突触强度的动态调节的基础。学习和记忆，因此控制 Pr 的分子机制的基础知识对于学习和记忆至关重要了解这些重要的神经过程。果蝇神经肌肉接头（NMJ）是一种广泛使用的神经肌肉接头。由于其可访问性和遗传工具包，用于研究 Pr 的模型突触在该系统中具有两个谷氨酸能运动。具有不同兴奋性和突触特性的神经元类别（称为 1b 和 1s）各自形成数百个 AZ 在每个 AZ 处，结构蛋白将 SV 定位在电压门控钙附近。 SV 膜上的钙敏感蛋白检测到钙流入，从而触发 SV 释放。然而，钙一旦进入细胞内也会很快被缓冲，使得 SV 和钙之间的距离最近的研究表明，Pr 的不同渠道是异质的。 AZ 由 1b 和 1s 组成，相邻 AZ 之间的差异可能高达 50 倍。这种异质性尚未得到充分证实，越来越多的证据表明结构 AZ 蛋白发挥着作用。通过调节 SV 相对于钙通道的位置，在调节 Pr 中发挥重要作用。发射损耗 (STED) 成像显示 AZ 结构蛋白的纳米级形态差异很大跨各个 AZ，表明 AZ 形态的异质性可以解释 Pr 的异质性此外，最近的数据表明平均 AZ 形态和 Pr 之间存在显着差异。由 1b 和 1s 形成的 AZ，表明这些神经元之间的基因表达差异可能是控制全细胞 AZ 形态的单细胞 RNA 测序表明 Toll-6 是差异最大的细胞之一。 1b和1s神经元之间表达的基因，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 AZ 特性之间的潜在差异为了确定 AZ 形态与 1b 和 1s 神经元之间的 Pr 的相关性，一种新颖的方法。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 将用于研究外源钙缓冲液。形态学在确定 SV 和钙通道之间的纳米域距离耦合中的重要性。确定 Toll-6 如何影响全细胞 AZ 形态以及 1b 和 1s 神经元中的 Pr、Toll-6 表达水平该项目将在 Troy Littleton 博士的实验室中进行。麻省理工学院 (MIT) 的 Picower 学习与记忆研究所 (PILM)。利特尔顿博士的实验室提供必要的设备以及 STED 显微镜和相关培训电生理学将由 Littleton 博士和 MIT 和 PILM 的高级实验室成员进行。优秀的科学和专业发展资源和机会。