Effect of Nanoscale Active Zone Morphology on Synaptic Vesicle Release Probability

调节单个突触小泡(SVs)从活动区(AZs)释放的概率(Pr)决定 突触前对突触强度的贡献。突触强度的动态调节被认为是基础 学习和记忆，所以控制PR的分子机制的基础知识对于 了解这些重要的神经元过程。果蝇神经肌肉接头(NMJ)是一种应用广泛的 模型突触由于其可访问性和遗传工具包而用于研究PR。在这个系统中，两个谷氨酸能马达 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 到个体的体壁肌肉上。在每个AZ，结构蛋白将SVS定位在电压门控钙离子附近 频道。钙离子内流是由SV膜上的钙敏感蛋白检测到的，从而触发SV释放。 然而，一旦进入细胞，钙也会迅速被缓冲，从而使SVS和钙之间的距离变得更远 渠道对决定公关极为重要。最近的研究表明，公关在不同的领域具有异质性 AZs由1b和1s组成，相邻AZs之间的差异可能高达50倍。然而，来源是 这种异质性还没有完全被发现。越来越多的证据表明，结构性AZ蛋白在 通过调节SVS相对于钙通道的位置，在调节Pr中发挥重要作用。超分辨率激发 发射耗竭(STED)成像显示AZ结构蛋白在纳米级形态上差异很大 表明AZ形态的异质性可以解释Pr在不同区域的异质性 AZS。此外，最近的数据表明，平均AZ形态和Pr在 AZs由1b和1s形成，这表明这些神经元之间的基因表达差异可能会 控制细胞范围的AZ形态。单细胞RNA测序表明，Toll-6是差异最大的 在1b和1s神经元之间表达基因，初步数据表明它能够影响AZ 形态和电生理学使其可能是AZ特性的潜在差异的潜在原因 这些神经元类。为了确定AZ形态如何与1b和1s神经元的PR相关，一项新的 基因编码的荧光释放传感器和STED显微镜将用于果蝇NMJ 比较单个AZ的纳米尺度AZ形态和Pr。外源性钙缓冲液将被用来研究 形态在确定SVS和钙通道之间的纳米结构域距离耦合中的重要性。 确定Toll-6对1b和1s神经元全细胞AZ形态和PR、Toll-6表达水平的影响 都会被单独的基因操纵。该项目将在特洛伊·利特尔顿博士的实验室中进行 麻省理工学院皮考尔学习和记忆研究所(PILM)。全 利特尔顿博士的实验室提供了必要的设备，并对STED显微镜和 电生理学将由Littleton博士和高级实验室成员进行。麻省理工学院和PILM还提供 优秀的科学和专业发展资源和机遇。

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.