Molecular determinants of synaptic diversity at the nanoscale

- 摘要- 突触是神经系统中进行通讯的基本单位， 传递是关键神经系统过程如学习、记忆和感觉适应的中枢。 此外，由于突触的功能障碍，许多神经系统疾病可能会发展。而 突触传递的电生理学研究已经存在了很长一段时间，只有最近才有 光学量子分析（OQA）工具的发展使得将形态和结构相关联成为可能。 元件到单个突触的传输特性。使用OQA的研究显示， 突触传递的多样性，甚至在相邻的突触之间，比以前认为的要多。问世 更高分辨率的OQA，如我们实验室开发的“OQSOR”，和超分辨率结构 成像方法开辟了一个令人兴奋的前沿，能够研究神经活动如何形成（并 突触的多样性，什么是分子决定因素和机制，设置这种多样性， 这些分子决定因素如何塑造突触多样性。 通过使用OQA，我们已经证明了突触多样性，如通过突触强度的差异（即， 动作电位诱发传输的概率; Pr）在单个 神经元与单个靶细胞形成突触。这种高度的异质性将我们引向中心假设 那就是突触的强度是由一个非常精确的，关键蛋白质的局部分布决定的。为了验证这一 假设我将使用模型神经元突触-果蝇幼虫神经肌肉接头 （NMJ）-在那里我将研究数百个突触在平行，在体内，并解决突触异质性 从功能和结构两个角度来看，在单个突触分辨率（50- 100 nm）。为了我的论文， 接受突触生理学、果蝇遗传学和高级超分辨率功能/结构成像方面的培训 和分析在我的论文的目标1中，我将建立在体内单一的功能性突触异质性的程度， 突触使用的神经元。具体来说，我已经完成了初步实验，调查 基础突触异质性和解决突触的基础强度（基础Pr）和 突触对更高频率的适应。通过超分辨率结构成像实验提出 对于目标2，我将研究突触中的一些关键蛋白质，并确定它是否是局部量， 它们之间的相对丰度（比率）和/或形成突触的突触内的纳米定位 多样性最后，对于目标3，通过慢性和急性操作，我将确定Unc-13 a在以下方面的作用： 塑造这种多样性。通过超分辨率结构和功能成像的结合， 这项工作将使我们对可能形成突触多样性的分子决定因素有更深入的了解。

-Abstract - Synapses are the fundamental units of communication in the nervous system and reliable synaptic transmission is central to key nervous system processes such as learning, memory, and sensory adaptation. Moreover, due to dysfunctions at the synapse, many neurological diseases may develop. While electrophysiological studies of synaptic transmission have been around for a long time, only the recent development of optical quantal analysis (OQA) tools has made possible to correlate morphological and structural elements to transmission properties of individual synapses. Studies using OQA have revealed a much larger diversity in synaptic transmission, even among neighboring synapses, than was previously thought. The advent of higher-resolution OQA, such as the one developed in our lab called “QuaSOR”, and super-resolution structural imaging methods opens up an exciting frontier of being able to investigate how neural activity shapes (and is shaped by) synaptic diversity, what are the molecular determinants and mechanisms the set this diversity, and how do these molecular determinants shape synaptic diversity. By using OQA, we've shown that synaptic diversity, as measured by difference in synaptic strength (i.e., probability of action potential evoked transmission; Pr ) is extremely heterogeneous (Pr: 0.01–0.5) within a single neuron synapsing onto a single target cell. This high degree of heterogeneity leads us to the central hypothesis of my thesis which is that synaptic strength is set by a very precise, local distribution of key proteins. To test this hypothesis I will use the model glutamatergic synapse–Drosophila melanogaster larval neuromuscular junction (NMJ)– where I will investigate hundreds of synapses in parallel, in vivo, and address synaptic heterogeneity from both functional and structural perspectives at single synapse resolution (50-100nm). For my thesis I am being trained in synaptic physiology, fly genetics, and advanced super-resolution functional/structural imaging and analysis. In Aim 1 of my thesis, I will establish the degree of functional synaptic heterogeneity in vivo single synapses using QuaSOR. Specifically, I have completed the preliminary experiments investigating the extent of basal synaptic heterogeneity and addressing the relationship between basal strength of synapses (basal Pr) and synaptic adaptation to higher frequencies. Through super-resolution structural imaging experiments proposed for Aim 2, I will investigate some of the key proteins at the synapse and determine whether it's the local quantities, the relative abundance between them (ratios), and/or the nanolocalization within the synapse that shape synaptic diversity. Finally, for Aim 3, through chronic and acute manipulations, I will determine the role of Unc-13a in shaping this diversity. Through the combination of super resolution structural and functional imaging, this proposed work will yield much needed insight into the molecular determinants that may shape synaptic diversity.