Molecular determinants of synaptic diversity at the nanoscale

-摘要- 突触是神经系统中交流的基本单位，是可靠的突触 信息传递是学习、记忆和感觉适应等关键神经系统过程的中心。 此外，由于突触功能障碍，可能会发展成许多神经疾病。而当 突触传递的电生理研究已经存在很长一段时间了，直到最近 光学量子分析(OQA)工具的发展使得将形态和结构联系起来成为可能 元素对单个突触的传输特性的影响。使用OQA的研究揭示了一个更大的 突触传递的多样性，甚至是相邻突触之间的差异，比之前认为的要多。《降临》 更高分辨率的OQA，例如我们实验室开发的称为“QuaSOR”的OQA，以及超分辨率结构 成像方法开辟了一个令人兴奋的前沿，能够研究神经活动是如何形成的(以及 突触多样性，决定这种多样性的分子决定因素和机制是什么？ 这些分子决定因素是如何塑造突触多样性的。 通过使用OQA，我们已经证明了突触多样性，通过突触强度的差异来衡量(即， 动作电位诱发传递的概率；Pr)在单个 神经元与单个靶细胞的突触。这种高度的异质性将我们引向中心假说 我的论点是，突触的强度是由非常精确的关键蛋白质的局部分布决定的。为了测试这一点 假设I将使用谷氨酸能突触-果蝇幼虫神经肌肉连接的模型 (NMJ)-我将在体内并行研究数百个突触，并解决突触的异质性 在单一突触分辨率(50-100 nm)下，从功能和结构两个角度观察。对于我的论文，我是 接受突触生理学、苍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。在我论文的目标1中，我将建立体内单个功能突触的异质性程度 使用QuaSOR的突触。具体地说，我已经完成了初步实验，调查了 基础突触异质性和处理突触基础强度(基础PR)和 突触适应更高的频率。通过超分辨率结构成像实验提出 对于目标2，我将研究突触上的一些关键蛋白质，并确定它是否是局部数量， 它们之间的相对丰度(比率)，和/或突触内形成突触的纳米定位 多样性。最后，对于目标3，通过慢性和急性操纵，我将确定UNC-13a在 塑造了这种多样性。通过超分辨率结构成像和功能成像的结合，这 拟议中的工作将产生对可能塑造突触多样性的分子决定因素的迫切需要的洞察。

-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.