Administrative Supplement (Diversity) to Generating functional diversity from molecular homogeneity at glutamatergic synapses

项目摘要 突触是神经系统中基本的通讯单位， 结构和功能的多样性有助于调整和校准信息传递。缺陷 突触的能力，以适当的多样化有助于病因的各种 神经发育、精神和神经变性疾病。一种产生 突触功能的多样性是通过分子异质性，其中不同的 基因在单个突触处表达以实现特定的功能特性。但 虽然很难解决，但越来越清楚的是，突触的显著多样性可以 是由有限的一组分子机器来实现的。原则上，果蝇的神经肌肉 连接（NMJ）是一个独特的强大的模型，以解决如何突触多样性产生给定的 复杂的遗传学、电生理学和成像方法。在这个系统中，两个 不同的运动神经元会聚到共同支配个体肌肉目标， 由强、弱输入共同驱动肌肉收缩的运动回路。但安 不能选择性地将传输与任一输入隔离已经成为 了解这个系统中突触的多样性在这里，我们建议使用一种独特的表达方式， 肉毒杆菌神经毒素（BoNT）选择性地沉默在强或弱突触传递 输入。初步数据表明，虽然每个神经元主要由相同的 活跃区的分子机制，一个核心组成部分，以前认为是功能 普遍存在于所有活动区域，实际上在强与弱的情况下， 突触我们将使用BoNT沉默，超分辨率成像，和最新的钙报告 靶向释放位点，以阐明活性区纳米结构和功能的差异 强突触和弱突触之间的区别我们还将利用CRISPR的新创新 诱变，以剖析八个核心活性区组分的专门功能， vs弱突触。最后，我们将询问这些核心活动区组件是如何 独特地靶向于强与弱突触的调节和重塑， 稳态突触可塑性总之，这些方法将揭示基本的见解， 如何通过可塑性建立和适应性修改突触多样性。 最终，这种理解将阐明异构化的关键机制。 通过有限的分子工具箱能够实现在放热释放位点的功能性质。

PROJECT SUMMARY Synapses are fundamental units of communication in the nervous system, where immense diversity in structure and function serve to tune and calibrate information transfer. Defects in the ability of synapses to properly diversify contribute to the etiology of a variety of neurodevelopmental, psychiatric, and neurodegenerative diseases. One means of generating diversity in synaptic function is through molecular heterogeneity, where combinations of distinct genes are expressed at individual synapses to enable specific functional properties. However, it has become increasingly clear, though difficult to resolve, that remarkable synaptic diversity can be achieved from a limited set of molecular machinery. In principle, the Drosophila neuromuscular junction (NMJ) is a uniquely powerful model to address how synaptic diversity is generated given the sophisticated genetic, electrophysiological, and imaging approaches. In this system, two distinct motor neurons converge to co-innervate individual muscle targets, where transmission from a strong and weak input together drive muscle contraction in the motor circuit. However, an inability to selectively isolate transmission from either input has been a major limitation towards understanding synaptic diversity in this system. Here, we propose to use expression of a unique Botulinum NeuroToxin (BoNT) to selectively silence transmission at strong or weak synaptic inputs. Preliminary data suggests that while each neuron is largely composed of the same molecular machinery at active zones, one core component, previously thought to function universally at all active zones, actually subserves dramatically different roles at strong vs weak synapses. We will use BoNT silencing, super resolution imaging, and the latest calcium reporters targeted to release sites to illuminate differences in active zone nanostructure and function between strong and weak synapses. We will also leverage new innovations in CRISPR mutagenesis to dissect the specialized functions of eight core active zone components at strong vs weak synapses. Finally, we will interrogate how these core active zone components are uniquely targeted for modulation and remodeling at strong vs weak synapses in the context of homeostatic synaptic plasticity. Together, these approaches will unlock fundamental insights into how glutamatergic synaptic diversity is established and adaptively modified through plasticity. Ultimately, this understanding will illuminate key mechanisms through which heterogeneous functional properties at glutamatergic release sites are enabled by a limited molecular toolkit.