Imaging triheteromeric NMDAR distribution and trafficking

NMDA型谷氨酸受体（NMDAR）的激活驱动信号传导和神经元可塑性， 大脑回路的连接和学习。然而，NMDAR过度激活可引发神经元细胞死亡， 神经变性和NMDAR功能减退是精神分裂症病因学的主要模型， 其他认知障碍。此外，NMDAR的突触下组织影响它们的可能性。 突触外受体被认为在细胞死亡中起关键作用 和基因表达。因此，神经元利用多种机制来控制神经元上NMDAR的丰度。 细胞表面，尤其是突触。这些机制是复杂的，部分原因是NMDAR是 四聚体，由两个专性GluN1亚基和两个通常来自GluN2家族的亚基形成。值得注意的是， 具有不同GluN2组成的NMDAR显示不同的生物物理特性，并且GluN2亚基 还指导不同的蛋白质相互作用和信号传导。因此，NMDAR的不同亚型极大地驱动 不同形式的生理可塑性，并与不同的疾病有关。因此，确定神经元如何 控制突触中特定NMDAR亚型的丰度一直是神经科学的长期目标。 不幸的是，我们对这些机制的理解受到了严重的限制，因为我们无法想象这些机制。 在神经元中的NMDAR的关键类别中，含有GluN1和两种不同的三聚体受体， GluN2亚基（最常见的是GluN2A+GluN2B）。三异聚体受体被认为是大多数 NMDAR在成人大脑中的表达，但目前还没有工具将其与神经元中的其他NMDAR亚型区分开来。 因此，它们在神经元中的分布仍然是个谜，控制它们的亚细胞机制 贩运人口几乎完全不为人知。为了克服这一点，我们在这里引入一个新的工具来可视化 神经元中的三异聚体NMDAR。我们的策略是基于双分子互补， GluN2A和GluN2B与修饰的荧光蛋白的两个部分互补以产生荧光 仅当组装含有GluN2A和GluN2B亚基的NMDAR时（分裂标记的NMDAR）。 初步数据表明，分裂标记受体在神经元内正常运输，并强烈积累 在突触内，全细胞记录表明，它们被谷氨酸激活， 标签的存在。从这些结果出发，我们建议开发和验证几个版本的 可用于不同实验的分裂标记的三异聚体NMDAR。我们将使用共焦和超分辨率 成像以提供神经元中三异聚体NMDAR分布的第一图。最后，由于 突触中的NMDAR周转对于确定突触强度和可塑性至关重要，我们将使用FRAP和 单分子追踪以提供三异聚体NMDAR的突触交换的第一测量。这 这项工作将填补我们对NMDAR知识的长期空白，并为研究健康神经元和疾病模型中三聚体NMDAR贩运的其他方面奠定必要的基础。

Activation of NMDA-type glutamate receptors (NMDARs) drives signaling and neuronal plasticity that mediates brain circuit wiring and learning. However, NMDAR overactivation can trigger neuronal cell death and is linked to neurodegeneration, and hypofunction of NMDARs is a leading model of the etiology of schizophrenia and other cognitive disorders. Further, the subsynaptic organization of NMDARs influences the probability of their activation during synaptic transmission, and extrasynaptic receptors are thought to play critical roles in cell death and gene expression. Thus, neurons utilize a variety of mechanisms to control the abundance of NMDARs on the cell surface and particularly in synapses. These mechanisms are complex in part because NMDARs are tetramers, formed from two obligatory GluN1 subunits and two subunits typically from the GluN2 family. Notably, NMDARs with different GluN2 compositions display different biophysical characteristics, and the GluN2 subunits also guide different protein interactions and signaling. Accordingly, different subtypes of NMDARs drive vastly different forms of physiological plasticity and are linked to different disorders. Thus, identifying how neurons control abundance of specific NMDAR subtypes in synapses has been a longstanding goal in neuroscience. Unfortunately, our understanding of these mechanisms has been crucially restricted by inability to visualize one of the key classes of NMDARs in neurons, the triheteromeric receptors, which contain GluN1 and two different GluN2 subunits (most commonly GluN2A+GluN2B). Triheteromeric receptors are thought to be the majority of NMDARs in adult brain, but there are as yet no tools to distinguish them from other NMDAR subtypes in neurons. Thus, their distribution with neurons remains mysterious, and the mechanisms controlling their subcellular trafficking remain almost totally unknown. To overcome this, we here introduce a new tool to visualize triheteromeric NMDARs in neurons. Our strategy is based on bimolecular complementation, and we tagged GluN2A and GluN2B with two parts of a modified fluorescent protein that complement to produce fluorescence only when an NMDAR is assembled containing both the GluN2A and GluN2B subunits (split-tagged NMDARs). Preliminary data demonstrate that split-tagged receptors traffic normally within neurons and accumulate strongly within synapses, and whole-cell recordings demonstrate that their activation by glutamate is unaltered by the presence of the tags. Proceeding from these results, we propose to develop and validate several versions of split-tagged triheteromeric NMDARs useful for different experiments. We will use confocal and super-resolution imaging to provide the first maps of triheteromeric NMDAR distribution in neurons. Finally, because the rate of NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, we will use FRAP and single-molecule tracking to provide the first measures of synaptic exchange of triheteromeric NMDARs. This work will fill longstanding gaps in our knowledge of NMDARs and lay necessary groundwork for investigation of other aspects of triheteromeric NMDAR trafficking in healthy neurons and disease models.