Imaging triheteromeric NMDAR distribution and trafficking

NMDA型谷氨酸受体(NMDAR)的激活驱动信号和神经元可塑性，从而介导 大脑回路连接和学习。然而，NMDAR的过度激活可以引发神经细胞死亡，并与 神经退行性变和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的突触下组织影响其 突触传递过程中的激活和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，神经元利用各种机制来控制NMDAR的丰度。 细胞表面，特别是在突触中。这些机制之所以复杂，部分原因是NMDAR 四聚体，由两个必需的GluN1亚基和两个典型的GluN2家族的亚基组成。值得注意的是， 不同GluN2组成的NMDAR表现出不同的生物物理特性，GluN2亚基 也引导不同的蛋白质相互作用和信号传递。因此，不同的NMDAR亚类型会极大地推动 不同形式的生理可塑性与不同的疾病有关。因此，确定神经元是如何 控制突触中特定NMDAR亚型的丰度一直是神经科学的一个长期目标。 不幸的是，我们对这些机制的理解因无法想象一种机制而受到严重限制 在神经元中NMDAR的关键类别中，三异构体受体包含GluN1和两个不同的 GluN2亚基(最常见的是GluN2A+GluN2B)。三异构体受体被认为是大多数 NMDAR在成人大脑中存在，但到目前为止还没有工具将它们与神经元中的其他NMDAR亚型区分开来。 因此，它们在神经元中的分布仍然是个谜，控制它们亚细胞的机制仍然是个谜。 人口贩运几乎仍然是完全未知的。为了克服这一点，我们在这里引入了一种新的可视化工具 神经元中的三异构体NMDAR。我们的战略是基于双分子互补，我们标记了 GluN2A和GluN2B与修饰的荧光蛋白的两部分互补产生荧光 仅当组装的NMDAR同时包含GluN2A和GluN2B亚基(拆分标记的NMDAR)时。 初步数据显示，分裂标记的受体正常地在神经元内运输并强烈积聚 在突触内，全细胞记录表明它们被谷氨酸激活不会被 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离标记的三异构体NMDAR。我们将使用共焦和超分辨率 成像，以提供神经元中三异构体NMDAR分布的第一张地图。最后，因为 突触中NMDAR的转换对于确定突触的强度和可塑性至关重要，我们将使用FRAP和 单分子跟踪提供了三异构体NMDAR突触交换的第一个测量方法。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异构体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.