Chemical labelling for visualizing native AMPA receptors in live neurons.
Chemical labelling for visualizing native AMPA receptors in live neurons.
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DOI：
10.1038/ncomms14850
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发表时间：
2017-04-07
影响因子：
16.6
通讯作者：
Hamachi I
中科院分区：
文献类型：
--
作者：
Wakayama S;Kiyonaka S;Arai I;Kakegawa W;Matsuda S;Ibata K;Nemoto YL;Kusumi A;Yuzaki M;Hamachi I
神经递质受体的位置和数量在突触后部位受到动态调节。然而，目前可用的可视化受体运输的方法需要将基因工程受体引入神经元，这可能会破坏原始受体的正常功能和处理。在这里，我们报告了一个强大的方法来可视化天然α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）型谷氨酸受体（AMPAR），这是必不可少的认知功能，没有任何遗传操作。这是基于由选择性配体-蛋白质识别驱动的共价化学标记策略，以使用化学AMPAR修饰（CAM）试剂将小荧光团拴系到AMPAR。CAM试剂的高穿透性使得能够在不影响受体功能的情况下可视化脑组织深处的天然AMPAR。此外，CAM试剂用于表征内源性AMPAR在培养的神经元和海马切片中的扩散动力学。这种方法将有助于阐明AMPAR贩运参与各种神经精神和神经发育障碍。可视化神经递质受体流量是了解大脑功能的基础，但目前的成像方法可能会损害这一过程。在这里，作者合成了小的荧光探针来标记细胞培养物和脑组织中的内源性谷氨酸受体，而不影响它们的功能。
The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders. Visualizing neurotransmitter receptor traffic is fundamental to understanding brain functions, but the current imaging methods can compromise the process. Here, the authors synthesize small fluorescent probes to label endogenous glutamate receptors in cell cultures and brain tissues, without affecting their function.