美国霍华德休斯医学研究所Kaspar Podgorski和Abhi Aggarwal共同合作，近期取得重要工作进展。他们最新研究开发出了用于突触传递成像的具有改善激活动力学和定位的谷氨酸指示剂。相关研究成果2023年5月4日在线发表于《自然—方法学》杂志上。

据介绍，荧光谷氨酸盐指示剂iGluSnFR能够对具有遗传和分子特异性的神经传递进行成像。然而，现有的iGluSnFR变体表现出低的体内信噪比、饱和激活动力学和突触后密度的排斥。

利用细菌、可溶性蛋白和培养神经元的多组分筛选，研究人员获得了具有改善信噪比和动力学的变体。他们开发了表面显示结构，可以改善iGluSnFR在突触后的纳米定位。由此产生的指示剂iGluSnFR3表现出快速的非饱和激活动力学，并报告了与培养神经元中的突触外信号相比，突触谷氨酸释放具有降低的饱和度和增加的特异性。

小鼠视觉皮层单个发作的同时成像和电生理学显示，iGluSnFR3瞬态报告单一动作电位具有高特异性。在振动感觉皮层第4层中，研究人员使用iGluSnFR3表征来自丘脑皮层神经元触摸诱发前馈输入的不同模式，以及L4皮层神经元树突棘的前馈和循环输入。

附：英文原文

Title: Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

Author: Aggarwal, Abhi, Liu, Rui, Chen, Yang, Ralowicz, Amelia J., Bergerson, Samuel J., Tomaska, Filip, Mohar, Boaz, Hanson, Timothy L., Hasseman, Jeremy P., Reep, Daniel, Tsegaye, Getahun, Yao, Pantong, Ji, Xiang, Kloos, Marinus, Walpita, Deepika, Patel, Ronak, Mohr, Manuel A., Tillberg, Paul W., Looger, Loren L., Marvin, Jonathan S., Hoppa, Michael B., Konnerth, Arthur, Kleinfeld, David, Schreiter, Eric R., Podgorski, Kaspar

Issue&Volume: 2023-05-04

Abstract: The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR’s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

DOI: 10.1038/s41592-023-01863-6

Source: https://www.nature.com/articles/s41592-023-01863-6