Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Allen Institute for Neural Dynamics, Seattle, WA, USA.
Nat Methods. 2023 Jun;20(6):925-934. doi: 10.1038/s41592-023-01863-6. Epub 2023 May 4.
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.
荧光谷氨酸指示剂 iGluSnFR 可实现具有遗传和分子特异性的神经递质传递成像。然而,现有的 iGluSnFR 变体表现出低的体内信噪比、饱和的激活动力学以及无法定位于突触后密度。通过在细菌、可溶性蛋白和培养神经元中的多测定筛选,我们产生了具有改进的信噪比和动力学的变体。我们开发了表面展示构建体,可改善 iGluSnFR 的纳米级定位到突触后。由此产生的指示剂 iGluSnFR3 表现出快速的非饱和激活动力学,并报告在培养神经元中,与 extrasynaptic 信号相比,突触谷氨酸释放的降低饱和和增加特异性。在小鼠视觉皮层的单个末梢中进行的同时成像和电生理记录表明,iGluSnFR3 瞬态以高特异性报告单个动作电位。在触须感觉皮层第 4 层,我们使用 iGluSnFR3 来表征来自丘脑皮质末梢的触发电传输入以及到 L4 皮层神经元树突棘的前馈和回传输入的不同模式。