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首页> 外文期刊>The European Journal of Neuroscience >Optical glutamate sensor for spatiotemporal analysis of synaptic transmission.
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Optical glutamate sensor for spatiotemporal analysis of synaptic transmission.

机译：光学谷氨酸传感器用于时空分析突触传递。

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Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high-resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS-based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.
机译：影像神经传递有望大大提高我们对突触传递机制和法规的了解。针对成像中枢神经系统中主要的兴奋性神经递质谷氨酸，我们开发了一种新型的光学谷氨酸探针，该探针由α-氨基-3-羟基-5-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体的配体结合域组成谷氨酸受体GluR2亚基和小分子荧光染料。我们预期这种荧光偶联物可能会报告谷氨酸结合引起的蛋白质构象变化后的微环境变化。经过100多种结合物的测试后，我们最终获得了一种名为E（谷氨酸）光学传感器（EOS）的结合物，当结合谷氨酸盐时，其离解常数为148 nm时，荧光强度最大变化了37％。通过将EOS固定在海马神经元培养物的细胞表面上，我们进行突触前激发后突触释放的谷氨酸的原位空间定位。结果表明，单次射击足以获得高分辨率的谷氨酸盐释放图像，表明该技术具有显着的灵敏度。此外，我们监测了佛波酯引起的突触前活动变化的时间过程，并发现突触前调制的异质性。这些结果表明，EOS通常可用于评估突触前调节和可塑性。这种基于EOS的谷氨酸成像方法可用于解决有关CNS中谷氨酸能神经传递的许多基本问题。

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