An excess of the excitatory neurotransmitter, glutamate, in the synaptic cleft during hypoxia/ischemia provokes development of neurotoxicity and originates from the reversal of Na+-dependent glutamate transporters located in the plasma membrane of presynaptic brain nerve terminals. Here, we have optimized an electrochemical glutamate biosensor using glutamate oxidase and developed a biosensor-based methodological approach for analysis of rates of tonic, exocytotic and transporter-mediated glutamate release from isolated rat brain nerve terminals (synaptosomes). Changes in the extracellular glutamate concentrations from 11.5 ± 0.9 to 11.7 ± 0.9 μΜ for 6 min reflected a low tonic release of endogenous glutamate from nerve terminals. Depolarization-induced exocytotic release of endogenous glutamate was equal to 7.5 ± 1.0 μΜ and transporter reversal was 8.0 ± 1.0 μΜ for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[14C]glutamate, spectrofluorimetric glutamate dehydrogenase and amino acid analyzer assays. The blood plasma glutamate concentration was also tested, and reliability of the biosensor measurements was confirmed by glutamate dehydrogenase assay. Therefore, the biosensor-based approach for accurate monitoring rates of tonic, exocytotic and transporter-mediated release of glutamate in nerve terminals was developed and its adequacy was confirmed by independent analytical methods. The biosensor measurements provided precise data on changes in the concentrations of endogenous glutamate in nerve terminals in response to stimulation. We consider that the glutamate biosensor-based approach can be applied in clinics for neuromonitoring glutamate-related parameters in brain samples, liquids and blood plasma in stroke, brain trauma, therapeutic hypothermia treatment, etc., and also in laboratory work to record glutamate release and uptake kinetics in nerve terminals.

中文翻译：

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一种用于监测脑神经末梢和血浆中谷氨酸释放的电流型谷氨酸生物传感器

缺氧/缺血期间突触间隙中过量的兴奋性神经递质谷氨酸盐会引发神经毒性的发展，并源于位于突触前脑神经末梢质膜中的 Na+ 依赖性谷氨酸盐转运蛋白的逆转。在这里，我们使用谷氨酸氧化酶优化了电化学谷氨酸生物传感器，并开发了一种基于生物传感器的方法学方法，用于分析离体大鼠脑神经末梢（突触体）的强直、胞吐和转运蛋白介导的谷氨酸释放率。细胞外谷氨酸浓度从 11.5 ± 0.9 到 11.7 ± 0.9 μM 持续 6 分钟的变化反映了内源性谷氨酸从神经末梢的低张力释放。去极化诱导的内源性谷氨酸的胞吐释放等于 7.5 ± 1。0 μM 和转运蛋白逆转为 8.0 ± 1.0 μM，持续 6 分钟。生物传感器数据与使用放射性标记的 L-[ 14 C] 谷氨酸、分光荧光谷氨酸脱氢酶和氨基酸分析仪测定获得的结果很好地相关。还测试了血浆谷氨酸浓度，并且通过谷氨酸脱氢酶测定证实了生物传感器测量的可靠性。因此，开发了一种基于生物传感器的方法，用于准确监测神经末梢中强直、胞吐和转运蛋白介导的谷氨酸盐释放率，并通过独立的分析方法证实了其充分性。生物传感器测量提供了神经末梢内源性谷氨酸浓度随刺激变化的精确数据。