In this study for the first time

In this study, for the first time: 1) the electrochemical glutamate oxidase-based biosensor was optimized for the determination of low micromolar glutamate concentrations; 2) the algorithm of the analysis of exocytotic and transporter–mediated glutamate release was developed; 3) the comparative analysis of the biosensor data with those obtained with the radiolabeled L-[14C]glutamate technique, spectrofluorimetric glutamate dehydrogenase-based assay and amino ptio analyzer was performed. One of the advantages of the biosensor is ability to measure the absolute values of endogenous glutamate in nerve terminals. The absolute values of the following parameters, i.e. the extracellular glutamate concentration, Ca2+-dependent glutamate release, and the total glutamate concentration in sonicated synaptosomes, measured using the biosensor, glutamate dehydrogenase assay and amino acid analyzer revealed comparability. The ambient glutamate level at 6 min time point was 11.5 μΜ (biosensor), 15 μΜ (glutamate dehydrogenase) and 12 μΜ (amino acid analyzer). Ca2+-dependent synaptosomal glutamate release was 15.5; 15; 14 μΜ, respectively (Fig. 5, Fig. 6). The total concentration of endogenous glutamate in nerve terminals after sonication determined by the biosensor, glutamate dehydrogenase and amino acid analyzer assays was almost similar (Fig. 5, Fig. 6) and corresponded to 60, 60, 63 μΜ respectively. The values of the ambient glutamate level and different manners of glutamate release were close, but not similar, in the biosensor and L-[14C]glutamate assays. The differences between from one side the L-[14C]glutamate assay and from the other side the biosensor, glutamate dehydrogenase and amino acid analyzer measurements can be due to the difference in the experimental protocols. The radiolabeled technique required preliminary loading of L-[14C]glutamate to nerve terminals (see Method section), whereas the other approaches allow measuring endogenous glutamate which was kept by nerve terminals during isolation, centrifugation and other experimental manipulations. The ratio [Glu release]transporter-mediated/[Glu]extracellular, [Glu release]exocytotic/[Glu]extracellular and also [Glu release]in Ca2+-supplemented media/[Glu]extracellular were characteristic parameters of the release efficiency and were near in all used methodological approaches. So, the adequacy of the developed glutamate biosensor-based methodological approach that allows measuring and calculating glutamate release efficacy in the synaptosomes has been proven. Comparative analysis of the data obtained with the biosensor, glutamate dehydrogenase and amino acid analyzer assays from one side, and radiolabeled L-[14C]glutamate from the other side revealed a difference, when the results were represented as a percentage of total concentration of glutamate in nerve terminals. To have corresponded data on the extracellular level of glutamate, exocytotic and transporter-mediate release in the biosensor and L-[14C]glutamate experiments, the putative total endogenous glutamate concentration in nerve terminals determined by the biosensor must be not 60 μΜ (Fig. 5, Fig. 6) but higher (approximately 80–100 μΜ). We suggested that this discrepancy can be explained by two facts. The first one was an incomplete disruption of the intrinsic compartments of synaptosomes even after repeated sonication, and thus not all endogenous glutamate was released to the incubation media and measured by the biosensor, amino acid analyzer and glutamate dehydrogenate assays. The rest of glutamate was still kept by the synaptic vesicles. The use of detergents and other approaches for synaptosome disruption to measure the total amount of endogenous glutamate was restricted in the biosensor application [[36], [37], [38]]. The second factor was an uncontrolled release of endogenous cytoplasmic glutamate dehydrogenase during synaptosome disruption and therefore metabolization of existed glutamate that in turn can decrease the total glutamate content before starting the biosensor, glutamate dehydrogenase and amino acid measurements. In contrast, the radiolabeled L-[14C]glutamate technique allowed measuring the total L-[14C]glutamate concentration without preliminary synaptosome disruption.