An amperometric glutamate biosensor for monitoring glutamate release from brain nerve terminals and in blood plasma

摘要

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 mu M 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 mu M and transporter reversal was 8.0 +/- 1.0 mu M for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[C-14]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. (C) 2018 Elsevier B.V. All rights reserved.