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

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 ±â€¯0.9 to 11.7 ±â€¯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 ±â€¯1.0 µΜ and transporter reversal was 8.0 ±â€¯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.

Dynamic Gradient of Glutamate Across the Membrane: Glutamate/Aspartate-Induced Changes in the Ambient Level of L-[14C]glutamate and D-[3H]aspartate in Rat Brain Nerve Terminals.

Extracellular/intracellular L-[14C]glutamate exchange and conservativeness of the extracellular level of L-[14C]glutamate was analyzed in isolated rat brain nerve terminals. L-Glutamate-, DL-threo-ß-hydroxyaspartate (DL-THA)-, and D-aspartate-induced increase in the ambient level of L-[14C]glutamate or D-[3H]aspartate was evaluated comparatively. 100 µM "cold" nonradiolabeled L-glutamate, DL-THA, D-aspartate extruded a quarter of radioactivity from L-[14C]glutamate-preloaded synaptosomes for 6 min. The similar results were obtained with L-glutamate-evoked extracellular/intracellular redistribution of D-[3H]aspartate. Contribution of presynaptic glutamate receptors to an increase in the extracellular L-[14C]glutamate level was evaluated using receptor agonists NMDA, AMPA, and kainate (100 µM), and it consisted of less than 5 % of total accumulated label. The existence of the efficient extracellular/intracellular glutamate exchange, and so dynamic glutamate gradient across the plasma membrane of nerve terminals was demonstrated. A two-substrate kinetic algorithm that included transporter reversal was considered. The extracellular level of L-[14C]glutamate and D-[3H]aspartate in nerve terminals depended on the amount of exogenous substrates of glutamate transporter available. Taking into account that L-glutamate, DL-THA, and D-aspartate are the substrates of glutamate transporters, and also the similarity in their effectiveness in the establishment of new extracellular level of the neurotransmitters, the central role of glutamate transporters in permanent glutamate turnover in nerve terminals was demonstrated.