Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor

The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.