Detection of glutamate released by neurons with an enzyme-based microelectrode: applications and limitations

Pharmacological evidence suggests that the neurotransmitter released by vertebrate photoreceptors is L-glutamate. To confirm and study this release, we developed a,glutamate-sensitive microelectrode (10-30 mu m tip diameter) consisting of a glass-encased Pt-wire whose exposed extremity was coated with a polypyrrole layer containing glutamate oxidase. Glutamate was detected amperometrically as hydrogen peroxide produced from oxidized L-glutamate (Pt-wire polarized at 500 mV against Ag/AgCl). Exposure to 100 mu M L-glutamate caused an increase in microelectrode current of about 50 pA; glutamate concentration changes down to about 1 mu M were detectable. Electrode sensitivity was 100-fold greater for L-glutamate than for other major amino-acids. However, oxidizable compounds (eg catecholamines) crossing the polypyrrole layer were also detected. We used this microelectrode to measure activity-dependent glutamate release from single presynaptic terminals of solitary photoreceptors from salamander retina; various strategies were followed, but we failed to detect any release of glutamate. In contrast, on a slice of insect retina where non-vesicular release of glutamate occurs, we measured a glutamate concentration of about 10 mu M close to the surface of the retina. Possible reasons for our failure to detect glutamate released from vertebrate photoreceptors are discussed. (C) 1997 Published by Elsevier Science Ltd.