Enzyme-Linked Sensitive Fluorometric Imaging of Glutamate Release from Cerebral

Abstract

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 μM. We used two types of Ca²⁺ channel inhibitors, MK-801 and ω-Conotoxin GVIA, which act to suppress Ca²⁺ transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when ω-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca²⁺ channels in the presynapse. Glutamate release and Ca²⁺ flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.