Synaptosomal Glutamate Release Assay

Synaptosomal pellets were analyzed for glutamate release using enzyme-linked fluorescence method previously described [24 (link),26 (link)]. As synaotosome is not amenable to electrical stimulation, a number of biochemical depolarization protocols have been developed, including the use of K⁺ channel blocker 4-AP or high external [K⁺] [24 (link)]. Therefore, synaptosomes were suspended in hepes-buffered medium containing 2 mM NADP⁺, 50 units of glutamate dehydrogenase, and 1.2 mM CaCl₂, and the synaptosome suspension was stimulated with either 1 mM 4-AP or 15 mM KCl. Increases in fluorescence due to production of NADPH was determined using a PerkinElmer LS55 spectrofluorimeter with excitation and emission wavelengths set at 340 nm and 460 nm, respectively. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg protein). Values quoted in the text and depicted in bar graphs represent the levels of glutamate cumulatively released after 5 min of depolarization, and are expressed as nmol/mg protein/5 min.