Synaptosomal Glutamate Release Assay

Glutamate release assay was performed using enzyme-linked fluorescent detection of the released glutamate [24 (link)]. Synaptosomes (0.5 mg/mL) were resuspended in the HEPES buffer medium and placed in a LS-55 spectrofluorimeter (PerkinElmer Life Sciences, Waltham, MA, USA) at 37 °C with stirring. Nicotinamide adenine dinucleotide phosphate (NADP⁺, 1 mM), glutamate dehydrogenase (50 units/mL), and CaCl₂ (1.2 mM) were added. After 3 min incubation, 4-aminopyridine (1 mM) was added to stimulate glutamate release. The fluorescence of NADPH was measured at 340 nm excitation and 460 nm emission and calibrated by glutamate standard (5 nmol). The released glutamate was expressed as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg). Release values quoted in the text represent levels of glutamate cumulatively released after 5 min depolarization and are indicated as nmol/mg/5 min.