Fluorometric Detection of Glutamate Release

Glutamate release from synaptosomes was detected through a glutamate dehydrogenase reaction, as described previously [48 (link),49 (link)]. Briefly, synaptosomes (0.5 mg/mL of final protein concentration) were incubated in HEPES-buffered medium containing glutamate dehydrogenase (20 U/mL) (Sigma, St. Louis, MO, USA), β-nicotinamide adenine dinucleotide (NAD⁺, 1 mM), and CaCl₂ (1.2 mM), at 37 °C for 5 min. In the presence of glutamate, glutamate dehydrogenase reduced NAD⁺ to NADH, a product that fluoresces (excitation and emission wavelengths of 340 and 460 nm, respectively). Fluorescence intensity of NADH was measured in a stirred thermostated cuvette (37 °C) using a PerkinElmer LS55 spectrofluorimeter. Endogenous glutamate released from the synaptosomes to the incubation medium was detected as an increase in NADH fluorescence. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg protein). Values quoted in the text and depicted in bar graphs represent the levels of glutamate that were cumulatively released after 5 min of depolarization, and are expressed as nmol/mg protein/5 min.