Release of N-Acetylaspartylglutamate on Depolarization of Rat Brain Slices

In a great number of investigations, evidence in favor of a neurotransmitter role of the N-terminal-blocked, acidic dipeptide N-acetylaspartylglutamate (NAAG) has been accumulating. In fact, in some systems of the mammalian brain, almost all of the classical criteria for neurotransmitters have been fulfilled by NAAG except for the demonstration of its release from nervous tissue on depolarization. For quantification of NAAG in superfusates of brain slices, we have developed an analytical procedure consisting of an ion exchange prepurification, followed by a derivatization procedure and gas chromatography-mass spectrometry with chemical ionization and selected ion monitoring. Deuterated NAAG was used as an internal standard to provide a high degree of reliability for the analytical method. Detection limits of less than 1 pmol were achieved. A statistically highly significant increase of NAAG concentration in superfusates from rat neocortex, piriform cortex/amygdala, and hippocampus on depolarization with 50 mM K+ could be demonstrated and was shown to be largely Ca2+ dependent. These results support the hypothesis that NAAG is a neurotransmitter. Especially with respect to the piriform cortex, the present demonstration of NAAG release is consistent with electrophysiological and immunohistochemical evidence for its neurotransmitter function at terminals of the lateral olfactory tract.