In vivo microdialysis and gas chromatography/mass spectrometry for studies on release of N-acetylaspartlyglutamate and N-acetylaspartate in rat brain hypothalamus

Microdialysis and gas chromatography/mass spectrometry wee used for the measurement of extracellular N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in rat hypothalamus. The sensitivity of the method for each of these compounds was approximately 5 pmol/36 μl of dialysate. Baseline NAA concentrations in dialysate were estimated to be approximately 25 pmol/36 μl, while that for NAAG was at or below the detection limit of 5 pmol/36 μl. In vivo and in vitro calibrations of microdialysis probes showed that the recovery for NAA was approximately 10 percent. For NAAG, the in vitro recovery was 6.3%, and in vivo recovery, 11%. Depolarization stimulation using 100 mM KCl in the microdialysis perfusate was employed to measure extracellular NAA and NAAG concentrations. Extracellular NAA was elevated to approximately 70 pmol/36 μl dialysate following depolarization. No significant elevation of NAAG was observed. By infusing known amounts of stable isotopically labeled NAAG-d₃ via the microdialysis probe and measuring the isotopically labeled catabolic product, NAA-d₃, in collected microdialysate, we were able to confirm the existence of one or more hydrolytic enzymes active towards NAAG in the hypothalamus. This finding suggests the possible involvement of active metabolic processes in the relationship between NAAG and NAA releases.