Extracellular glutamate (GLU(ECF)) was collected by microdialysis from the corticostriatal region of awake rats, at the basal level and after elevation by perfusion of GLU uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid. Concurrently, [2,5-(13)C]glucose was infused intravenously to 13C-enrich brain GLU predominantly at C5. The 13C enrichment of GLU(ECF) was measured, after tert-butyldimethylsilylation, by gas-chromatography/mass-spectrometry. Excellent signal-to-noise ratios of the analyte signals at three selected ion-pairs were achieved at approximately 20 pmol. The fractional 13C enrichment of basal dialysate GLU C5, collected during 0.75-1.25 h of [2,5-(13)C]glucose infusion, was 0.263+/-0.01, very close to the enrichment of whole-brain (predominantly intracellular) GLU C5 measured in parallel NMR study. The result strongly suggests that the dialysate GLU consists predominantly of neurotransmitter GLU, which was 13C-enriched in, and released from, neurons by exocytosis and had diffused to the dialysis probe; the label is undiluted by 12C-GLU(ECF) present before the enrichment. Hence, our result supports the view, proposed on the basis of Ca(2+)- and tetrodotoxin-sensitivity of dialysate GLU, that basal dialysate GLU in awake non-stimulated brain mainly represents neurotransmitter GLU. Isotope labeling provides a novel method for determining the extent to which dialysate GLU reflects synaptic GLU(ECF), and for measuring its turnover under physiological or pathological conditions.
Copyright 2002 Elsevier Science B.V.