In vivo microdialysis and gas-chromatography/mass-spectrometry for 13C-enrichment measurement of extracellular glutamate in rat brain

Abstract 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 13 C-enrich brain GLU predominantly at C5. The 13 C 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 ∼20 pmol. The fractional 13 C 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 13 C-enriched in, and released from, neurons by exocytosis and had diffused to the dialysis probe; the label is undiluted by 12 C-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.