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- 13C]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 ∼20 pmol. The fractional 13C enrichment of basal dialysate GLU C5, collected during 0.75–1.25 h of [2,5- 13C]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.