13C enrichment of extracellular neurotransmitter glutamate in rat brain: Combined mass spectrometry and NMR studies of neurotransmitter turnover and uptake into glia in vivo

13C-enrichment analysis of glutamate in the extracellular fluid (GLU E C F : 2-3 μM) by gas-chromatography/mass-spectrometry (GCMS) was combined with in vivo N MR observation of whole-brain GLU (∼10 mM) to study neurotransmitter uptake. Brain GLU C5 was 1 3 C-enriched by intravenous [2,5- 1 3 C]glucose infusion. GLU E C F was collected by microdialysis from the corticostriatal region of awake rats. The 1 3 C-enrichment of basal dialysate GLU C5 during 0.75-1.25 hr of infusion was 0.263 ′ 0.01, very close to the enrichment of whole-brain GLU C5. The result strongly suggests that dialysate GLU consists predominantly of neurotransmitter GLU. For selective 1 3 C-enrichment of neurotransmitter GLU, the whole-brain 1 3 C-enrichment was followed by [ 1 2 C]glucose infusion to chase 1 3 C from the small glial GLU pool. This leaves [5- 1 3 C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. The uptake of synaptic [5- 1 3 C]GLU E C F into glia and metabolism to glutamine (GLN) were monitored in vivo by NMR observation of [5- 1 3 C, 1 5 N]GLN formed during 1 5 NH 4 Ac infusion. The rate of GLN synthesis, derived from neurotransmitter GLU E C F (which provided 80-90% of the substrate) was 6.4 ′ 0.44 μmol/g/hr. Hence, the observed rate represents a reasonable estimate for the rate of glial uptake of GLU E C F , a process that is crucial for protecting the brain from GLU excitotoxicity.