Detection of Neuronal Glutamate in Brain Extracellular Space In Vivo Using Microdialysis and Metabolic Labeling with Glutamine

Extracellular glutamate (Glu) concentration measured in the brain using microdialysis sampling is regulated differently from that expected for classical neurotransmitters; e.g., the basal Glu concentration is not affected by blocking action potentials. Additionally, other sources, such as glial cells, contribute to Glu extracellular concentration making it difficult to interpret detected changes. We have found that infusing 2.5 μM ¹³C₅-glutamine (Gln) through a microdialysis probe inserted in the rat cortex results in collection of 144 ± 35 nM (<i>n</i> = 11) ¹³C₅-Glu in dialysate. The recovered ¹³C₅-Glu was reduced by 33% by infusion of 20 mM α-(methylamino)isobutyric acid and 58% by 500 mM riluzole, inhibitors of glutamine transport into neurons. The ¹³C₅-Glu measured was reduced by 62% with tetrodotoxin (TTX), a sodium channel blocker, and 59% with (1<i>S</i>,3<i>R</i>)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a metabotropic glutamate agonist, while endogenous Glu remained unchanged. These results support the hypothesis that the measured ¹³C₅-Glu is derived from neurons via the Gln-Glu shuttle. To further investigate regulation of ¹³C₅-Glu, we applied a stressor (tail pinch), observing a 155% increase in dialysate ¹³C₅-Glu concentration. This effect was blocked by infusion of TTX indicating neuronal release. Local infusion of l-<i>trans</i>-pyrrolidine-2,4-dicarboxylic acid (PDC), a Glu uptake inhibitor, increased both endogenous Glu and ¹³C₅-Glu concentrations, consistent with reverse transport and spread of neuronal release. Taken together, these experiments show that metabolic labeling of Glu via Gln delivered through a microdialysis probe allows differentiation of neuronal and other sources of Glu in the brain. The results support the concept of compartmentalized Glu in the brain.