A range of behaviours are elucidated via ionotropic glutamate receptors (iGluR). In this work, we examined the acute activation of iGluRs by a range of receptor ligands and effectors to see whether distinguishable metabolic sequelae were elucidated by the activity. We used a guinea-pig brain cortical tissue slice model using targeted receptor ligands ((RS)-(tetrazol-5-yl)glycine (TZG),, as well as compounds (quinolinic acid and kynurenic acid (KynA)) involved in some neuroinflammatory responses. The data were derived using 13 C and 1 H NMR spectroscopy, and analysed by metabolomic approaches and multivariate statistics. The metabolic effects of agonists at the three major classes of iGluR were easily separated from each other using this method. The classical N-methyl-D-aspartate receptor agonist TZG and the antagonist CGS 19755 produced excitatory and inhibitory metabolic responses, respectively, while the blocker MK-801 resulted in a significant decrease in net metabolism and produced the largest decrease in all metabolite pool sizes seen by any glutamatergic ligand we have studied. Quinolinic acid and KynA produced similar acute metabolic responses, which were unlike those to TZG or CGS 19755, but similar to that of D-Ser. D-Ser was highly stimulatory of net flux into the Krebs cycle. These data show that the metabolic response to iGluR perturbation in vitro is a sensitive discriminator of function.

Metabotropic glutamate receptors (mGluR) modulate neuronal function. Here, we tested the effect on metabolism of a range of Group I and II mGluR ligands in Guinea pig brain cortical tissue slices, applying 13 C NMR spectroscopy and metabolomic analysis using multivariate statistics. The effects of Group I agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) depended upon concentration and were mostly stimulatory, increasing both net metabolic flux through the Krebs cycle and glutamate/glutamine cycle activity. Only the higher (50 lM) concentrations of CHPG had the opposite effect. The Group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), consistent with its neuroprotective role, caused significant decreases in metabolism. With principal components analysis of the metabolic profiles generated by these ligands, the effects could be separated by two principal components.Agonists at Group II mGluR [(2S,2¢R,3¢R)-2-(2¢,3¢-dicarboxycyclopropyl)glycine (DCG IV) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC)] generally stimulated metabolism, including glutamate/glutamine cycling, although this varied with concentration. The antagonist (2S)-a-ethylglutamic acid (EGLU) stimulated astrocyte metabolism with minimal impact on glutamate/glutamine cycling. (RS)-1-Aminophosphoindan-1-carboxylic acid (APICA) decreased metabolism at 5 lM but had a stimulatory effect at 50 lM. All ligand effects were separated from control and from each other using two principal components. The ramifications of these findings are discussed. Keywords:13 C NMR spectroscopy, glutamate, glutamate/ glutamine cycle, metabolism, metabolomics, metabotropic glutamate receptors. Metabotropic glutamate receptors (mGluR) are a family of G-protein-coupled glutamate receptors, whose activation produces changes with a relatively slow time course, and which are not directly linked to rapid and large changes in neuronal membrane conductance. There are currently eight known mGluR genes classified into three groups based on sequence homology, coupling mechanisms and pharmacological properties (Nakanishi 1992). Group I mGluR comprises mGluR1 and mGluR5, and transduce signals through phospholipase C activity. Group II mGluR comprises mGluR2 and mGluR3, and Group III mGluR comprises mGluR4, 6, 7 and 8. Both Groups II and III are coupled to adenylyl cyclase.

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na(+)-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC50 to approximately 13 and to approximately 2 microM for, respec tively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [3H]L-glutamate uptake in "mini-slices" of guinea pig cerebral cortex (IC50 to approximately 12 microM). In slices (350 microM) of guinea pig cerebral cortex, 5 microM L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 microM L-CCG III decreased incorporation of 13C from [3-13C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the "missing" L-glutamate. At 50 microM, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.