Effects of γ‐Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and γ‐Aminobutyric acid metabolism in the anesthetized rat In vivo

Abstract

GABA clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters. The relative flows through these two pathways for GABA released from neurons remains unclear. In this study we determined the effect of tiagabine, a selective inhibitor of neuronal GABA transporter-1 on the rates of glutamate and GABA metabolism, and GABA re-synthesis via the GABA-glutamine cycle. Halothane anesthetized rats were administered tiagabine (30 mg/kg, i.p.), and 45 min later received an intravenous infusion of either [1,6-13C2]glucose (in vivo) or [2-13C]acetate (ex vivo). Non-treated rats served as controls. Metabolites and 13C enrichments were measured with 1H-[13C]-NMR spectroscopy and referenced to their corresponding end-point values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the 13C turnover data measured in vivo during [1,6-13C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P>0.05) from control in tissue amino acid levels or 13C enrichments from [2-13C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMRglc(ox)Glu: control, 0.27±0.05 vs. tiagabine, 0.22±0.04 μmol/g/min; ↓11%, Vcyc(Glu-Gln): control, 0.23±0.05 vs. tiagabine, 0.21±0.04 μmol/g/min) and GABAergic neurons (↓18-25%, CMRglc(ox)GABA: control, 0.09±0.02 vs. tiagabine, 0.07±0.03 μmol/g/min; Vcyc(GABA-Gln): control, 0.08±0.02 vs. tiagabine, 0.07±0.03 μmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P>0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects.

Glutamine-Glutamate Cycle Flux Is Similar in Cultured Astrocytes and Brain and Both Glutamate Production and Oxidation Are Mainly Catalyzed by Aspartate Aminotransferase

“…The transport of glutamate and its precursor glutamine from blood to the brain is very slow and changes in plasma concentration of glutamine have only little influence on brain glutamate content [1,2]. The neurotransmitters glutamate and GABA must therefore be synthesized within the brain.…”

“…Subsequently, glutamine is transferred to neurons in the glutamine-glutamate cycle together with astrocytically-accumulated, previously-released transmitter glutamate (see below). Most glutamine is accumulated by glutamatergic neurons, but some (about 20% depending on the brain region [2,4,33]) is taken up by GABAergic neurons where it is converted by glutamate decarboxylase (GAD) to GABA as previously reviewed [5,6,16,34]. …”

PRESS timings for resolving 13C4‐glutamate 1H signal at 9.4 T: Demonstration in rat with uniformly labelled 13C‐glucose

“…Measuring brain 13 C 4 ‐Glu with MRS during an infusion of a 13 C‐labelled substrate such as [U‐ 13 C 6 ]‐Glc provides a measure of V TCA from the Glu C4 FE time course . Such measures have been performed in rat brain at 9.4 T . Techniques that require system multinuclear capability were used.…”

“…Indirect 13 C studies at 9.4 T have primarily involved POCE based techniques; however, our approach is closer to that of Ref. .…”

“…The 13 C measurements can also be obtained indirectly ( 1 H‐[ 13 C] MRS) by measuring signal from the 13 C‐coupled protons of interest, thereby exploiting the higher sensitivity of the proton ( 1 H) nucleus. Indirect 1 H‐[ 13 C] MRS techniques, such as adiabatic carbon editing and decoupling (ACED)‐STEAM, a variation of the stimulated echo acquisition mode (STEAM) sequence, and techniques incorporating the proton‐observed carbon‐edited (POCE) sequence, have been used in vivo to obtain 13 C 4 ‐Glu time courses . The techniques require multinuclear capability because of the application of both 13 C and 1 H pulses.…”