Role of the GLT‐1 subtype of glutamate transporter in glutamate homeostasis: the GLT‐1‐preferring inhibitor WAY‐855 produces marginal neurotoxicity in the rat hippocampus

Abstract

Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.

Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (–)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake

“…Methodological differences in the study by Bonde et al (2003), such as the use of serum-free medium during slice maturation and the presence of PI from the very beginning of the experiment, might explain the discrepancy. Previous data actually support the possibility that EAATs can still take up Glu even under ischemic conditions (Rao et al, 2001;Mitani and Tanaka, 2003;Selkirk et al, 2005). It has also been reported that the function of GLT1 may change from neuroprotective (uptake mode) to neurodegenerative (release mode) depending on ischemic conditions (Mitani and Tanaka, 2003).…”

Molecular and cellular mechanisms of excitotoxic neuronal death

“…In a series of experiments, Bonde and colleagues have shown that blocking glutamate transporters with TBOA under normal conditions in rat hippocampal slice cultures results in marked necrotic neurodegeneration, presumably due to increased glutamate in the synaptic cleft, as the effect is blocked by glutamate receptor antagonists [238]. In addition, TBOA exacerbates ischemia in rat hippocampus [239].…”

Glutamate uptake block triggers deadly rhythmic bursting of neonatal rat hypoglossal motoneurons

“…In principle, this seems likely because glutamate uptake is already expressed in the neonatal brain and readily demonstrated even in primary cultures (Danbolt, 2001). In addition, TBOA neurotoxicity is reported to occur when this agent is bath-applied to neuronal cultures from neonatal animals as much as when it is microinjected into the rat adult brain in vivo (Selkirk et al 2005), indicating a widespread potential for neurotoxicity when glutamate uptake is blocked.…”