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.

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.

Over-expression of the human EAAT2 glutamate transporter within neurons of mouse organotypic hippocampal slice cultures leads to increased vulnerability of CA1 pyramidal cells.

Excitatory amino acid transporters (EAATs) maintain the balance between pathological and physiological conditions by limiting the extracellular concentration of glutamate within the CNS and thus preventing excitotoxic injury. The loss of EAAT2 has been associated with the development of neurological diseases such as amyotrophic lateral sclerosis. It has therefore been suggested that the over-expression of specific EAATs may provide some degree of neuroprotection. However, the inability to isolate and study the function of the different EAAT isoforms in a cell type-specific manner has made it difficult to determine the exact contribution of individual EAATs toward neuroprotection or neurodegeneration in the context of excitotoxic injury. To address this question, we transduced hippocampal slice cultures from 1-week-old C57B/6 mice with recombinant adeno-associated virus carrying an EAAT2 gene expression cassette. EAAT2 gene expression was driven in neurons with the neuron-specific enolase promoter. Using this model system, we were able to induce a significant increase in the expression of functional EAAT2. Consequently, a significant increase in CA1 neuronal damage was observed in slices over-expressing EAAT2 in neurons following an acute exposure to exogenous glutamate. These data suggest that the increased expression of EAAT2 within neurons may contribute to neurodegeneration.

Effects of a selective agonist and antagonist of CRF2 receptors on cardiovascular function in the rat.

The aim of the present study was to investigate the effects of activation or blockade of the CRF(2) receptor subtype on cardiovascular function in conscious rats following systemic i.v. administration of the CRF(2) receptor peptide agonist urocortin 2 given alone and the selective CRF(2) receptor peptide antagonist antisauvagine-30 given alone. Urocortin 2 caused a dose-dependent reduction in mean arterial blood pressure and a dose-dependent increase in heart rate. Pretreatment with antisauvagine-30 blocked the hypotensive effect of urocortin 2. Antisauvagine-30 failed to produce any statistically significant effects on mean arterial blood pressure and heart rate at doses that completely blocked the effects of urocortin 2. These data verify the cardiovascular effects of selective CRF(2) receptor activation, but find no evidence for an endogenous CRF(2)-mediated tone.

Ischemic preconditioning reduces infarct volume after subdural hematoma in the rat.

The aim of the present study was to investigate the effects of ischemic preconditioning on infarct volume in a rat model of subdural hematoma (SDH). Ischemic preconditioning was induced by 15 min transient middle cerebral artery (MCA) occlusion followed 3 days later by the injection of 300 microl of autologous venous blood into the subdural space. Preconditioning significantly reduced the volume of cortical infarction (by 26%, P<0.001) 24 h after SDH induction, but not brain swelling (P>0.05) relative to sham-operated non-preconditioned animals. These data support the view that ischemic preconditioning reduces ischemic brain damage in this rat model of SDH.