Abstracts

Rationale: Four GAT proteins have been identified and designated GAT1-4 according to mouse nomenclature. Both GATs located within the synapse (GAT1) that facilitate re-uptake of GABA into the presynaptic neuron, and GATs on astrocytes in close proximity to the synapse (GAT3 and GAT4) are thought mostly to contribute to the phasic GABA currents. Whereas, GAT2 located extrasynaptically at asymmetric glutamatergic synapses are believed to contribute to the tonic GABA current. It has previously been shown that combining tiagabine a selective GAT1 inhibitor and EF1502 an inhibitor of GAT1 and GAT2 offer a synergistic anticonvulsant effect in audiogenic seizure susceptible Frings mice. This synergistic effect was found to be mediated by GAT2 presumably by activation of extrasynaptically GABAA receptors (J Pharmacol Exp Ther. 2005;312:866-874.). Based on the synergistic interaction between GAT1 and GAT2, the present study assessed various other interactions between GAT1, GAT2, GAT3, and GAT4. Therefore, SNAP-5114 an inhibitor of GAT3 and GAT4 was combined with tiagabine and EF1502. In addition, this study investigated whether EF1502 and tiagabine would be able to raise the extrasynaptic concentration of GABA sufficiently to displace THIP, an agonist at α4 containing GABA receptors. Methods: The effect of EF1502, SNAP-5114, tiagabine, and THIP on audiogenic seizures in the Frings mouse was evaluated using the isobologram method. Interactions were defined as being either synergistic, additive, or antagonistic. The indirect effect of EF1502 and tiagabine on THIP displacement was also evaluated in CF1 mice using minimal rotarod impairment at a fixed speed of 6 rpm and analysed by Two-Way ANOVA. Results: Combination studies employing SNAP-5114 and tiagabine suggested an additive interaction; whereas, the combination using SNAP-5114 and EF1502 was synergistic in one out of three dose mixtures, and additive in the two of three dose combinations. In testing the ability of tiagabine and EF1502 to raise the extrasynaptic GABA concentration, the combination study employing tiagabine and THIP only suggested an additive interaction; whereas the combination between EF1502 and THIP was antagonistic. Furthermore, the minimal motor impairment produced by THIP was only reversed significantly by EF1502 at a dose of 15 mg/kg. Both EF1502 at 5 and 10 mg/kg and tiagabine at 0.5 and 1 mg/kg, corresponding to 1x and 2x of the respective anticonvulsant ED50 dose in Frings mice, were unable significantly to displace THIP. Conclusions: Inhibition of GATs at different synaptic and extrasynaptic sites can produce an anticonvulsant effect. However, it seems that only inhibitors of extrasynaptic GATs (i.e., GAT2) sufficiently increase the extrasynaptic GABA concentration which is necessary to produce a synergistic anticonvulsant effect with synaptically or close to synaptically acting GAT inhibitors (i.e., at GAT1 and GAT4, respectively).