Abstracts

Rationale: Calcineurin (CAN) inhibitors have been reported to show anticonvulsant effect on experimental models of epilepsy. Seizures may be accompanied by modifications in brain CAN activity both in human studies and experimental models of epilepsy. In order to investigate if the anticonvulsant effect of CAN inhibition is mediated by changes in neurotransmitter extracellular concentrations, we have studied the effect of anticonvulsant doses of the CAN inhibitor ascomycin (ASC) on extracellular amino acid levels in the rat hippocampus.Methods: Rat hippocampus was continuously perfused with an ASC solution (100μM) through CMA/12 microdialysis probes at a flow rate of 2 μl/min during 4 hours with continuous EEG recording. After 2 hours, a picrotoxin solution (100-300μM) was perfused during 5 min. Samples from the microdialysate were collected and analyzed by HPLC using pre-column derivatization with 6 aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection. In a second series of experiments, glutamate (100 μM) and glycine (100 μM) were perfused together with ASC to investigate the relationship among the anticonvulsant effect and extracellular amino acid levels.Results: 100 μM ASC significantly increased picrotoxin seizure threshold in 70% of the animals studied . Mean threshold increase was 36,75%. ASC induced also a significant decrease on extracellular glycine levels (58.4±6.1% of basal control levels, p<0.01 by ANOVA, fig 1) with no effect on GABA (106.3±8.6%), glutamate (94.9±12.2%) and aspartate (89.8±11.2% levels. 100 μM glutamate did not modify the effect of ASC on picrotoxin threshold, but 100 μM glycine prevented the anticonvulsant action of ASC.Conclusions: The molecular mechanisms of the anticonvulsant effect of CAN inhibitors are unknown. We have shown that ASC microperfusion into the rat hippocampus increases picrotoxin seizure threshold and decreases extracellular glycine levels. The anticonvulsant effect of ASC is reversed by increasing glycine but not glutamate levels, indicating that the observed changes in glycine concentrations might be related to the anticonvulsant action of ASC.