Changes in extracellular amino acid concentrations in the rat hippocampus after in vivo actin depolymerization with latrunculin A.

The effect of latrunculin A microperfusion on hippocampal extracellular concentrations of glutamate, aspartate, glycine and GABA, as measured by in vivo microdialysis, was investigated. Latrunculin A (4 microg/ml) was perfused for three consecutive days (8h a day) to promote in vivo F-actin depolymerization. Intrahippocampal latrunculin A microdialysis induced seizures during the second and third day of perfusion, and the animals started showing spontaneous seizures 1 month after lartrunculin A administration. Hippocampal glutamate levels were significantly increased during the first day of latrunculin A microperfusion without significant changes during the second and third day of perfusion. Aspartate levels were significantly increased during the first and second days of treatment. The rise on glutamate and asparate levels was partially reversed by perfusion of NMDA antagonist MK-801. Glycine concentrations were significantly increased during the 3 days of latrunculin A microdialyis, but no significant effect was observed on baseline GABA levels. One month after latrunculin A microperfusion, no significant differences in glutamate and aspartate extracellular concentrations were detected as compared to controls, however, significant increases in glycine and GABA extracellular concentrations were observed. The immediate increases in glutamate, aspartate and glycine levels indicate a modulatory effect of the F-actin cytoskeleton on extracellular concentrations of glutamate, aspartate and glycine. The chronic elevations in GABA and glycine levels are more likely to be related with long-term epileptogenesis processes. Our results suggest that the in vivo biochemical study of actin-dependent processes seems to be a promising approach to the neuropathology and neuropharmacology of epileptic seizures.

Analysis of neuroactive amino acids from microdialysate samples by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method.

A sensitive and rapid reversed-phase high-performance liquid chromatographic method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection is reported. By directly derivatizing microdialysate samples with AQC, an automatic and rapid simultaneous measurement of aspartate, serine, glutamate, glycine and histidine was developed. Excellent linearity (r2 > or = 0.998) was achieved for the standard mixture used for the validation experiments. Within-day and between-day precision was less than 6.2%, and the accuracy ranged from 95 to 105.2% in standards. This method is suitable for single run analysis of a high number of small volume microdialysate samples from rat hippocampus. Amino acids from microdialysate samples were quantified with RSD for reproducibility below 2%, and at approximately 0.1% for retention time.