Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin and 5 mM HPA-β-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue.

Three different fluorescent tags have been compared for the quantitative analysis of aspartate and glutamate in brain microdialysate samples. Separation conditions have been optimized to achieve short analysis time using reversed polarity separation in coated capillary. Method validation has revealed similar quantification limit of 0.1 μM of analytes using either of the labels, although LOD values were different: 7.8-9.8 nM for 4-fluoro-7-nitro-2,1,3-benzoxadiazole, 3.5 nM for fluorescein-5-isothiocyanate and 1.3-1.5 nM for carboxyfluorescein succinimidyl ester derivatives. The almost two orders of magnitude difference between LOD and LOQ values is likely due to the unreliable derivatization reaction at low sample concentration. Based on the superior stability, FITC derivatization was used for the analysis of biological samples. The applicability of the method has been demonstrated by analyzing basal and potassium evoked amino acid concentrations in individual brain microdialysate samples.

It has long been proposed that L: -aspartate (Asp) is an excitatory neurotransmitter similar to L: -glutamate (Glu) but with distinct signaling properties. The presence of Asp in excitatory synapses of the medial striatum/nucleus accumbens of domestic chicks suggests that Asp plays a role of neurotransmitter also in the avian brain. Neurotransmitters are released from the presynaptic bouton mostly by Ca(2+) dependent exocytosis. We used in vivo microdialysis to monitor the simultaneous changes of the extracellular levels of Asp and Glu in the medial striatum of young post-hatch domestic chicks. Microdialysis samples were collected from freely moving birds at 5 min intervals and analysed off-line using capillary electrophoresis. Event-related elevations of extracellular Glu and Asp concentrations in response to handling stress and to high KCl (50 mM) were observed. Increase of Glu and Asp on handling stress was 200 and 250 %, whereas on KCl stimulation the values were 300 and 1,000 %, respectively, if stress was applied before high KCl, and 150 and 200 %, respectively, in the absence of stress. In most cases, the amino acids showed correlated changes, Asp concentrations being consistently smaller at resting but exceeding Glu during stimulation. Using Ca(2+) free medium, the KCl triggered elevation of Glu was reduced. When KCl stimulation was combined with tetrodotoxin infusion, there was no significant elevation in Asp or in Glu suggesting that most of the extracellular excitatory amino acids were released by synaptic mechanisms. The results support the suggestion that Asp is co-released with Glu and may play a signaling role (as distinct from that of glutamate) in the striatum of birds.

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D-aspartate (D-Asp) modulates adult neural plasticity and embryonic brain development by promoting cell proliferation, survival and differentiation. Here, developmental changes of the excitatory amino acids (EAAs) L-Glu, L-Asp and D-Asp were determined during the first postembryonic days, a time window for early learning, in selected brain regions of domestic chickens after chiral separation and capillary electrophoresis. Extracellular concentration (ECC) of EAAs was measured in microdialysis samples from freely moving chicks. ECC of D-Asp (but not L-EAAs) decreased during the first week of age, with no considerable regional or learning-related variation. ECC of L-Asp and L-Glu (but not of D-Asp) were elevated in the mSt/Ac in response to a rewarding stimulus, suggesting importance of Asp-Glu co-release in synaptic plasticity of basal ganglia. Potassium-evoked release of D-Asp, with a protracted transient, was also demonstrated. D-Asp constitutes greater percentage of total aspartate in the extracellular space than in whole tissue extracts, thus the bulk of D-Asp detected in tissue appears in the extracellular space. Conversely, only a fraction of tissue L-EAAs can be detected in extracellular space. The lack of changes in tissue D-Asp following avoidance learning indicates a tonic, rather than phasic, mechanism in the neuromodulatory action of this amino acid.