Monitoring D-serine dynamics in the central nervous system by capillary electrophoresis.

摘要

Neurochemical dynamics in central nervous system (CNS) tissue were measured directly using capillary electrophoresis (CE). The primary focus was investigating the mode of action of the putative neuromodulator, D-serine. An online microdialysis-capillary electrophoresis (MD-CE) instrument was assembled to monitor neurotransmitter release. Online MD-CE eliminates the need for sample collection and improves the temporal resolution relative to traditional microdialysis methods. Once constructed, the system was used to measure multiple neurotransmitters from retinal homogenates. Unlike traditional methods for homogenate analysis, online MD-CE requires relatively little tissue, no sample preparation and has a much higher sample throughput. A novel microliter perfusion chamber was designed to measure neurochemical release from CNS tissue. Analysis of the perfusate was performed with the online MD-CE instrument. Performance of the perfusion chamber was characterized using the vertebrate retina. Detectable basal levels of numerous chemicals, including the neurotransmitters GABA and glutamate were detected in the perfusate. However, detectable levels of D-serine were of released. Elevated levels of GABA, glutamate, L-serine and taurine were measured in the perfusate during high potassium stimulations, demonstrating the ability of the MD-CE system to monitor changes in the perfusate with the application of pharmacological agents. While the retina did not release detectable levels of D-serine in the perfusion chamber, D-serine uptake by the retina was measured using a commercial CE instrument. D-serine uptake was found to be Na+-dependent and inhibited by L-alanine, L-cysteine and L-threonine. These characteristics are consistent with transport mediated by system ASC transporters (ASCT). D-serine release was studied using mouse cortical slices release that release detectable levels of D-serine into the perfusate. The pharmacology of D-serine dynamics was examined to test the current hypothesis that activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptors induces D-serine release. While kainate (KA) did induce release of D-serine, the release was not blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA/KA receptor antagonist, indicating that the effect is not specific to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA receptors. Further investigation revealed that like D-serine uptake in the retina, regulation of extracellular D-serine concentrations in the mouse cortex is consistent with ASCT, either by competitive inhibition of uptake or increased release through hetero-exchange.