We have developed an online high-speed capillary electrophoresis (CE) assay that is capable of measuring changes in the in vivo concentration of primary amine containing neurotransmitters every 10 seconds. Neurotransmitters are sampled using microdialysis. Dialysate is derivatized online with OPA, a fluorogenic reagent that reacts with primary amines. Derivatized dialysate is coupled online with high-speed CE using a flow-gate interface. High-speed CE is used to separate and quantify neurotransmitters present in the dialysate. Laser induced fluorescence detection is performed off column in a high-sensitivity sheath-flow cuvette. The temporal resolution of this method is 10 seconds, a 120-fold improvement over previous microdialysis assays. The online approach eliminates difficulties associated with collecting, storing, derivatizing and analyzing dozens or even hundreds of dialysate fractions.
The putative neuromessenger D-serine will be used as an example of the usefulness of the online microdialysis-CE technique. A neuromessenger role for D-serine has only recently been proposed. This was somewhat surprising considering that until as recently as fifteen years ago it was thought that higher organisms only used L-amino acids. Recent studies now suggest that D-serine and glutamate co-activate the NMDA receptor. The NMDA receptor has been implicated in stroke, epilepsy, Alzheimers, Parkinsons, schizophrenia and learning, suggesting that D-serine may play an important role in understanding these phenomena. We have used microdialysis-CE to study D-serines role in the mammalian brain on a time scale not previously possible. The effect of pharmacological agents including exocytosis initiators and glutamate receptor agonists and antagonists will be will be shown. These experiments offer insight into D-serines role and its mechanism of release.