Several amino acids in the central nervous system (CNS) work as neurotransmitters and neuromodulators that are critical for cell to cell signaling and neuronal network function. Besides L-amino acids, there are several D-amino acids found in the neurons of animals but the presence and function of these D-amino acids are not well understood, partly because of analytical challenges in their measurement. The chemical complexity, great excess of L-amino acids, and small volume samples contribute to these challenges. Here we use several capillary electrophoresis (CE) approaches to quantify D-glutamate (D-Glu) and D-aspartate (D-Asp) and confirm our identifications of these D-amino acids using both enzyme and antibody treatments. Our samples (here the Aplysia californica CNS) are treated separately with D-aspartate oxidase, an enzyme that oxidizes D-Glu and D-Asp, with antibodies for D-Glu and D-Asp, or with saline. Next, the treated samples are derivatized with amine-reactive fluorogenic reagent, naphthalene-2,3-dicarboxaldehyde (NDA), followed by analysis via CE with laser-induced fluorescence (LIF). CE-LIF is well suited to these measurements because it requires small sample volumes and its high sensitivity. Chiral separation of derivatized amino acids is achieved with chiral selector, alpha-cyclodextrin. Our results clearly demonstrate the presence of D-Glu and D-Asp in Aplysia CNS, and the enzyme and antibody treatments confirm these assignments. We are now examining how D-Glu and D-Asp change in Aplysia neurons in response to chemical and electrical stimulations to correlate electrophysiological activity of the neurons and release of the D-amino acids.
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