Analysis of D-Aspartate as a Signaling Molecule in the Aplysia Californica Central Nervous System Using Capillary Electrophoresis and Radioisotopic Labeling

Abstract

A second cell-cell signaling molecule investigated is D-aspartate (D-Asp). D-Asp is present in the nervous systems of both vertebrates and invertebrates, and its biosynthesis has been observed in mammalian cells. We suspect that D-Asp acts as a classical neurotransmitter in the central nervous system. The mollusk Aplysia californica has shown high levels of D-Asp in the neural ganglia; and this, along with its relatively simple invertebrate system, makes Aplysia a viable model system for studying the function and neurochemistry of D-Asp. Using Aplysia californica , we have investigated the localization of D-Asp as well as its synthesis from L-Asp. We developed a technique utilizing off-line CE coupled with radionuclide detection, which enables high sensitivity characterization of L- to D-Asp conversion. Furthermore, these capillary electrophoretic techniques, along with liquid scintillation counting and MALDI-MS analysis, have allowed us to address other criteria required to determine whether D-Asp acts as a classical neurotransmitter, including release from cells, sodium dependence of uptake, response to D-Asp by tissues, and the D-Asp stimulated release of cardiomodulatory peptides from the R3-14 cells. These techniques developed provide robust methods for the analysis of neurotransmitters and elucidation of their chiral moieties in complex mass-limited biological samples.