Analysis of D-aspartate as a signaling molecule in the Aplysia californica central nervous system using capillary electrophoresis and radioisotopic labeling

Characterizing the neurochemistry occurring in single neurons is important in order to understand the functioning of neural systems. Several important neurotransmitter systems have been investigated. First, the catecholamines, which are derived from tyrosine and include dopamine, octopamine, and norepinephrine, are of particular interest because of their widespread distribution in central and peripheral tissues, broad array of biological action, and critical role in diverse pathologies. We have built a novel sheath-flow CE-LINF system that exploits the respective advantages of PMT and CCD-based detectors, by employing a series of dichroic beamsplitters that disperses the signal based on the emission wavelength and transmits it to one of three PMTs. This instrument also uses a hollow-cathode HeAg metal vapor laser (224 nm) that is able to excite the S0→S2 transition of catecholamines, which translates into a higher level of fluorescence generated per unit of input power for catecholamines.; 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.