Measuring D-amino acids in the central nervous system using capillary electrophoresis.

摘要

Understanding what molecules are used by neurons and glia for cell-to-cell signaling is important as this knowledge provides insights into neuronal network function within the brain. Even after decades of research, novel signaling molecules remain to be uncovered in the nervous system. Since their discovery in the central nervous system, D-amino acids (D-AAs) have drawn intense research interest because several of them are involved in signal transmission processes and brain development. However, a lack of appropriate analytical tools has prevented more refined studies to elucidate the roles of D-AAs in intercellular signaling. Here new and improved analytical platforms are described with the aim of sampling the complex neuronal microenvironment, accomplishing chiral separations and quantifying low abundant D-AAs reliably.;Among D-AAs found across the metazoan, two D-AAs are studied extensively, namely D-serine (D-Ser) and D-aspartate (D-Asp). D-Ser can be synthesized endogenously by serine racemase which converts L-Ser to D-Ser. D-Ser can affect the activity of N-methyl-D-aspartate (NMDA) receptor, which is an important receptor involved in learning and memory formation. D-Asp levels correlate with embryonic development stages and D-Asp concentrations drop to much lower levels in adult animals. Through the past ten years of studying D-Asp within Sweedler group, we have determined its neuronal localization, biosynthesis, transport, and demonstrated several of the physiological responses it elicits within Aplysia californica (A. californica)..;Capillary electrophoresis (CE) is an ideal method to characterize D-AAs in the central nervous system due to the compatibility of CE with small size sample and the low detection limits it provides when coupled with laser-induced fluorescence (LIF) as a detection method. Through a variety of CE measurements, two main questions are answered in this thesis. First, is D-Asp released from A. californica neurons and is it released in a stimulation-dependent manner? Second, is D-Ser released from glia via a vesicular release pathway? The first question was answered by selectively extracting and quantitatively measuring the extremely diluted D-Asp from the releasates of A. californica ganglia. Our results indicate a stimulation-dependent release of endogenous D-Asp, which is the last criterion needed to be satisfied to show that D-Asp meets the requirements of a traditional neurotransmitter within A. californica. The second question was addressed by quantifying the amino acid contents in intact and leaked glial vesicles. Various CE methods were incorporated to validate the quantitation results. These analytical measurements clearly show the enrichment of D-Ser, along with another signaling molecule L-glutamate (L-Glu), inside glial vesicles. Together with the results of biochemical assays from our collaborator, our data demonstrate D-Ser being stored in glial vesicles and released from glia via vesicular release pathway.