The goal of the research is to identify the peptidergic neurons for individual stereoisomers of several identified D-amino acid-containing neuropeptides and to explore the occurrence of unidentified neuropeptides or peptide isomers containing D-amino acids in the neuronal model system, Aplysia californica. The biosynthesis release of these D-amino acid-containing neuropeptides under influence of stimulation (e.g. depolarization anaerobic condition, and pharmacological effects) will also be studied The research requires quantification of intact peptide stereoisomers present in mass/volume limited neuronal samples. Therefore, a significant portion of the research will be the development of suitable analytical methodology. Research will be focused on direct chiral capillary electrophoresis (CE) coupled with wavelength-resolved laser induced native fluorescence (LINF) and capillary gel electrophoresis (CGE) coupled with mass spectrometry (MS). Experiments will include: analysis of D/L amino acids both free and bound in single neurons or neuron clusters; localization of each stereoisomer of certain identified D-amino acid-containing neuropeptides at the cellular level; and determination of the peptide stereoisomers synthesized in released from individual peptidergic neurons or neuron clusters under the influence of stimulation. The hypotheses are: some unidentified D-amino acids and neuropeptide containing them can be discovered through analyzing single neurons or neuron clusters where they are synthesized or bound to; the D-amino acid-containing neuropeptides under study are synthesized in particular groups of neurons, the neuronal distributions are connected with their biological and neurological properties; the amount ratio of the peptide stereoisomers synthesized/released changes with stimulation. The successful development and implementation of the analytical methods proposed in the research will allow us to quantitatively study certain chirality issues of neurochemistry in unprecedented details. In leading to a description of the occurrence, cellular distribution, and biosynthesis/release of individual stereoisomers of D-amino acids and neuropeptides containing them, this research will contribute to our basic understanding of the nervous system.
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