As our understanding of the nervous system increases, the questions posed by neuroscientists become more complex and require more sophisticated analytical schemes to answer them. A major challenge of contemporary neurobiology is to understand the mechanisms of cellular communication. Essential to an understanding of how neurons communicate is complete information concerning the neurotransmitters and neuropeptides present in and released from individual neurons. The methods currently employed for the assay of such molecules are not sensitive enough to quantitate the neuropeptides found within small subsections of individual neurons nor to detect the release of neuropeptides from a single neuron under most conditions. The goals of this proposal are to develop the analytical instrumentation and methodology capable of identifying and quantifying neuropeptides from single cells or cellular processes, and to use the model neuronal system Aplysia californica both to test these new methods, as well as to elucidate fundamental aspects of peptidergic transmission. Two approaches will be used - matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis. A significant portion of the research involves improving sampling techniques to allow mass spectrometry to study small neurons, sections of neurons and the release of material from specific cellular regions. In addition, the biological activity of a number of novel neuropeptides will be characterized and new neuropeptides identified. By using the advances in separation science and mass spectrometry developed as part of this research, significant gains can be made in our understanding of the processing, distribution, and release of intercellular signaling molecules. In leading to a description of the subcellular dynamics of neuronal signaling, this work will contribute to the basic understanding of the nervous system.
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