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 mechanism of cellular communication. Essential to an understanding of how neurons communicate is complete information concerning 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 within small subsections of individual neurons nor to detect the release of neurotransmitters from a single neuron under most conditions. One long term objective of this research program is to develop and implement new analytical instrumentation and methodology to allow the identification and quantitation of the classical transmitters and neuropeptides found in individual identified neurons, as well as the release of these compounds under a variety of stimulation paradigms. Two approaches will be used -- microseparations (including capillary electrophoresis and dynamic channel electrophoresis), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A significant portion of the research involves improving the sampling techniques compatible with these methods needed to sample 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 should be identified. Once the instrumentation and methodology are in place, neurotransmitter distribution and release will be studied using the marine mollusk Aplysia californica as the neuronal model system. 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 neuromodulatory compounds. 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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