The focus of this proposal is to examine key regulators of neuropeptide biosynthesis, secretion and mRNA expression in neuronal cells using cultured superior cervical ganglion (SCG) neurons as models. Neuronal peptide production levels are frequently much lower than levels of classical transmitters and as a result, few neuronal systems are amenable to extensive peptide biochemical studies. Despite its many important physiological roles, the regulation of neuropeptide Y (NPY) expression has not been well studied. Levels of NPY expression in the SCG are at least 10-fold higher than other neuropeptides, and are amenable to detailed biochemical and physiological studies. The factors and environmental cues that coordinately or independently regulate SCG NPY and catecholamine (CA) expression will be investigated. Several questions will be addressed, including (1) What are the regulators of neuronal NPY expression? (2) Are NPY and CA expression altered by the same regulatory factors? (3) How does neuronal phenotype affect NPY expression? (4) Do specific target tissues regulate neuronal NPY expression? (5) What are the cellular mechanisms of altered neuropeptide expression? Several parameters will be used to characterize the cultures including NPY content using radioimmunoassay, NPY biosynthesis using biosynthetic labeling techniques, NPY mRNA expression using Northern blot analysis, and CA content and biosynthesis using biosynthetic labeling and HPLC/electrochemical detection techniques. Using these methods, the effects of neuronal input onto SCG cells, that is stimulation of second messenger system, calcium flux, depolarization, and specific receptor-mediated stimulation on SCG NPY production will be examined. Alterations in NPY levels and biosynthesis will be correlated to CA production levels. Cultured SCG neurons are unique because they exhibit plasticity in neurotransmitter phenotype expression. For example, SCG neurons synthesize primarily dopamine in culture; addition of ascorbate to the culture, however, leads to norepinephrine biosynthesis, while ascorbate plus glucocorticoid leads to epinephrine production. Specific target cell factors. change SCG neurotransmitter expression from catecholaminergic to cholinergic phenotype; how these changes alter NPY expression will be of interest. The effects of direct cell-to-cell interaction on SCG NPY and CA production will be assessed in coculture experiments using cultured target cells. The effects of appropriate SCG target cells (cells innervated by SCG), including pineal gland, salivary gland, iris and vascular smooth muscle cells, on neurotransmitter/neuropeptide expression will be compared to those by inappropriate target cells. Morphological studies will establish the cellular basis for changes in culture NPY levels (alterations in number of cells expressing NPY or in peptide content/cell), and examine the subcellular disposition of NPY and neuronal synaptic contacts on target tissues. These studies will have significant implications in establishing how complex and integrated signals direct normal neuronal development and physiological functions.
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