The peptidergic neuronal phenotype is distinct from that of conventional neurons in that the production and secretion of neuropeptides requires continual transcription, translation, packaging in the golgi, and axonal transport of the large dense core (secretory) vesicles (LDCVs) to nerve terminals before neurosecretion can occur. In contrast, other neurotransmitter secreting neurons (including catecholamine neurons) are able to replenish their vesicular neurotransmitter stores in the nerve terminal by local transport mechanisms after secretion.We are examining the underlying cell biological mechanisms by studying magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system and dopamine-secreting neurons in the mesencephalon as representatives of these two phenotypes. Our goals are:(1)to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP in the hypothalamus, and gene expression of tyrosine hydroxylase in the mesencephalon and (2) to target the gene expression of specific molecules to these neurons both in vivo and in vitro, in order to perturb and visualize the neurosecretory processes in both phenotypes. Our previous work led us to propose the intergenic region( IGR) hypothesis, which states that the 3.6-kbp IGR between the mouse OT and VP genes contains the critical enhancer sites for cell-specific expression. Support for this hypothesis has come from our recent cloning and sequencing of the human IGR, and its comparison with the mouse sequence, which has rationalized the design of various OT and VP mouse gene constructs which are now being evaluated for cell-specific expression in transgenic mice and in hypothalamic organotypic cultures. In addition, by using this information we have been able to target green fluorescent protein (GFP) to LDCVs and have studied the calcium-dependent secretion of OT-GFP & VP-GFP fusion proteins from individual pituitary nerve terminals in transgenic mice and PC-12 cells using an fluorescence imaging approach. We also completed our differential analysis of gene expression in OT and VP neurons by employing single-cell (RT-PCR-derived) oxytocin and vasopressin neuronal cDNA libraries. Several of these differentially expressed genes have been cloned and represent isoforms of known enzymes. Others are paternally expressed genes of varied function (e.g., peg-3). The biological significance of this differential gene expression is presently under active study. In a new initiative, we have studied the cell-specific expression of tyrosine hydroxylase in the CNS using a 9kbp upstream region of the gene, coupled to an EGFP reporter. Transgenic mice containing this construct are presently under production and analysis of cell-specific expression will soon be underway. Given success, this transgenic model will be of immense value for the study of catecholamine systems (e,g., substantia nigra and locus coeruleus) involved in Parkinsons Disease. Two tissue culture models are currently under development in our lab, and we have studied the effects of various growth factors on the survival & process outgrowth of OT, VP and dopamine neurons in our postnatal dissociated neuronal and organotypic cultures of hypothalamus and mesencephalon, respectively, and found that both GDNF and BDNF were very effacacious as trophic factors but in the mesencephalon require cAMP pretreatment. Our future plans are to continue to analyse the OT , VP and tyrosine hydroxlase gene promoters in these models in order to identify the cell-specfic enhancers and to study calcium-dependent secretion from dendrites and nerve terminals in the hypothalamic VP and OT neurons and from dopamine neurons in the substantia nigra.
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