Peptidergic neurons require """"""""on-line"""""""" transcription, translation, packaging into large dense core (secretory) vesicles (LDCVs) via the golgi, and axonal transport of the LDCVs to nerve terminals in order to secrete neuropeptides. In contrast, catecholaminergic (CA) neurons produce and after secretion replenish their vesicular CA stores in the nerve terminal by local enzymatic and membrane transport mechanisms. We are studying these mechanisms in peptidergic oxytocin (OT) and vasopressin (VP) magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), and CA-secreting neurons in the central nervous system (CNS). Our principal goals are: (1) to elucidate the mechanisms that are involved in the cell-specific gene expression of OT and VP in the hypothalamus, and the gene expression of tyrosine hydroxylase in the CNS and (2) to target the gene expression of diverse molecules to these specific neurons both in vivo and in vitro, in order to perturb and visualize their neurosecretory processes. We have proposed 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 in the MCNs. Our recent cloning and sequencing of the human IGR and its comparison with the mouse sequence provided support for this hypothesis and has lead to the identification of 26 conserved sequences as putative enhancer sites in the IGR. In addition, this rationalized the design of several OT and VP mouse gene constructs which are now being completed and evaluated for cell-specific expression in transgenic mice and by the use of biolistics in hypothalamic organotypic cultures. We recently found that 10ng/ml CNTF added to the organotypic culture medium increased the survival of the VP-MCNs by 6-fold and the OT-MCNs by 3-fold. Corresponding changes were found in both VP-and OT-mRNA levels in the SON as a result of the CNTF treatment. Further experiments indicated that the CNTF treatment did not increase OT- and VP-gene expression, and that the increase of OT and VP mRNA observed in the SON was due to the increase in VP MCN number. This rescue of especially the more vulnerable VP-MCN phenotype by CNTF, will for the first time allow for long-term molecular and physiological studies of both MCN phenotypes in vitro. As a by-product of these studies, we have been able to target green fluorescent protein (GFP) to LDCVs and have studied the calcium-dependent secretion of OT-GFP fusion proteins from individual pituitary nerve terminals in transgenic mice using a fluorescence imaging approach. We completed an analysis of gene expression in OT- and VP-MCNs by differentially screening single-cell cDNA libraries. Most of the differentially expressed genes were of unknown function, and in situ hybrization studies confirmed their cell-specific expression. Three of these genes were expressed exclusively in the HNS. Some of these appear to be transcriptional-activating factors, and others encode proteins involved in other cell functions. The roles of these differentially expressed genes are presently under active study. We also studied the cell-specific expression of tyrosine hydroxylase in the CNS using a 9kbp upstream region of the gene, coupled to an EGFP reporter. Four transgenic mouse lines which express EGFP in a cell-specific manner in catecholaminergic neurons in the CNS have been produced. Analysis of EGFP expression in these mice shows that dopamine neurons in the arcuate nucleus and norepinephrine neurons in the locus coeruleus are particularly robust. In addition, we developed organotypic tissue culture models of CA neurons in the CNS, and have successfully produced healthy slice-explants from the forebrain to the hindbrain which contained classical CA neuronal systems. CA neurons in these slice explants were successfully transfected with constructs containing various length tyrosine hydroxlase gene promoters by particle mediated gene transfer, indicating that these cultures will be superb models for the study of the physiological regulation of gene expression in CA neurons in the CNS.
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