The goals of this project are to study the molecular mechanisms that underlie the gene expression of neuropeptides, their biosynthesis, axonal transport and calcium-dependent, regulated secretion. The biological systems under study are the magnocellular oxytocin (OT) and vasopressin (VP) neurons in the hypothalamo-neurohypophysial system. One of our approaches is to harness the regulatory elements in the OT and VP genes in transgenic mice in order to target specific molecules to these cells in vivo. We have hypothesized that these regulatory elements are located in an intergenic region (IGR) between the two genes. In the past year, we made AVP and OT gene constructs that contained either the total 3.5 kb IGR or a 2.1 kb IGR fragment and a CAT reporter in either exon I or exon III of the genes. Preliminary results from transgenic mice which have integrated these constructs into their genomes indicate that CAT inserts into exon I prevent expression of the genes, whereas the inserts into exon III are permissive. Furthermore, for the VP transgene the 2.1 kb IGR fragment permits cell-specific expression, whereas the 3.5 kg IGR contains a repressive element for VP expression. We have also studied the gene expression of VP and OT in in vitro model systems as an alternative to transgenesis. For this purpose, we have developed an """"""""organotypic"""""""" stationary slice explant hypothalamic culture system. Yields of magnocellular OT and VP neurons are very high in these cultures, and we have demonstrated that the OT and VP cells in these cultures robustly express c-fos. We enhanced neuropeptide expression in response to depolarization (by high potassium or veratridine treatment). They are also readily transfected by adeno- associated viral (AAV) vectors containing lac-Z driven by CMV promoters. Particle-mediated gene transfer (i.e., biolistics) was also performed successfully in these cultures and was used to quantitatively analyze the N-type calcium channel I-1B promoter in CNS slice cultures. Current efforts are to use the OT- and VP-CAT constructs described above (for transgenesis) in viral and biolistic transfections of the slice-cultures in an effort to speed up analysis of the OT and VP cell-specific cis- elements. Finally, we have developed competition-based RT-PCR methods with DNA or RNA mimics in order to quantify OT, VP, and synaptotagmin isoform mRNAs, and various I1, I2 and J calcium channel subunit mRNAs in the cultures and in single cells from the intact CNS. We have had preliminary success using this method for determination of gene expression profiles (of multiple mRNA species) in individual identified OT and VP neurons under various experimental conditions in vivo and in vitro.
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