This proposal requests funds to continue to investigate pre and post synaptic aspects of the vasopressin (VP) neuronal system of the rat brain. Expression of the VP gene by neurons of the bed nucleus of the stria terminalis (BnST) and medial amygdala (MA) is dependent on gonadal steroids, is sexually dimorphic, and this appears to be determined in the early post-natal period. We hypothesize that estrogen and androgens act directly on these neurons to regulate VP gene transcription. Although estrogen appears to be critical for VP gene expression, the VP gene lacks classical estrogen response elements which have been shown to mediate transcriptional effects of the estrogen receptor, but doe contain a cyclic AMP response element/AP-1 site. The proposed studies will examine the possibility that the effects of E2 on the VP gene are due an interaction with fosjun AP-1 dependent transcriptional effects, or involve phosphorylation of the cAMP response element binding protein (CREB). These studies will be carried out in vivo using immunohistochemical methods to detect each of the transcription factors, and antisense oligonucleotides to block expression of fos and jun related proteins, as well as in in vitro systems using transfected cells. The roles of E2 and androgens in development of the neurons will be studied, and we will test the idea that their effects are related to their ability to enhance the expression of gene products such as the Growth Associated Protein (GAP-43) which are important in neuronal growth, morphology, and survival. The proposed studies will investigate in detail brain VP receptor pharmacology, anatomy, and ontogeny. Evidence from this and other labs suggests that brain VP receptors are similar to a V1a subtype found primarily in hepatic and vascular smooth muscle cells. Use of multiple probes suggests that the mRNA expressed in brain differs in structure and distribution from the hepatic receptor. In addition, VP receptors expressed in the developing brain are pharmacologically distinct from V1a type as well. Thus, a combination of molecular and anatomical approaches to determine the nature of VP receptors expressed in developing and adult rat brain. In addition, previously isolated genomic clones containing a VP V1 a gene and are currently being sequenced to determining the general structural organization of this gene to test for possible mechanisms such as RNA splicing which could account for differences in mRNA species. Isolation of this genomic clone as well as one for the oxytocin (OXY) receptor gene makes it possible to sequence the 5'-flanking region of the putative genes to identify potential modes of regulation of their expression. In support of this, several glucocorticoid response elements are present in both the VP and OXY receptor clones we have studied. The present proposal will examine various aspects of hormonal regulation of receptor genes in vivo an in in vivo.
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