Cellular Biology of Oxytocin and Vasopressin Gene Expres

Neurological Disorders And Stroke

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Abstract

Research activity in the Molecular Neuroscience Section, LNC, NINDS, DIR in FY 05 primarily focused on two objectives: 1) To investigate the physiological and molecular factors that regulate oxytocin (OT) and vasopressin (VP) gene expression in magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), and 2) to study the mechanisms that underlie the regulation of the circadian rhythm of VP gene expression in the suprachiasmatic nucleus (SCN. The OT and VP genes are very selectively expressed in the central nervous system , and are expressed in only five nuclei located in the forebrain. In the HNS, the only neuronal phenotypes present in the rat supraoptic nucleus (SON) are the OT and VP producing MCNs. The relative simplicity of this central nucleus makes it an excellent model for the study of cell-specific neuropeptide gene expression. We found that about 300 bp in the 5' flanking regions in the mouse OT and VP genes and a 178 bp domain immediately downstream of exon III of the VP gene was sufficient to direct OT and VP gene expression specifically in the hypothalamus. We identified four conserved sequences (motifs) in the downstream 178bp domain that might be candidates for the putative enhancers in the IGR that regulate OT- and VP-gene hypothalamic-specific expression. However, our most recent deletion experiments have suggested that this 178 bp domain must be intact to generate OT- and VP-gene expression in the hypothalamus, thereby precluding the identification of simple enhancer elements in this downstream domain. We adopted a new approach to identify candidate genes that are selectively expressed in the MNCs in the HNS, and that also undergo very large changes in expression under physiological stimulation. Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin (OT) gene expression in the supraoptic nucleus (SON). We therefore examined the effect of sustained hypoosmolality on global gene expression in the OT and VP MCNs in the HNS, in order to identify genes associated with the MCN?s adaptation to this physiological condition. Using laser microdissection of the SON, T7 based linear amplification of its RNA, and a 35,319 element cDNA microarray, we compare gene expression profiles between SONs in normoosmolar (control), dDAVP-treated normoosmolar, and hypoosmolar rats. We found 4,959 genes with statistically significant differences in expression between normosmolar control and the hypoosmolar SONs, with 1,564 of these differing in expression by more than two-fold. These genes serve a wide variety of functions, and most were up-regulated in gene expression in hypoosmolar compared to control SONs. Of these, 90 were preferentially expressed in the SON, and 44 coded for transcription-related factors, of which 15 genes were down-regulated and 29 genes were up-regulated in the hypoosmolar rat SONs. None of these transcription-related factor genes significantly changed in expression following sustained dDAVP-treatment alone, indicating that these changes were associated with the hypoosmolar state and not due solely to a decreased activity in the SON. Quantitative in situ hybridization histochemistry was selectively used to confirm and extend these microarray observations. The results indicated that the hypoosmolar state is accompanied by a global but selective increase in expression of a wide variety of regulatory genes in the SON. Several novel genes, such as C1q domain containing 1, which showed a very high and specific expression in the MCNs, were also shown to be osmotically regulated to the same extent or even more than vasopressin (VP) itself. In addition, various genes which were not preferentially expressed in the SON, such as specific members of the cytokine family, were also greatly osmotically regulated, suggesting that there might be important roles for these genes in MCN function in the SON. Another gene that we found that is preferentially and abundantly expressed in the VP or OT MCNs is Paternally Expressed Gene 3 (Peg3), an imprinted gene expressed exclusively from the paternal allele that encodes a Kruppel-type zinc finger-containing protein involved in maternal behavior . We also found robust expression in the VP-MCNs of an RNA, which we designated APeg3 in that it is transcribed in the antisense direction to the 3' untranslated region of the Peg3 gene. The APeg3 mRNA is about 1kb in size, and the full-length sequence of APeg3, as was determined by 5' and 3' RACE, contains an open reading frame that predicts a protein of 93 amino acids, and is predominantly expressed in VP-MCNs. Both Peg3 and APeg3 gene expression in the VP-MCNs increase during systemic hyperosmolality in vivo, demonstrating that both of these genes are osmoregulated.Studies of neuropeptide gene expression in the central nervous system in vivo are predominantly performed by in situ hybridization histochemistry (ISHH), using exon-specific probes. These probes measure the steady-state levels of mRNA, which reflect both gene transcription and variable mRNA degradation processes in the neuron. In contrast, intron-specific probes provide measures of heteronuclear RNA (hnRNA) levels in the nucleus of the neuron, and because of the rapid turnover of this intermediate form of RNA, this determination of the hnRNA level closely approximates the transcription rate of the gene. The study of vasopressin (VP) gene transcription has been made possible by the availability of an effective intronic probe for vasopressin, and this probe has been widely used in studies of the regulation of the VP gene in vivo and in vitro. In contrast, the few efforts made to produce and use an oxytocin (OT) intronic probe have been unsuccessful, its inadequacy usually attributed to the small sizes of the introns in the OT gene. We designed an intron-specific riboprobe directed at detecting OT hnRNA in neurons. Our results, as expected, show that the signal of the OT intronic probe is specifically localized in the nucleus of the OT neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN). By using this intronic OT riboprobe, we have been able to study changes in OT as well as VP transcription in the rat hypothalamus during rat acute osmotic stimulation, the estrus cycle, and lactation.Organotypic cultures of rat hypothalamus were used to study the effects of various neurotransmitters (Ionotropic excitatory amino acid agonists, NMDA; AMPA, kainate, the metabotropic glutamate agonist, DHPG, and VIP) and potassium depolarization on the transcription of VP in the suprachiasmatic nucleus (SCN. Using tetrodotoxin (TTX), a blocker of spontaneous neural activity in these cultures, we could determine whether the effects are transsynaptic. Using our VP heteronuclear RNA (hnRNA) assay, we found that transynaptic communication within the SCN is necessary to maintain normal level of VP transcription during daytime and that calcium channels, predominantly the L-type, are significantly involved in this process. We also showed that either VIP, a critical neurotransmitter in the SCN that is coupled to adenylate cyclase activation, or forskolin, a direct activator of adenylate cyclase, could induce transcription of VP in the SCN in the presence of TTX. These effects were also mimicked by a VIP agonist, RO 25-1553 , which selectively activates the VPAC2 receptor, which is abundantly found in the SCN.