The Molecular Neuroscience Section, LNC, NINDS, DIR in FY 06 continued to study: 1) 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) 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. The relative simplicity of the supraoptic nucleus (which contains only two neuronal phenotypes, the OT and VP MCNs), and it?s profound response to systemic perturbations, makes it an excellent model for the study of the regulation of cell-specific neuropeptide gene expression. We first studied the effect of sustained hypoosmolality on global gene expression in the OT and VP MCNs in the rat HNS, in order to identify genes associated with the MCN?s adaptation to this physiological condition. We used laser microdissection of the SON, T7 based linear amplification of its RNA, and a 35,319 element cDNA microarray, to 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. Of these, 123 genes were preferentially expressed in the SON as compared to the whole hypothalamus. Among these, 89 genes were also profoundly regulated by systemic osmotic perturbations. The above data was obtained using two spotted DNA arrays, whereas most gene expression profiles are determined using high-density Affymetrix oligonucleotide arrays. Using the Affymetrix oligonucleotide array platform, we confirmed our previous findings obtained using the two spotted DNA arrays, and found previously known as well as novel genes in the SON that were changed in expression during hypoosmolality. Of particular interest, are genes that were not previously known to be present in the SON prior to array studies. These include C1q domain containing 1, Rho GDP dissociation inhibitor (Rho GDI beta), encephalopsin, VGF nerve growth factor inducible, dihydropyrimidase, and solute carrier 12 (member 1). Each of these genes had either very high expression ratios and/or very large changes in gene expression in response to osmotic perturbation, and all of these genes decreased in expression in the SON in the hypoosmolar state. One of these genes, C1q domain containing 1 (also known as EEG-1), has been identified as a novel growth-inhibitor gene involved in terminal differentiation of human erythrocytes, where it is designated as EEG-1. As a result of this analysis, we found that C1q domain containing 1 was very high in expression ratio and it also decreased five-fold in expression during hypoosmolality. In addition, its expression during chronic hyperosmolar conditions was found to increase to 926% of control levels in MCNs, indicating a high level of osmoresponsiveness. Double-label ISHH indicated that C1q domain containing 1 gene is primarily expressed in VP-MCNs. The fact that this gene?s expression is profoundly altered during systemic osmotic perturbations, suggests that it may also be involved in regulation of the VP and/or OT MCNs. An important value of array studies is that they provide an archive of genes that are present in tissues of interest. For this reason, we have made the data from this study available to all investigators via the NINDS internet website URL address: http://matra.ninds.nih.gov/data/Gainer/Publications/.? ? Studies of neuropeptide gene transcription in the central nervous system (CNS), can be performed by in situ hybridization histochemistry (ISHH) by using intron-specific probes that measure pre-mRNA or heteronuclear RNA (hnRNA) levels in the neuron. The study of oxytocin gene transcription by this approach has been difficult, due to the absence of a good intronic probe for OT. Therefore, we developed a reliable intron-specific probe for the study of OT heteronuclear RNA (hnRNA) levels by in situ hybridization histochemistry (ISHH). Using this riboprobe, we demonstrate strong and specific signals in neurons confined to the supraoptic (SON) and paraventricular (PVN) nuclei of the rat hypothalamus. We used this new intronic OT probe, together with other well established intronic and exonic OT and VP probes, to reevaluate OT and VP gene expression in the hypothalamus under two classical physiological conditions, acute osmotic stimulation and lactation. We found that MCNs in 7-8 day lactating female rats exhibit increased OT but not VP gene transcription. Since VP mRNA is increased during lactation, this suggests that decreased VP mRNA degradation during lactation may be responsible for this change. In contrast, while there was the expected large increase in VP hnRNA after acute salt loading, there was no change in OT hnRNA, suggesting that acute hyperosmotic stimuli produce increased VP but not OT gene transcription. This study provided evidence for new and unexpected, differential responses of the OT and VP MCNs under these conditions. Hence, we suggest that the use of both exon- and intron-specific probes which distinguish the changes in hnRNA and mRNA levels, respectively, can provide insight into the relative roles of transcription and mRNA degradation processes in changes in gene expression evoked by physiological stimuli. ? ? Vasopressin (VP) transcription in the rat suprachiasmatic nucleus (SCN) in organotypic culture was studied by in situ hybridization histochemistry (ISHH) using an intron-specific VP heteronuclear RNA (hnRNA) probe. The circadian peak of VP gene transcription in the SCN in vitro is completely blocked by a 2 hour exposure to tetrodotoxin (TTX) in the culture medium, and this TTX inhibition of VP gene transcription is reversed by exposure of the SCN to either forskolin, or potassium depolarization. This suggests that an intrinsic, spontaneously active neuronal mechanism in the SCN is responsible for the cAMP- and depolarization-dependent pathways involved in maintaining peak VP gene transcription. We evaluated a variety of neurotransmitter candidates, membrane receptors, and signal-transduction cascades that might constitute the mechanisms responsible for the peak of VP gene transcription. We find that Vasoactive Intestinal Peptide (VIP) and a VPAC-2 receptor-specific agonist, Ro-25-1553, are the most effective ligands tested in evoking a cAMP-MAP kinase signal transduction cascade leading to an increase in VP gene transcription in the SCN. In addition, a second independent pathway involving depolarization activating L-type voltage-gated calcium channels and a Ca-dependent kinase pathway (inhibited by KN62) rescues VP gene transcription in the presence of TTX. In the absence of TTX these independent pathways appear to act in a cooperative manner to generate the circadian peak of VP gene transcription in the SCN.These experiments on the SCN in TTX lead us to a dualistic view of the regulation of VP gene transcription in TTX, by both a VIP-cAMP-MAP kinase pathway and a depolarization calcium ion influx-CaCAM kinase pathway. This is consistent with the hypothesis that neural activity affects VP gene transcription by activation of each of these pathways. Since KN62 and PD98059 (and U0126) inhibitors when used alone had similar effects as TTX, suggests that these two pathways may be acting in a cooperative fashion to regulate VP gene transcription.
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