Vasopressin hnRNA Research Articles

Neurotransmitter regulation of c- fos and vasopressin gene expression in the rat supraoptic nucleus

Acute increases in plasma osmotic pressure produced by intraperitoneal injection of hypertonic NaCl are sensed by osmoreceptors in the brain, which excite the magnocellular neurons (MCNs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in the hypothalamus inducing the secretion of vasopressin (VP) into the general circulation. Such systemic osmotic stimulation also causes rapid and transient increases in the gene expression of c- fos and VP in the MCNs. In this study we evaluated potential signals that might be responsible for initiating these gene expression changes during acute hyperosmotic stimulation. We use an in vivo paradigm in which we stereotaxically deliver putative agonists and antagonists over the SON unilaterally, and use the contralateral SON in the same rat, exposed only to vehicle solutions, as the control SON. Quantitative real time-PCR was used to compare the levels of c- fos mRNA, and VP mRNA and VP heteronuclear (hn)RNA in the SON. We found that the ionotropic glutamate agonists (NMDA plus AMPA) caused an approximately 6-fold increase of c- fos gene expression in the SON, and some, but not all, G-coupled protein receptor agonists (e.g., phenylephrine, senktide, a NK-3-receptor agonist, and α-MSH) increased the c- fos gene expression in the SON from between 1.5 to 2-fold of the control SONs. However, none of these agonists were effective in increasing VP hnRNA as is seen with acute salt-loading. This indicates that the stimulus-transcription coupling mechanisms that underlie the c- fos and VP transcription increases during acute osmotic stimulation differ significantly from one another.

Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus.

In this study, we test the hypothesis that there are differential splicing patterns between the expressed oxytocin (OT) and vasopressin (VP) genes in the rat supraoptic nucleus (SON). We quantify the low abundance, intron-containing heteronuclear RNAs (hnRNAs) and the higher abundance mRNAs in the SON using two-step, quantitative SYBR Green real-time reverse transcription (RT)-PCR and external standard curves constructed using synthetic 90 nt sense-strand oligonucleotides. The levels of OT and VP mRNA in the SON were found to be similar, approximately 10(8) copies/SON pair, whereas the copy numbers of VP hnRNAs containing intron 1 or 2 and the OT hnRNA containing intron 1 are much lower, i.e., approximately 10(2)-10(3) copies/rat SON pair. However, the estimated copy number of the intron 2-containing OT hnRNA is much larger, approximately 10(6) copies/SON pair. The relative distributions of all the OT and VP RNA species were invariant and independent of the physiological status of the rats (e.g., osmotically stimulated or lactating rats). Using intron-specific riboprobes against hnRNAs, we demonstrate by fluorescence in situ hybridization strong signals of OT hnRNA containing intron 2 predominantly in the cytoplasm, in contrast to the localization of the VP hnRNA found only in the nuclei. Taken together, these data support the view that the splicing patterns between OT and VP gene transcripts are different and show that there is a selective cytoplasmic retention of OT intron 2.

Differential Kinetics of Oxytocin and Vasopressin Heteronuclear RNA Expression in the Rat Supraoptic Nucleus in Response to Chronic Salt Loading In vivo

Previous studies have shown that the secretion of oxytocin and vasopressin from the posterior pituitary always accompanies systemic hyperosmotic stimuli in rats, and that oxytocin and vasopressin mRNAs consistently increase in response to prolonged hyperosmotic stimuli. Hence, it has been widely interpreted that oxytocin and vasopressin secretion and gene expression are closely coupled. In the present study, we used both vasopressin and oxytocin intron- specific probes to measure vasopressin and oxytocin heteronuclear RNA (hnRNA) levels, respectively, by in situ hybridisation in the rat supraoptic nucleus (SON) in conjunction with radioimmunoassays of vasopressin and oxytocin peptide levels in plasma and in the posterior pituitary in normally hydrated rats and after 1-5 days of salt loading. Increased oxytocin secretion in response to hyperosmotic stimuli exceeded vasopressin secretion at every time point studied. Vasopressin hnRNA in the SON increased to near maximal levels within minutes after the hyperosmotic stimulus, and was maintained throughout all 5 days of salt loading. By contrast, oxytocin hnRNA did not significantly change from control levels until approximately 2 days after hyperosmotic stimulation, and was not maximal until 3 days. In summary, increases in oxytocin gene transcription in response to osmotic stimuli are dramatically delayed compared to increases in vasopressin gene transcription under the same conditions. These data indicate that oxytocin gene transcription is not as closely correlated with pituitary peptide secretion as is vasopressin gene transcription, and suggests that there is a fundamental difference in excitation-secretion-transcription coupling mechanisms that regulate these two closely related genes in the rat magnocellular neurones in the SON.

Studies of oxytocin and vasopressin gene expression in the rat hypothalamus using exon- and intron-specific probes

To develop a comprehensive approach for the study of oxytocin (OT) and vasopressin (VP) gene expression in the rat hypothalamus, we first developed an intronic riboprobe to measure OT heteronuclear RNA (hnRNA) levels by in situ hybridization histochemistry (ISHH). Using this 84-bp riboprobe, directed against intron 2 of the OT gene, 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 magnocellular neurons in 7- to 8-day lactating female rats exhibit increased OT but not VP hnRNA. Since VP mRNA is increased during lactation, this suggests that decreased VP mRNA degradation during lactation may be responsible for this change. In contrast, whereas 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. Hence, 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.

Androgen inhibits, while oestrogen enhances, restraint-induced activation of neuropeptide neurones in the paraventricular nucleus of the hypothalamus.

The hormonal response to stress is enhanced by oestrogen but inhibited by androgens. To determine underlying changes in activity of neuropeptide neurones in the paraventricular nucleus of the hypothalamus (PVN), we examined the effect of oestrogen and androgen treatment on restraint-induced c-fos mRNA, corticotropin-releasing hormone (CRH) heteronuclear RNA, and arginine vasopressin hnRNA expression in the PVN. Male rats were gonadectomized and injected with oestradiol benzoate (EB) or dihydrotestosterone propionate (DHTP; s.c., daily for 4 days). Rats were stressed by restraint for 10 min or 30 min before killing. Other rats were stressed for 30 min and then returned to their home cage for 20 min before killing. Corticosterone and adrenocorticotropic hormone responses to restraint stress were significantly greater in EB-treated rats and lower in DHTP-treated rats at the 30-min timepoint compared to controls. c-fos mRNA increases following stress were augmented by EB but inhibited by DHTP. CRH hnRNA expression increased significantly in the PVN in response to restraint stress, and this increase was augmented by EB treatment, but decreased by DHTP treatment. Vasopressin hnRNA expression was also increased in response to stress, and this increase was attenuated by DHTP. These findings indicate that gonadal hormones influence the reactivity of the hypothalamic-pituitary adrenal axis to stress.

Adenoviral-mediated over-expression of Brn2 in the rat paraventricular nucleus: no effect on vasopressin or corticotrophin releasing factor RNA levels

We have used an over-expression strategy to test the hypothesis that the Class III POU transcription factor Brn2 is rate limiting in the control of the level of expression of the vasopressin (VP) gene in the paraventricular nucleus of the rat hypothalamus. Knockout studies in mice have suggested that Brn2 may contribute to the control of the level of VP gene expression in the adult hypothalamus. However, we show here that in heterologous cell lines, Brn2 transactivates neither the proximal promoter of the rat VP gene, nor a novel reporter construct consisting of the rat VP structural gene and 3 and 2 kbp of upstream and downstream flanking sequences. We hypothesised that this maybe due either to the lack of cis-acting elements within the confines of the reporter vectors used, or to the absence in heterologous cells, of factors required for Brn2 activity. As no cell lines exist that correspond to VP neurons, we devised an adenoviral vector delivery strategy that enabled efficient over-expression of Brn2 in the paraventricular nucleus of the intact rat. Localised over-expression of Brn2 had no effect on VP hnRNA levels. Neither did we detect corticotrophin releasing factor (CRF) mRNA up-regulation by Brn2 over-expression in vivo. This was unexpected as Brn2 transactivates the proximal CRF promoter in vitro. Whilst Brn2 is required for the development of the hypothalamic structures that express VP and CRF, these data suggest that this transcription factor is not required, or is not rate limiting, for expression in the adult.

Lipopolysaccharide endotoxin potentiates the effect of osmotic stimulation on vasopressin synthesis and secretion in the rat hypothalamus.

Vasopressin secreted by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei is essential for water balance. In this study, we examined magnocellular neurone responses to osmotic stimulation in vehicle-injected controls or rats receiving an intraperitoneal (i.p.) injection of 250 microg/100 g of lipopolysaccharide (LPS), 3 h or 6 h earlier. LPS injection had no effect on plasma vasopressin concentrations in control rats but it caused marked and transient potentiation of the responses to a single i.p. injection of hypertonic saline (five- and two-fold, 3 and 6 h after LPS, respectively). The enhancement of plasma vasopressin responses was independent of plasma sodium concentrations or changes in blood pressure. Basal vasopressin mRNA expression in the paraventricular and supraoptic nuclei decreased slightly 6 h after LPS injection, without changes in vasopressin transcription as indicated by vasopressin heteronuclear (hn) RNA levels. Parvocellular neurones showed expected increases in vasopressin hnRNA expression following LPS injection and a further increase after i.p. hypertonic saline injection (due to the painful component). In contrast to magnocellular vasopressin mRNA expression, the effects of LPS and hypertonic saline injections in parvocellular neurones were additive and not synergistic. Light microscopic immunohistochemical examination revealed an increase in size of vasopressin but not oxytocin axonal terminals in the neural lobe 3 h after LPS injection. Osmotic stimulation caused marked depletion of vasopressin immunoreactivity in axonal terminals of the neural lobe in both control and LPS-pretreated rats. The changes in vasopressin axon terminals were accompanied by induction of interleukin (IL)-1 beta and IL-6 in the posterior pituitary. The data show that endotoxemia causes morphological and functional alterations of the hypothalamic neurohypophyseal system, resulting in facilitation rather than inhibition of vasopressin synthesis, and secretion in response to osmotic stimulation.

Differential regulation of corticotropin-releasing hormone and vasopressin transcription by glucocorticoids.

CRH and vasopressin (VP), the main regulators of pituitary ACTH secretion, co-exist in parvocellular cells of the PVN, but their levels of expression are regulated differentially during manipulations of the hypothalamic pituitary adrenal (HPA) axis. The effects of glucocorticoids on this system was studied using in situ hybridization with intronic and exonic probes to measure changes in CRH and VP messenger RNA (mRNA) and heteronuclear (hn) RNA in 48-h adrenalectomized (ADX) rats receiving injections of corticosterone (2.8 mg/100 g, ip) or vehicle. We also determined the time course of changes in VP expression following the first 72 h of ADX. Levels of VP heteronuclear (hn) RNA and the number of parvocellular cells containing VP hnRNA remained very low in sham operated rats, whereas biphasic changes were observed after ADX. Grain density levels increased 11.5-fold over sham-operated controls by 6 h, declined to 2-fold by 18 h, to increase again to 10- and 20-fold by 48 and 72 h, respectively. In 48-h ADX rats, vehicle injection increased CRH hnRNA levels transiently (11-fold the basal by 15 and 30 min), returning to basal at 60 min, whereas VP hnRNA levels increased progressively up to 28-fold the basal by 2 h. Corticosterone injection had no significant effect on vehicle-induced increases in CRH hnRNA, in spite of marked elevations in circulating corticosterone. In contrast to CRH, VP hnRNA levels increased only transiently by 15 min, and then decreased below basal (near sham-ADX levels) by 2 h. The data show that in normal conditions the responsiveness of parvocellular neurons to stress is under marked inhibition by the low resting levels of glucocorticoids, and that the sensitivity of CRH and VP transcription to glucocorticoid feedback is markedly different.

Vasopressin and corticotropin-releasing hormone gene responses to novel stress in rats adapted to repeated restraint.

Stress-responsive neurons of the hypothalamic paraventricular nucleus (PVN) show functional plasticity and adapt to repeated restraint (RR) stress. To investigate whether neuronal adaptation to the homotypic stress also affects their response to a heterotypic stressor, we used in situ hybridization with intronic and exonic probes to measure primary transcript (hnRNA) and messenger RNA (mRNA) levels for CRH and vasopressin (VP) in the PVN of control and RR rats after the heterotypic stress of i.p. hypertonic saline injection (ipHS). Two weeks of daily restraint blunted plasma corticosterone and parvocellular CRH, but not VP, transcript responses to a further restraint episode. IpHS increased circulating corticosterone in both groups, but levels were higher in RR rats. CRH hnRNA increased within 15 min and returned to baseline by 1 h in both naive and RR rats. CRH mRNA increased more slowly in both groups, peaking at 2 h, with RR rats showing greater responses at this time. Parvocellular VP hnRNA reached a peak 2 h after ipHS in naive rats, but more rapidly (1 h) and to higher levels in RR rats. The number of parvocellular neurons expressing VP hnRNA increased approximately 5-fold after ipHS in both groups. Basal VP mRNA levels and the number of parvocellular cells expressing VP mRNA were elevated in RR rats. Both ipHS and naive rats showed an increase in VP mRNA transcripts after ipHS, with RR rats showing greater levels at 2 and 4 h. Magnocellular cells in both PVN and supraoptic nuclei showed increases in VP hnRNA within 15 min. The data demonstrate VP responses in parvocellular neurons of both control and RR rats, whereas profound inhibition of CRH transcription is selective for the homotypic stressor with CRH responsiveness to the heterotypic stress preserved or increased.

Transcriptional responses of the vasopressin and corticotropin-releasing hormone genes to acute and repeated intraperitoneal hypertonic saline injection in rats

The contribution of corticotropin releasing hormone (CRH) and vasopressin (VP) to the adaptation of ACTH responses to chronic stress was studied by analysis of CRH and VP expression in the hypothalamic paraventricular nucleus (PVN) of rats receiving acute or chronic i.p. hypertonic saline injection (ipHS), a stress model in which the HPA axis is not desensitized after repeated stimulation. Repeated ipHS for 14 days had no effect on CRH hnRNA levels but increased CRH mRNA levels by 42.2%. Parallel with preserved plasma corticosterone responses to repeated ipHS, CRH hnRNA responses and CRH mRNA response to the last injection in repeatedly stressed rats were identical to those in naive rats (8.6-fold increase by 15 min, returning to basal level by 1 h). Parvocellular VP hnRNA responses to a single ipHS were slower and more prolonged than for CRH (7.1-, 11.5-, 9.8- and 4.6-fold by 1, 2, 4 and 6 h), and VP mRNA levels increased by 4 h and remained elevated 12 h later. Parvocellular VP hnRNA was at basal levels after 14 days ipHS, but VP mRNA levels remained elevated as during acute stimulation. Despite high basal mRNA levels, VP hnRNA responses to the last repeated ipHS were minor, suggesting increases in mRNA stability. This study shows that conserved pituitary ACTH responsiveness to a homotypical repeated stress is associated with the ability of parvocellular PVN neurons to increase CRH transcription after repeated stimulation.

In situ hybridization analysis of vasopressin gene transcription in the paraventricular and supraoptic nuclei of the rat: regulation by stress and glucocorticoids.

Hypothalamic arginine vasopressin-containing neurons are prime elements in central circuits regulating the hypothalamo-pituitary-adrenocortical stress response. It is known that release and synthesis of vasopressin are cued by stressful stimuli. The present study was designed to assess effects of stress on vasopressin transcription and mRNA expression in defined populations of vasopressin neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Intron- and exon-directed in situ hybridization analyses were used to examine stress regulation of vasopressin heteronuclear (hn) gene transcription and mRNA levels. Actions of glucocorticoids on vasopressin induction were tested using adrenalectomized rats implanted with subcutaneous pellets delivering a constant, physiological dose of corticosterone. Pellet implantation into adrenalectomized rats allows for normal pituitary-adrenal tone in the absence of the ability to mount glucocorticoid stress responses. Elevation of vasopressin heteronuclear (hn) RNA in the medial parvicellular PVN was observed in both normal and adrenalectomized-corticosterone replaced rats as early as 30 minutes after stress initiation. In control rats, vasopressin hnRNA levels returned to baseline by 120 minutes. In contrast, vasopressin hnRNA remained elevated 120 minutes post-restraint in adrenalectomized-corticosterone replaced rats, indicating that the glucocorticoid stress response acts to rapidly inhibit vasopressin transcription. Significant changes in post-stress vasopressin mRNA levels were observed in the parvicellular PVN of control rats 90 minutes following restraint induction, returning to normal expression profiles by 120 minutes. Adrenalectomized-replaced rats showed elevated vasopressin mRNA expression at all time points examined. No changes were observed in magnocellular vasopressin-containing nuclei at any time point, suggesting that magnocellular vasopressin is not induced by this particular stress paradigm. Thus, in parvicellular paraventricular nucleus neurons the vasopressin gene is rapidly induced by stress. Restraint-induced up-regulation of vasopressin transcription is limited by glucocorticoid secretion, consistent with direct actions of glucocorticoid negative feedback on the vasopressin gene in parvicellular neurons.