Vasopressin mRNA Levels Research Articles

Neuropeptide gene expression in hypothalamic magnocellular neurons of normal and hypophysectomized rats: A combined immunohistochemical and in situ hybridization study

Hypothalamic magnocellular neurons of the paraventricular and supraoptic nuclei contain several peptides and non-peptide putative neurotransmitters co-existing with vasopressin and oxytocin. However, the functional role of these substances is still unknown. In the present paper the temporal course of changes in the expression of vasopressin, oxytocin, galanin, cholecystokinin, dynorphin and tyrosine hydroxylase in magnocellular hypothalamic neurons of rats subjected to hypophysectomy was examined. Following different survival times the animals were processed either for immunohistochemistry with antibodies against the above mentioned peptides or for in situ hybridization with synthetic oligonucleotide probes complementary to the mRNAs encoding for the peptides. The results obtained showed a marked rise in vasopressin mRNA levels at two days followed by a decrease up to 36 days of survival. Oxytocin mRNA responded to the lesion with a transient decrease, with its lowest values between five and seven days. This was followed by a recovery which almost reached normal values at 36 days of survival. The results also showed a marked, transient activation of the synthetic pathway for galanin and cholecystokinin. The numbers of cells expressing these peptides were maximal between five and seven days, and the respective mRNA levels were significantly increased at these survival times. This was followed by a decrease in the amount of galanin- and cholecystokinin-like immunorcactivity as well as in the levels of their respective mRNAs. Dynorphin-like immunoreactivity showed a course similar to that of galanin and cholecystokinin in operated animals. However, the amounts of dynorphin mRNA were significantly increased at two days, but were followed by a reduction at five days and remained low throughout the different survival times tested. The experiments performed with the tyrosine hydroxylase antibodies and probe showed undetectable levels of the enzyme and its mRNA in normal and hypophysectomized animals. These results demonstrate that, in magnocellular hypothalamic neurons, expression of several peptides occur in differential ways after hypophysectomy. The possibility is discussed that these changes represent part of the mechanisms underlying the process of degeneration and regeneration known to occur in magnocellular hypothalamic neurons after hypophysectomy.

Differential steroid hormone and neural influences on peptide mRNA levels in CRH cells of the paraventricular nucleus: A hybridization histochemical study in the rat

The three major classes of neurons in the paraventricular nucleus (PVH) provide a rich model for studying hormonal and neural influences on multiple neuropeptides expressed in individual cells. A great deal of previous work has examined this problem at the immunohistochemical level, where hormonal and neural influences on peptide levels have been established. In situ hybridization methods were used here to determine whether these effects are accompanied by measurable changes in neuropeptide mRNA levels. In the first series of experiments, the time-course of corticosterone replacement effects on corticotropin-releasing hormone (CRH) mRNA levels in parvicellular neuroendocrine cells of adrenalectomized animals were determined, and a dose-response curve was established. CRH mRNA hybridization remains maximal with plasma levels of steroid up to about 50 ng/ml, then declines sharply between about 60-130 ng/ml, and is just detectable at higher levels. We confirmed that corticosterone decreases vasopressin mRNA levels in this cell group and showed that levels of preproenkephalin mRNA are also decreased, whereas no significant changes in cholecystokinin, beta-preprotachykinin, and angiotensinogen mRNA levels could be detected. Thus, corticosterone decreases some neuropeptide mRNA levels and has no influence on others in this cell group. Tyrosine hydroxylase mRNA hybridization is also unaffected in this part of the nucleus. In a second group of experiments, the cell-type specificity of corticosterone influences was examined. It was found that while the hormone depresses CRH mRNA levels in parvicellular neurons, it increases such levels in PVH neurons with descending projections, in certain magnocellular neurosecretory neurons, and in a part of the central nucleus of the amygdala, whereas no influence was detected in the rostral lateral hypothalamic area. Furthermore, the stimulatory effects of corticosterone have different threshold levels in different cell groups. Thus, in different types of neurons, corticosterone may increase, decrease, or have no influence on CRH mRNA levels. In contrast, while corticosterone depresses vasopressin mRNA levels in parvicellular CRH neurons, it has no obvious effects on vasopressin mRNA levels in magnocellular or descending neurons; as with CRH, the effects of corticosterone on vasopressin mRNA levels are cell-type specific. In a third series of experiments it was shown that glucocorticoid receptor and mineralocorticoid receptor mRNAs are found in all three cell types in the PVH and that corticosterone tends to produce modest increases in mRNA levels for both receptors. Finally, it was shown that unilateral catecholamine-depleting knife cuts do not change mRNA levels for any of the neuropeptides (or steroid hormone receptors) examined here, although dramatic changes in neuropeptide levels themselves have been shown.4+

Quantitation of vasopressin and oxytocin mRNA levels in the brain

Publisher Summary Recent developments in molecular biology and the isolation of the two different but closely related genes for vasopressin (VP) and oxytocin (OT) made it possible to develop methods for the quantitation of the messenger RNA (mRNA) levels of these two neuropeptides. These mRNA levels are a reflection of the gene transcription rates, and they also provide a useful index for the biosynthetic capacity of neurons to synthesize a given peptide. The use of the vasopressin and oxytocin systems for studies on neuropeptide gene regulation is very advantageous, because the vasopressinergic and oxytocinergic neurons are clearly grouped into distinct nuclei of the brain that are linked to peripheral or central functions of these neuropeptides. Just as the solution hybridization assay, dot-blot analysis can provide accurate levels of VP mRNA and OT mRNA but not information on the nature and integrity of the RNA. Although the dot-blot assay requires a more extensive deproteination of RNA samples and takes more time to obtain results, it has the advantage that it has a higher sensitivity due to the steep standard curve.

Differential responses in vasopressin and oxytocin gene expression in distinct hypothalamic nuclei after hypothalamoneurohypophyseal disconnection and vasopressin substitution.

A possible feedback of circulating vasopressin (VP) on the expression of its gene in the hypothalamoneurohypophyseal system was studied in rats with depleted stores of neurohypophyseal hormones. The neural lobe of the pituitary gland was disconnected by anterolateral deafferentation of the basal hypothalamus using a Halasz knife cut. One group of lesioned animals was substituted continuously with VP using mini-osmopumps. After 1 week, the lesion had caused a complete disappearance of neurohypophyseal hormones from the neurointermediate pituitary and induced a 2-fold increase in water intake and an increase in plasma osmolality of approximately 7 m0sm/kg. VP mRNA levels of the supraoptic nucleus (SON), paraventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) were measured in punched tissue by Northern blot analysis and dot-blot analysis. VP mRNA levels of the SON and PVN were reduced by 50% due to the deafferentiation, which might have been caused by degeneration. VP substitution of the lesioned animals normalized water intake but not plasma osmolality. In the VP-substituted animals, VP mRNA levels of the SON were 2-fold reduced compared to the nonsubstituted lesioned animals. The VP mRNA levels of the PVN were not affected by VP substitution. The VP mRNA levels of the SCN increased 2-fold after the lesion but were not influenced by VP substitution. Concomitant determination of oxytocin (OT) mRNA showed that OT mRNA levels in the PVN and SON were not affected by the deafferentiation and did not respond to VP substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin and oxytocin gene expression in the supraoptic and paraventricular nucleus of the spontaneously hypertensive rat (SHR) during development of hypertension

To study the regulation of hypothalamic vasopressin (VP) and oxytocin (OT) gene expression in relation to the development of hypertension, levels of VP mRNA and OT mRNA were determined in spontaneously hypertensive rats (SHR). Differences in VP and OT mRNA content of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of 4- and 10-week-old SHR and Wistar-Kyoto controls (WKY) were quantitated by dot-blot and Northern blot analysis. VP and OT pituitary content and VP plasma levels were measured by radioimmunoassays. VP mRNA levels were approximately 2-fold and 3-fold higher in the SON and PVN of 4-week-old SHR, respectively, as compared to controls. The OT mRNA levels were approximately 35% lower in both nuclei of the SHR. There was no difference in VP and OT pituitary content between 4-week-old SHR and WKY, but VP plasma levels were higher in SHR. In the 10-week-old SHR VP mRNA levels were still approximately 30–40% higher and the OT mRNA levels were approximately 40% lower in both nuclei when compared to age-matched WKY. Pituitary VP and OT contents were respectively 1.5-fold higher and 20% lower in the 10-week-old SHR than in 10-week-old WKY. VP plasma levels were still elevated in the SHR. The data indicate that in the hypothalamo-neurohypophyseal system of the SHR the VP system is in a higher state of activity, while the OT system is lower in activity.

Diurnal variation in vasopressin and oxytocin messenger RNAs in hypothalamic nuclei of the rat

Diurnal changes in the expression of the vasopressin (VP) and oxytocin (OT) genes in the supraoptic (SON), paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the rat were investigated by dot-blot and in situ hybridization of the VP and OT mRNAs. A significant diurnal variation in VP mRNA level was measured in the SCN, with highest levels around 17.00 h and lowest levels around midnight. No variations in levels of VP mRNA and OT mRNA were detected in SON and PVN. The data indicate that the regulation of the VP gene in the SCN is independent of that in the magnocellular nuclei.

Oxytocin and vasopressin gene expression during gestation and lactation

Levels of rat hypothalamic oxytocin (OT) and vasopressin (AVP) mRNA were determined during gestation and lactation using a densitometric hybridization assay. Pregnancy induced a gradual rise in OT mRNA, reaching, by day 18, a level exceeding control by a factor of 2.5. Throughout the lactation period OT mRNA remained elevated at levels corresponding to 3 times that of control. Surprisingly, the dynamics of AVP mRNA accumulation paralleled closely the profile observed for OT mRNA throughout the time period studied. We conclude that in late pregnancy and lactation the expression of both, the OT and the AVP gene is stimulated in parallel by mechanisms operating at a pretranslational level, involving increased gene transcription or mRNA stabilization, or both. Further characterization of the two mRNA species revealed that both mRNAs are endowed with very long poly(A) tails of > 200 nucleotides, under conditions of both, normal and stimulated states of gene expression. The role, if any, of the prolonged poly(A) tails in mRNA stability remains to be determined.

The vasopressin gene is expressed prior to regulation in the supraoptic nuclei of fetal rats

The development of the regulation of vasopressin (prepropressophysin) mRNA in the supraoptic nuclei (SON) of rats was studied using quantitative in situ hybridization. On day 21 of gestation, vasopressin mRNA levels in the SON were increased by osmotic stimulation, with significant positive correlation between vasopressin mRNA levels and plasma osmolality. On day 19 of gestation, vasopressin mRNA levels in the SON were not significantly increased by osmotic stimulation, and no correlation between vasopressin mRNA levels and plasma osmolality was noted. The results suggest that the mechanisms for osmotic regulation of vasopressin mRNA levels develop between days 19 and 21 of gestation, a time course consistent with the initiation of afferent innervation of the SON.

Oxytocin and vasopressin gene expression in the hypothalamo-neurohypophyseal system of the rat during the estrous cycle, pregnancy, and lactation.

To study the state of hypothalamic expression of the oxytocin (OT) and vasopressin (VP) genes in different conditions of the female rat, OT and VP mRNA levels were determined during the estrous cycle, pregnancy, and lactation. OT and VP mRNA levels of the supraoptic nucleus were quantified by Northern blot and dot-blot analysis. OT and VP contents of the pituitary gland were determined by RIA. During the estrous cycle, OT mRNA levels of the supraoptic nucleus were significantly increased 1.5- to 2-fold at estrous relative to the other periods of the cycle. No significant cyclic variation was observed in VP mRNA levels. The OT content of the pituitary gland was significantly decreased at estrus and also the VP contents showed a cyclic variation with the lowest levels occurring at estrus and metestrus. At the last day of gestation a 3- and 2-fold increase of OT mRNA and VP mRNA, respectively, was measured, but no changes were observed at gestational day 12, 15, and 18. In rats lactating for 15 days, OT and VP mRNA levels were approximately 3-fold higher than during the estrous cycle. Concomitantly the OT and VP contents of the pituitary gland were decreased by approximately 50%. It is concluded that both OT and VP gene expression are stimulated just before birth and during lactation indicating that both genes are sensitive to common factors. A dissociation of the regulation of the OT and VP genes occurs in the estrous cycle.

Regulation of vasopressin and oxytocin synthesis in anterior pituitary and peripheral tissues.

Recent studies have demonstrated the presence of immunoreactive oxytocin (OT) and vasopressin (VP), OT and VP receptors and physiological functions for these two hormones in a variety of peripheral tissues, including anterior pituitary gland. The objectives of this study were to determine if (i) OT and VP genes are expressed in rat testis and anterior pituitary gland and (ii) if osmotic stimulation known to modify the regulation of OT and VP genes in hypothalamus, would modify the expression of these genes in rat testis and anterior pituitary gland. Using oligonucleotide probes (courtesy of Drs. M. Brownstein and W. Scott Young, NIMH) corresponding to the VP gene or OT gene and specific fractions of human OT and VP genes (courtesy of Dr. J. Battey, NCI) subcloned in the pGEM-3 riboprobe system, and Northern blot and slot blot techniques, OT and VP mRNAs were found in rat testis and anterior pituitary gland. When adult male rats (SD) were either deprived of drinking water or offered 2% salt solution as a sole source of drinking fluid for 72 hrs, both OT and VP mRNA levels were increased in hypothalamus, anterior pituitary gland and testis. Our data suggest that testis and anterior pituitary gland could also be sites of synthesis of OT and VP and that the same stimulus may regulate these genes in various tissues.

Vasopressin messenger ribonucleic acid in supraoptic and suprachiasmatic nuclei: appearance and circadian regulation during development.

The developmental appearance and regulation of hypothalamic vasopressin (prepropressophysin) mRNA was studied using quantitated in situ hybridization techniques. Vasopressin mRNA levels in the supraoptic nuclei were reliably detected on day 16 of gestation, while mRNA in the suprachiasmatic nuclei (SCN) was detectable on day 21. These developmental patterns correlate well with the immunohistochemical appearance of prepropressophysin translation products previously reported in these nuclei. A prominent day-night rhythm of vasopressin mRNA levels was evident in the SCN on day 21 of gestation; the rhythm was also present on postnatal days 2 and 11. No such rhythm was present in the supraoptic nuclei at any developmental stage examined. These results demonstrate regulated expression of the vasopressin gene during fetal life. Vasopressin mRNA levels in the SCN are already under specific circadian control when the nuclei are morphologically immature and lacking many of the connections found in adult animals.

Oxytocin Gene Expression in Discrete Hypothalamic Magnocellular Cell Groups Is Stimulated by Prolonged Salt Loading

The effect of prolonged osmotic stimulation on the oxytocin (OT) mRNA levels of OT-producing neurons was investigated in separate hypothalamic nuclei of the rat. After drinking 2% NaCl for 2 weeks, a 2-fold increase in the OT mRNA content was found by Northern blot analysis of microdissected supraoptic nucleus (SON) and paraventricular nucleus (PVN). The same samples showed a similar change in vasopressin (VP) mRNA levels. This treatment resulted in a 70% depletion of both OT and VP from the neurointermediate lobe of the pituitary gland and a marked increase in OT and VP plasma levels. Analysis of the OT mRNA concentration by quantitative in situ hybridization showed that the SON, PVN, and anterior commissural nucleus responded similarly, with a 1.5-fold increase in OT mRNA after 7 days of osmotic stimulation. These results demonstrate that OT gene expression in the magnocellular neurons of the SON, PVN, and anterior commissural nucleus is sensitive to this osmotic stimulus, as is the VP gene of magnocellular neurons in the SON and PVN. The responsiveness of OT-producing neurons may point to a role for OT in the regulation of water and electrolyte balance.

Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry

35S-labeled synthetic oligodeoxyribonucleotide probes were used to measure levels of vasopressin (VP) and oxytocin (OT) mRNAs in rat hypothalamus by quantitative in situ hybridization histochemistry (ISHH). VP and OT mRNA-containing cells were seen in the paraventricular (PVN) and supraoptic (SON) nuclei. VP mRNA was found to increase five-fold in the parvocellular region of the PVN after adrenalectomy while no changes occurred in magnocellular VP or OT mRNA levels. In the Brattleboro rat, VP mRNA levels were decreased and OT mRNA levels increased in the magnocellular regions. RNA species containing the VP introns were present at one fortieth of the level of processed VP mRNA in control rats. We also performed ISHH followed by immunohistochemistry on the same sections. We found that VP and its encoding mRNA were always located together as were OT-neurophysin and its encoding mRNA. In this study, we extend previous work by showing the characteristic distributions in the PVN and SON of VP and OT mRNA-containing cells and by measuring neuropeptide mRNA changes.

Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by solution hybridization assays.

Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magnocellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on recombinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was less than 1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdissected supraoptic nucleus (SON) mean (+/- SEM) basal levels of 1.37 +/- 0.18 pg VP mRNA and 1.95 +/- 0.14 pg OT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 +/- 0.02 pg VP mRNA and 1.77 +/- 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85-fold increase in VP mRNA levels of the SON and a 1.6-fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.

Vasoressin gene expression is attenuated in the fetal Brattleboro rat

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.

Vasopressin mRNA regulation in individual hypothalamic nuclei: a northern and in situ hybridization analysis

The present study examines the relative levels of vasopressin (AVP) mRNA within the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei of the rat hypothalamus, and details the rates at which these levels change over the course of a 6 d salt-loading regimen. The quantitation of vasopressin mRNA was achieved by using three different procedures: (1) cell-free translation in rabbit reticulocyte lysate or (2) Northern analysis of poly(A)RNAs isolated from micro-punch dissected SON, PVN, and SCN, and (3) in situ hybridization histochemistry. The former involved the quantitative immunoprecipitation of the neurophysin precursors containing arginine8-vasopressin (AVP) or oxytocin, and the latter two techniques employed a radiolabeled synthetic oligodeoxynucleotide complementary to the 3' region of the AVP mRNA. Both the cell-free studies and the Northern gel analyses detected a sevenfold increase of AVP mRNA in the SON, a fivefold increase in the PVN, and no significant change in the SCN following 6 d of salt-loading. After the initiation of salt-drinking, these increases were shown to occur between 24 and 48 hr in the SON and between 48 and 72 hr in the PVN. The in situ hybridization studies revealed the anatomically correct hybridization of either 32P- or 3H-labeled AVP oligonucleotide to magnocellular perikarya within both the SON and PVN. Autoradiographic grains could be shown to be confined to the cytoplasm of these cells, and could be co-localized with immunoreactivity directed against the carboxy terminus of the AVP percursor. Comparison of x-ray level autoradiograms of control and 6 day salt-loaded SON revealed up to a sevenfold increase in specific signal in the salt-loaded sections. It is concluded that the response of AVP mRNA to osmotic stimuli in the three hypothalamic nuclei is heterogeneous, and that this heterogeneity can be explained by separating AVP neurons into two systems: one responsible for eliciting the antidiuretic actions of AVP via plasma AVP levels, and the other involved in CNS activities not directly involved with antidiuresis.

Glucocorticoid sensitivity of vasopressin mRNA levels in the paraventricular nucleus of the rat.

Immunocytochemical studies have shown that adrenalectomy produces changes in the content and distribution of [arginine-8]vasopressin (AVP) immunoreactivity in the paraventricular nucleus of the hypothalamus. The purpose of this study was to determine whether manipulation of adrenal hormones affects the levels of AVP mRNA. In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes and immunocytochemistry were used to detect the distribution of AVP mRNA and AVP-immunoreactive perikarya. AVP mRNA is codistributed with AVP immunoreactivity in the posterior magnocellular subdivision of the paraventricular nucleus and its accessory nuclei, the supraoptic nucleus and the suprachiasmatic nucleus. In adrenalectomized rats, the density and distribution of the hybridization signal were increased in the paraventricular nucleus; a 2-fold increase in the area comprising the signal was observed. At the cellular level, silver grains were detected in corticotropin-releasing-factor-immunoreactive neurons throughout the medial parvocellular subdivision of the paraventricular nucleus. No changes were seen in the distribution of AVP mRNA in the supraoptic or suprachiasmatic nuclei. Treatment with dexamethasone prevented the increase in AVP mRNA produced by adrenalectomy. In contrast, adrenalectomy did not alter the hybridization signal obtained with a probe for alpha-tubulin mRNA. These results suggest, at the cellular level, that adrenalectomy induces a glucocorticoid-sensitive stimulation of AVP mRNA synthesis in the central nervous system. Thus, considerable plasticity in gene expression is retained in the hypothalamus of the adult rat.