Oxytocin mRNA Levels Research Articles

Profound Impairment in Social Recognition and Reduction in Anxiety-Like Behavior in Vasopressin V1a Receptor Knockout Mice

Considerable evidence suggests that arginine vasopressin (AVP) is critically involved in the regulation of many social and nonsocial behaviors, including emotionality. The existence of two AVP receptors in the brain, namely the V1a and V1b subtypes, and the lack of clear pharmacological data using selective agonists or antagonists, make it difficult to determine which receptor is responsible for the AVP-mediated effects on behavior. Here we report the behavioral effects of a null mutation in the V1a receptor (V1aR) in male mice. Male mice lacking functional V1aR (V1aRKO) exhibit markedly reduced anxiety-like behavior and a profound impairment in social recognition. V1aRKO performed normally on spatial and nonsocial olfactory learning and memory tasks. Acute central administration of AVP robustly stimulated stereotypical scratching and autogrooming in wild-type (WT), but not V1aRKO males. AVP and oxytocin (OT) mRNA and OT receptor-binding levels were similar in WT and V1aRKO mice. Given the current findings, the V1aR may provide a novel potential pharmacological target for social and affective disorders including autism, and anxiety disorders.

Six-hour selective REM sleep deprivation increases the expression of the galanin gene in the hypothalamus of rats

The effect of short-term selective REM sleep deprivation (RSD) on the gene expression of galanin in the rat hypothalamus was examined using in situ hybridization histochemistry. Monitoring an electroencephalogram (EEG) and electromyogram (EMG) on an on-line computer screen, as the RSD rats entered REM sleep, they were gently stroked on their backs using a brush to wake them during the RSD period. Galanin mRNA levels in the preoptic area (POA) were significantly increased by RSD for a period of 6 h. RSD had no significant effect on the mRNA levels of corticotrophin-releasing factor (CRF), arginine vasopressin (AVP), oxytocin (OXT) or orexins. These results suggest that 6-h selective RSD may not be sufficient to induce the activation of the hypothalamo-pituitary adrenal axis, and that the expression of the galanin gene in the hypothalamus reacts more readily against the loss of REM sleep in comparison to other hypothalamic neuropeptides such as arginine vasopressin, oxytocin and orexins.

Reduced sodium appetite and increased oxytocin gene expression in mutant mice lacking β-endorphin

Central opioid and oxytocinergic systems have been involved in the regulatory control of sodium appetite. In addition, previous studies support the existence of a functional interaction between opioid peptides and oxytocinergic pathways, and suggest that β-endorphin neurons would modulate the activity of central oxytocinergic pathways, its pituitary secretion and sodium appetite. To investigate the role of this opioid peptide in the control of oxytocin (OT) synthesis and sodium appetite regulation we used mice with gene dosage-dependent variations in brain β-endorphin content, expressing either 100%, 50%, or 0% of normal β-endorphin content. Our results show that β-endorphin knockout (KO) and heterozygous (HT) mutant mice consume approximately a 50% less 2% NaCl solution compared with wild type mice (WT), after furosemide and low sodium diet treatment. These data suggest that β-endorphin may facilitate induced sodium appetite, giving new evidence about the role of β-endorphin on sodium appetite behavior. Our data also indicate that OT mRNA levels evaluated by in situ hybridization significantly increased within the hypothalamic paraventricular nucleus of WT animals after induced sodium ingestion, giving support to former evidence indicating an inhibitory role for central OT in the control of sodium appetite. Moreover, β-endorphin mutated mice have similar higher levels of OT mRNA expression after the different conditions analyzed: basal, control or experimental, compared with WT mice. Both control HT and KO mice showed higher OT mRNA expression levels than control WT group and these levels did not change after induced sodium intake. Taken together, our data suggest that the reduced sodium ingestion observed in β-endorphin deficient mice could be due to a higher expression of the OT gene. This conclusion would support the hypothesis that OT inhibits sodium intake and provides new evidence about β-endorphin modulation of OT synthesis and sodium appetite.

Estrogen receptor-β regulates transcript levels for oxytocin and arginine vasopressin in the hypothalamic paraventricular nucleus of male mice

Estrogen receptor (ER)-β, unlike ER-α, is localized in the hypothalamic paraventricular nucleus (PVN) which also contains neuropeptide synthesizing neurons, such as oxytocin (OT), arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH). Although it is known that some ER-β containing neurons co-express OT and AVP, but not CRH, in the PVN, it is not yet determined whether ER-β activation may indeed play a role in estrogenic regulation on syntheses of these neuropeptides. In the present study, we tested this hypothesis by comparing the effects of estrogen on the levels of OT, AVP and CRH messenger RNA (mRNA) between ER-β knockout (βERKO) and wild type (WT) control male mice. Mice were gonadectomized and implanted with either a pellet containing estradiol benzoate (2.5–5.0 μg/day) or a placebo pellet for 21 days. In situ hybridization histochemistry revealed that estrogen treatment resulted in a significant increase in OT transcripts (151.6±6.0%) and a decrease in AVP transcripts (77.8±5.2%) in the PVN of WT mice, compared to the placebo control group. This estrogenic regulation of OT and AVP mRNA levels in the PVN was completely abolished in βERKO mice. Similar genotype differences in the effects of estrogen on the numbers of OT- and AVP-containing cells were found in immunocytochemical studies performed in a separate set of mice. On the other hand, the expression of CRH mRNA in the PVN was not affected by estrogen treatment in either WT or βERKO mice. Furthermore, estrogen did not cause any changes in the levels of OT or AVP mRNA, regardless of genotype, in the supraoptic nucleus where, unlike in rats, ER-β containing neurons are rarely found in mice. Finally, estrogen significantly increased OT mRNA levels in both βERKO and WT in the medial preoptic area, which contains both ER-α and ER-β. These results suggest that ER-β activation may play a critical role in estrogenic regulation of OT and AVP gene expression in the PVN.

Prolonged alcohol intake leads to irreversible loss of vasopressin and oxytocin neurons in the paraventricular nucleus of the hypothalamus

Previous data revealed that numerous neurons in the supraoptic nucleus degenerate after prolonged ethanol exposure, and that the surviving neurons increase their activity in order to prevent dramatic changes in water metabolism. Conversely, excess alcohol does not induce cell death in the suprachiasmatic nucleus, but leads to depression of neuropeptide synthesis that is further aggravated by withdrawal. The aim of the present study is to characterize the effects of prolonged ethanol exposure on the magnocellular neurons of the paraventricular nucleus (PVN) in order to establish whether or not magnocellular neurons display a common pattern of reaction to excess alcohol, irrespective of the hypothalamic cell group they belong. Using conventional histological techniques, immunohistochemistry and in situ hybridization, the structural organization and the synthesis and expression of vasopressin (VP) and oxytocin (OXT) in the magnocellular component of the PVN were studied under normal conditions and following chronic ethanol treatment (6 or 10 months) and withdrawal (4 months after 6 months of alcohol intake). After ethanol treatment, there was a marked decrease in the number of VP- and OXT-immunoreactive magnocellular neurons that was attributable to cell death. The surviving neurons were hypertrophied and the VP and OXT mRNA levels in the PVN unchanged. Withdrawal did not alter the number of VP- and OXT-producing neurons or the gene expression of these peptides. These results substantiate the view that after prolonged ethanol exposure numerous neurons of the hypothalamic magnocellular system degenerate, but the mRNA levels of VP and OXT are not decreased due to compensatory changes undergone by the surviving neurons.

Forced swimming stimulates the expression of vasopressin and oxytocin in magnocellular neurons of the rat hypothalamic paraventricular nucleus.

Previous studies have shown that a 10-min forced swimming session triggers the release of both vasopressin and oxytocin into the extracellular fluid of the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) in rats. At the same time oxytocin, but not vasopressin, was released from the axon terminals into the blood. Here we combined forced swimming with in situ hybridization to investigate whether (i) the stressor-induced release of vasopressin and oxytocin within the PVN originates from parvo- or magnocellular neurons of the nucleus, and (ii) central release with or without concomitant peripheral secretion is followed by changes in the synthesis of vasopressin and/or oxytocin. Adult male Wistar rats were killed 2, 4 or 8 h after a 10-min forced swimming session and their brains processed for in situ hybridization using 35S-labelled oligonucleotide probes. As measured on photo-emulsion-coated slides, cellular vasopressin mRNA concentration increased in magnocellular PVN neurons 2 and 4 h after swimming (P < 0.05). Similarly, oxytocin mRNA concentration was significantly increased in magnocellular neurons of the PVN at 2 and 8 h (P < 0.05). We failed to observe significant effects on vasopressin and oxytocin mRNA levels in the parvocellular PVN and in the SON. Taken together with results from previous studies, our data suggest that magnocellular neurons are the predominant source of vasopressin and oxytocin released within PVN in response to forced swimming. Furthermore, in the case of vasopressin, central release in the absence of peripheral secretion is followed by increased mRNA levels, implying a refill of depleted somato-dendritic vasopressin stores. Within the SON, however, mRNA levels are poor indicators of the secretory activity of magnocellular neurons during stress.

Chronic Hypoosmolality Induces a Selective Decrease in Magnocellular Neurone Soma and Nuclear Size in the Rat Hypothalamic Supraoptic Nucleus

AbstractThe magnocellular neurones of the hypothalamo‐neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two‐fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10–20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for βIII‐tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus.

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality.

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10-20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na(+)-K(+)-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

The neurosteroid allopregnanolone modulates oxytocin expression in the hypothalamic paraventricular nucleus.

Virgin, ovariectomized rats exposed to 2 wk of sequential estradiol (E(2)) and progesterone (P) followed by P withdrawal have increased hypothalamic oxytocin (OT) mRNA and peptide levels relative to sham-treated animals. This increase is prevented if P is sustained. In the central nervous system, P is metabolized to the neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), which exerts effects by acting as a positive allosteric modulator of GABA(A) receptor/Cl(-)-channel complexes. In the present study, ovariectomized rats that received sequential E(2) and P for 2 wk followed by P withdrawal were administered allopregnanolone at the time of P withdrawal. Hypothalamic and plasma allopregnanolone concentrations, serum E(2) and P concentrations, and hypothalamic OT mRNA levels were measured at death. Steroid-induced increases in OT mRNA were attenuated in animals treated with allopregnanolone at the time of P withdrawal. The results suggest that allopregnanolone plays an important modulatory role in steroid-mediated increases in hypothalamic OT.

The effects of progesterone on oxytocin mRNA levels in the paraventricular nucleus of the female rat can be altered by the administration of diazepam or RU486.

Oxytocin (OT) facilitates the onset of maternal behaviour in the late pregnant rat, enhances uterine contractility at parturition, and elicits milk ejection during lactation. If the rising estradiol (E2 and declining progesterone (P) of late pregnancy is reproduced in a virgin ovariectomized rat by implanting E2- and P-filled capsules for 2 weeks followed by removal of P-containing implants 36-48 h prior to death, OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of the rat. Both E2 administration and P withdrawal are necessary to increase OT mRNA, but the mechanisms of these effects are not understood. P may work within the PVN although P receptors are reported to be sparse or non-existent in the PVN or outside the PVN on PR-containing neurones that project to OT-containing neurones or via membrane bound receptors that are known to bind neurosteroids and gamma aminobutyric acid (GABA). To determine the mechanism through which P may inhibit or P withdrawal may increase OT mRNA levels, virgin ovariectomized (OVX) rats received sequential E2 and P via Silastic implants for 14 days. On day 13, prior to removal of P capsules on day 14, the rats were given the benzodiazepine agonist, diazepam, or saline injections subcutaneously (s.c.) twice daily until death on day 16. OT mRNA levels were increased in the steroid-treated group that received saline but not diazepam. In experiment 2, P capsules were removed on day 14 or pharmacological P withdrawal was induced by injecting RU486 injections s.c. twice daily until death 48 h later. OT mRNA levels were increased in the steroid-treated group that received RU486. Subsequent studies demonstrated the expression of PR mRNA within the rat PVN. The data suggest that gonadal steroids may influence PVN OT mRNA levels by modulating the GABA(A) receptor or by directly altering gene transcription via the PR.

Hypothalamic oxytocin in the developing ovine fetus: interaction with pituitary–adrenocortical function

Oxytocin (OT) stimulates corticotroph function in adult sheep, however, there is little information on OT synthesis and its potential involvement in hypothalamo-pituitary-adrenal (HPA) function in the fetus. The objectives of this study were to examine developmental changes in hypothalamic OT synthesis and to investigate the actions of OT on fetal corticotroph function. Hypothalami were removed at various stages of pre- and post-natal development. OT mRNA levels were measured using in situ hybridization. For in vitro studies, fetal pituitaries were removed on days 129 and 138 of gestation. Anterior pituitary cells were dispersed and cells were treated with different concentrations and combinations of OT, corticotrophin-releasing hormone (CRH), vasopressin (AVP) and cortisol. OT mRNA was present in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by day 60 of gestation, and levels significantly increased at term. OT mRNA was present in parvocellular and magnocellular fields of the PVN. In vitro, OT stimulated adrenocorticotropin (ACTH) output in a dose-dependent fashion, but had no effect on cellular pro-opiomelanocortin (POMC) mRNA levels. There was no significant difference in corticotroph responsiveness to secretagogues between cells harvested at gestation day 129 or gestation day 138. Simultaneous exposure to CRH and OT stimulated increases in ACTH output that were significantly greater than for OT or CRH alone. However, no similar synergistic interaction existed between OT and AVP. Cortisol attenuated OT-stimulated ACTH output. In conclusion, hypothalamic OT mRNA increases at term and OT can stimulate ACTH output from fetal corticotrophs. Together, these data indicate that OT may be involved in the regulation of ACTH secretion in fetal sheep in late gestation.

Thyroid hormones and estrogen affect oxytocin gene expression in hypothalamic neurons.

The oxytocin (OT) gene promoter has a composite hormone response element, such that several members of the steroid/thyroid hormone superfamily of nuclear receptors can interact at this response element in vitro. To investigate this in brain tissue, parallel to foregoing behavioural experiments, we used in situ hybridization histochemistry to seek interactions between estrogen and thyroid hormones on OT mRNA in the hypothalamus. In ovariectomized (OVX) rats, high doses of triiodothyronine (T3) elevated OT mRNA levels in the paraventricular (PVN) nucleus, while treatment with estradiol benzoate (EB) alone had no significant effect. In contrast, animals that were thyroidectomized (TX) in addition to OVX had dramatically elevated levels of OT gene expression in the PVN following EB treatment. That is, endogenous thyroid hormones interfered with EB-induction of gene expression. Moreover, in both OVX and TX/OVX animals, OT gene expression was reduced to values equivalent to controls when T3 was given together with EB. Particular subdivisions of the PVN responded differentially to T3 and EB treatment, demonstrating marked heterogeneity of OT-containing neurons in this nucleus. Thus, parallel to and perhaps related to the manner in which thyroid hormones reduced estrogen-stimulated behaviour, endogenous or exogenous thyroid hormones interfered with estrogen stimulation of OT mRNA. These data demonstrate competition between nuclear proteins, transcription factors, in hypothalamic neurons.

Increased Expression of Magnocellular Vasopressin mRNA in Rats with Deoxycorticosterone-Acetate Induced Salt Appetite

The neuropeptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated in the genesis of hypertension due to deoxycorticosterone acetate (DOCA)-salt treatment of uninephrectomized rats. In this work, we studied if DOCA treatment of intact rats in doses arousing a salt appetite (a prehypertensive state), modulated mRNA for AVP and OT in the hypothalamus. Male Sprague-Dawley rats were offered both tap water and 3% NaCl in separate bottles and received vehicle or subcutaneous injections of 10 mg DOCA on alternate days for 7 days (4 injections) or 17 days (9 injections). They developed a preference for 3% NaCl solutions 24–48 h after treatment. Brain slices from rats killed on the 8th or 18th day were exposed to ³⁵S-labeled probes encoding prepro-AVP mRNA or OT mRNA, respectively. Expression of these mRNAs was measured in the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) and magnocellular cells of the supraoptic nucleus (SON). No changes were obtained in neuropeptide mRNA levels in the parvocellular division of the PVN between control and the two groups of DOCA-treated rats. However, DOCA-treated animals presented an increased number of grains per cell for AVP mRNA in the magnocellular division of the PVN and in magnocellular cells of the SON, as shown by group mean comparisons and frequency histograms. No changes were detected for OT mRNA. In a second series of studies, control or DOCA-treated rats were offered 3% NaCl or water as the only choice. Animals drinking 3% NaCl showed increased AVP and OT mRNA levels, whether they received DOCA or not. However, AVP mRNA levels in both nuclei were higher in DOCA-treated rats drinking 3% NaCl than in controls drinking salt solution. In comparison, control and DOCA-treated rats drinking water showed lower levels of AVP mRNA. OT mRNA levels in the SON remained unchanged in the same groups. The results suggest that in the magnocellular cells of the PVN and SON, increments in AVP mRNA are obtained following increments in salt intake produced by either mineralocorticoid treatment or exclusive salt drinking. In rats offered salt solution and water to drink, DOCA effects on AVP mRNA developed before changes occurred in serum sodium levels. Because combined DOCA + salt treatment induced a higher response in terms of AVP mRNA expression, we suggest that AVP could be a target of the central effects of the mineralocorticoid.

Cell-specific gene expression in oxytocin and vasopressin magnocellular neurons.

The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neurohypophysial system (HNS) have been the most studied of all the neuroendocrine cell-types. Despite this, our understanding of the mechanisms that underly the cell-specific expression of the peptide genes in these neurons has remained obscure. Part of the reason for this may be related to the close apposition of the OT and VP genes in the chromosomal locus, the genes being separated by as little as 3.5 kb in the mouse, and their interactions which are critical for cell-specific expression of the genes. Recent studies using intact rat OT and VP constructs in transgenic mice, and rat and mouse VP genes with CAT inserts in exon III as reporters in transgenic rats and mice, respectively, have suggested the presence of cell-specific enhancer elements in the 3' downstream (intergenic region, IGR) region of the VP gene. Evidence in favor of this view is presented from transgenic mouse studies on the expression of mouse OT- and VP-CAT gene constructs. Oxytocin and vasopressin phenotypes in the magnocellular neuronal population have traditionally been assessed by either immunocytochemical or in situ hybridization histochemical methods leading to the view that these genes are never coexpressed. However, more sensitive methods show that most OT cells also express some VP mRNA, and most VP cells contain some OT mRNA. A third phenotype containing equivalent levels of both OT and VP mRNA can also be found under some conditions, thereby complicating our analysis of cell-specificity. A continuing problem hindering studies of the regulation of OT and VP gene expression in neurons, is the absence of an appropriate cell line to examine these issues. We have found that stationary slice-explant cultures allow for excellent preservation of highly differentiated magnocellular neurons in long-term culture, and that these cultures can be used for physiological and pharmacological studies and analysis of gene expression.

Time course of induction of oxytocin messenger ribonucleic acid levels in the hypothalamic paraventricular nucleus of ovariectomized rats following gonadal steroid administration

The nonapeptide oxytocin (OT) is important for uterine contractility at parturition, milk ejection during lactation, and the induction of maternal behavior. OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of late pregnant and lactating rats and are modulated by the steroid milieu that accompanies these states. Specifically, sequential exposure to estradiol (E 2) and progesterone (P) followed by P withdrawal 48 hrs prior to sacrifice increases PVN, and to a lesser but significant degree, SON OT mRNA. To better define the time course of induction of OT mRNA levels following P withdrawal, ovariectomized Sprague-Dawley rats were treated with empty or steroid-filled capsules. On day 1, animals received an E 2-filled or empty capsule, followed by P-filled or empty capsules on day 3. On day 14, P-filled or empty capsules were removed and animals were sacrificed 24, 36, or 48 hrs later. The hypothalamic PVN were analyzed for OT mRNA by in situ hybridization histochemistry. Significant differences in PVN OT mRNA were found among the groups (P < 0.0001, Kruskal-Wallis). Animals in the 48 hr (P = 0.007) and 36 hr (P = 0.005), but not the 24 hr, steroid-treated groups had significantly increased OT mRNA relative to their respective sham-treated cohorts (Mann-Whitney U test). The relative abundance of PVN OT mRNA differed among the steroid-treated groups (Kruskal-Wallis, P < 0.0003), with highest levels at 48 hr. We conclude that increases in PVN OT mRNA occur by 36 hrs, and are highest at 48 hrs, after P withdrawal in the E 2-primed rat. Future studies will determine if OT-mediated changes in behavior or physiology that surround parturition are related to these changes in OT mRNA.

The duration of estradiol and progesterone exposure prior to progesterone withdrawal regulates oxytocin mrna levels in the paraventricular nucleus of the rat

The nonapeptide oxytocin (OT) is important for milk ejection during lactation, uterine contractility at parturition, and the onset of maternal behavior. Sequential exposure to estradiol (E2) and progesterone (P) followed by P withdrawal increases OT mRNA in the paraventricular nucleus (PVN), and to a lesser degree the supraoptic nucleus (SON), of the rat 48 hours after the P is removed. Although increases in PVN OT mRNA are not accompanied by changes in posterior pituitary OT peptide content, the PVN contains OT neurons that project to both the posterior pituitary (magnocellular group) and extra pituitary sites (parvocellular groups). Steroid-induced increases in OT mRNA occur in both the magnocellular and the parvocellular regions of the PVN. The latter are believed to contribute to CNS release of OT which may be important for certain behaviors including the onset of maternal behavior. The same steroid sequence that increases PVN OT mRNA also induces maternal behavior in virgin ovariectomized rats. Exposure of animals to E2 and P for 2 weeks resulted in the shortest latency to the onset of maternal behavior in ovariectomized rats, whereas exposure for 6 days was associated with a longer latency. In this study we questioned if the duration of E2 and P exposure prior to P withdrawal is an important regulator of PVN OT mRNA levels. We compared OT mRNA levels in the PVN of virgin ovariectomized rats administered no steroid or sequential E2 and P for 2 weeks versus 6 days. On day 1 animals received steroid-filled or empty capsules followed by P-filled or empty capsules on day 3. In one steroid-treated group, E2 and P were continued for 6 days and in the other group for 14 days prior to P removal. Animals were sacrificed 48 hours after P removal. Levels of OT mRNA were compared among 6 day and 2 week steroid-treated animals and sham-treated animals. The relative abundance of OT mRNA was significantly increased, P < 0.05, in animals receiving the 2-week, but not the 6-day, steroid treatment compared to sham-treated animals. Pituitary OT peptide content was not significantly different among the three groups. We conclude that the duration of steroid exposure may be an important regulator of the level of OT mRNA in the PVN of the rat.

The effects of short photoperiod, pinealectomy, and melatonin treatment on oxytocin synthesis and release in the male syrian hamster

The pineal gland has been shown to affect plasma oxytocin (OT) levels, but the mechanism of this action is not apparent. In the present study, the ability of the photoperiod to affect plasma OT levels, neurointermediate lobe (NIL) OT content, and hypothalamic OT mRNA levels was studied in male Syrian hamsters. In addition, the ability of pinealectomy to prevent and melatonin (MEL) to mimic the short photoperiod-induced changes were also determined. Exposure to short days (SD) led to the expected decrease in testes weight and plasma PRL levels, but plasma OT levels were unchanged. However, NIL OT content was increased in the SD-exposed animals. Hypothalamic OT mRNA levels were not significantly altered by SD exposure. Pinealectomy blocked the effects of SD on testes weight, whereas afternoon MEL injections mimicked the effects of SD. In long day (LD)-exposed hamsters, pinealectomy induced a decrease in NIL OT content without altering hypothalamic OT mRNA levels. In SD-exposed animals, NIL OT content was not affected by pinealectomy. Melatonin injections had no significant effect on NIL OT content or hypothalamic OT mRNA levels. The data from the present study suggest that exposure of male Syrian hamsters to short photoperiods influences some aspects of OT synthesis and/or transport to produce its increased accumulation in the NIL, but does not affect OT release. These changes are apparently not the result of SD-induced changes in MEL secretion, but conceivably could be related to the previously documented effects of SD on hypothalamic catecholamine turnover.

Sequential estrogen and progesterone (P) followed by P withdrawal increases the level of oxytocin messenger ribonucleic acid in the hypothalamic paraventricular nucleus of the male rat

We previously reported that alterations in gonadal steroid hormones can influence oxytocin (OT) messenger ribonucleic acid (mRNA) expression in the hypothalamic paraventricular nucleus (PVN) of the steroid-treated ovariectomized rat, the pregnant rat, and the lactating rat. Specifically, OT mRNA in the PVN of the female rat was increased by sequential estrogen and progesterone (P) followed by withdrawal of P. In this study we investigated whether this same steroid paradigm increases OT mRNA levels in the castrate rat. Castrate rats were administered either sequential estrogen and P followed by P withdrawal or no steroid treatment prior to sacrifice. PVN OT mRNA was measured by Northern blot hybridization and pituitary OT peptide content by radioimmunoassay (RIA). The steroid-treated rats had increased OT mRNA levels compared to the sham treated rats (p < 0.04), but pituitary OT peptide content was not significantly altered. We conclude that sequential estrogen and P followed by P withdrawal increases PVN OT mRNA in the castrate, as well as the ovariectomized, rat.

Hypothalamic-pituitary corticotroph function after shunting of magnocellular vasopressin and oxytocin to the hypophyseal portal circulation.

Surgical pituitary stalk compression (PSC) causes neural lobe denervation and development of ectopic magnocellular terminals in the external zone of the median eminence, resulting in shunting of magnocellular vasopressin (VP) and oxytocin (OT) to the pituitary portal circulation. To determine the effects of PSC on hypothalamic and pituitary function, VP and CRH receptors and POMC messenger RNA (mRNA) in the pituitary, and CRH, VP, and OT mRNA levels in the PVN were examined in 7-day PSC and sham-operated rats. Immunohistochemical staining revealed marked accumulation of immunoreactive VP in the swollen pituitary stalk and the external zone of the distal median eminence of PSC rats. Plasma ACTH and corticosterone levels were significantly increased by PSC, an effect that was attenuated by minipump infusion of desamino-[D-Arg8]-vasopressin (DDAVP) given to correct the diabetes insipidus. [3H]VP binding to anterior pituitary membranes from PSC rats was reduced by 50% compared to that in sham-operated controls, but VP V1b receptor mRNA levels measured by in situ hybridization were unchanged. Pituitary CRH receptors measured by binding autoradiography and CRH receptor mRNA levels did not change after PSC. POMC mRNA was unchanged in the anterior pituitary lobe, but markedly increased in the intermediate lobe of PSC rats, with or without DDAVP infusion. In the hypothalamic supraoptic and paraventricular nuclei, PSC resulted in reduction of VP mRNA (-83%) and OT mRNA (-38%) levels, probably reflecting retrograde degeneration of magnocellular neurons. This decrease was more pronounced for VP mRNA than for OT mRNA CRH mRNA levels in parvicellular cells of the paraventricular nuclei of PSC rats were reduced by 55%. Correction of the diabetes insipidus by DDAVP treatment further decreased hypothalamic VP mRNA levels, but restored CRH mRNA to control levels. The data show that continuous exposure of the pituitary to high VP levels from ectopic magnocellular fibers in the median eminence results in VP, but not CRH, receptor loss. Pituitary VP receptor down-regulation and decreased hypothalamic CRH expression are likely to contribute to the reduced corticotroph responsiveness to VP reported in this experimental model.