Intranuclear Oxytocin Release Research Articles

Oxytocin release in magnocellular nuclei: neurochemical mediators and functional significance during gestation

When released from dendrites within the supraoptic (SON) and paraventricular (PVN) nuclei (intranuclear release) during suckling, oxytocin exerts autocrine and paracrine effects on oxytocin neurons that are necessary for the unique timing and episodic pattern of oxytocin release into the systemic circulation that is characteristic of lactation. Recent reports have shown that stimulation of central noradrenergic and histaminergic receptors are both necessary for intranuclear release of oxytocin in response to suckling. In addition, in vitro studies indicate that excitatory amino acids may also be critical for central oxytocin secretion, although in vivo experiments have not provided direct support for this hypothesis. In addition to a critical role in intranuclear oxytocin release during lactation, norepinephrine has also been shown to stimulate central oxytocin during gestation. Stimulation of central oxytocin receptors during gestation appears critical for normal systemic oxytocin secretion in responses to suckling during the subsequent period of lactation. Oxytocin receptor blockade during pregnancy alters normal timing of systemic oxytocin release during suckling and reduces milk delivery. Several adaptations occur in the central oxytocin system that are necessary for determining the unique response characteristic observed during parturition and gestation. Central oxytocin receptor stimulation during gestation has been implicated in pregnancy-related morphological changes in magnocellular oxytocin neurons, disinhibition of oxytocin neurons to GABA, and adaptations in membrane response characteristics of oxytocin neurons. In conclusion, intranuclear oxytocin release during gestation and lactation are critical for establishing, and then evoking the unique pattern of systemic oxytocin secretion in response to the suckling offspring necessary for adequate milk delivery. Furthermore, activation of central noradrenergic receptors appears to be critical for release of central oxytocin in both of these reproductive states.

Differential sensitivity of intranuclear and systemic oxytocin release to central noradrenergic receptor stimulation during mid- and late gestation in rats.

A number of changes occur in the oxytocin (OT) system during gestation, such as increases in hypothalamic OT mRNA, increased neural lobe and systemic OT, and morphological and electrophysiological changes in OT-containing magnocellular neurons, suggestive of altered neuronal sensitivity, which may be mediated by ovarian steroids. Because central norepinephrine (NE) and histamine (HA) are potent stimulators of OT release during parturition and lactation, the present study investigated the effects of central noradrenergic and histaminergic receptor activation on systemic (NE, HA) and intranuclear (NE) OT release in pregnant rats and in ovariectomized rats treated with ovarian steroids. Plasma OT levels in late gestation were significantly higher compared with all other groups, and neither adrenergic nor histaminergic receptor blockade decreased these elevated levels. Furthermore, the alpha-adrenergic agonist phenylephrine, but not histamine, stimulated systemic OT release to a significantly greater extent in late gestation than in midpregnant, ovariectomized, or steroid-treated females. Although basal extracellular OT levels in the paraventricular nucleus, as measured with microdialysis, were unchanged during pregnancy or steroid treatment, noradrenergic receptor stimulation of intranuclear OT release was significantly elevated in midgestation females compared with all other groups. These studies indicate that sensitivity of intranuclear and systemic OT release to noradrenergic receptor activation differentially varies during the course of gestation.

Prolactin and oxytocin interaction in the paraventricular and supraoptic nuclei: effects on oxytocin mRNA and nitric oxide synthase.

We investigated the contribution of prolactin and oxytocin to the increase in staining for NADPH-d and oxytocin mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) observed at the end of pregnancy, or following a steroid-priming regimen that mimics the hormonal profile of late pregnant females. Ovariectomized rats received chronic implants of silastic capsules containing oestrogen and progesterone followed by progesterone removal. In experiment 1, oxytocin antagonist (OTA) was administered to rats to investigate whether intranuclear oxytocin release was necessary for NADPH-d staining. In experiments 2a and b, rats received concurrent treatment with bromocryptine (0.5 mg/day) to suppress endogenous prolactin release, and either systemic prolactin (0.5 mg once daily), or prolactin (2 micro g/ micro l), or vehicle infused twice a day into the third ventricle, or chronic oxytocin infusion (24 ng/day) for 3 days following progesterone removal. Brains were then processed for NADPH-d histochemistry. In experiment 3, the interaction of prolactin and oxytocin on oxytocin mRNA within the SON and PVN was examined. NADPH-d staining in the SON and PVN was reduced by the highest dose of the OTA, and by bromocryptine treatment. Central prolactin and oxytocin replacement completely restored NADPH-d staining in bromocryptine-treated rats. Finally, both bromocryptine and the OTA suppressed oxytocin mRNA expression and prolactin replacement restored expression levels to that of controls. Together, these data suggest that the increased capacity to produce nitric oxide in the SON and PVN during late pregnancy is dependent on prolactin stimulating oxytocin gene mRNA and hence intranuclear oxytocin release.

Endogenous opioid regulation of stress-induced oxytocin release within the hypothalamic paraventricular nucleus is reversed in late pregnancy: a microdialysis study

Oxytocin secretion into blood in response to swim stress is differentially regulated by endogenous opioids in virgin and pregnant rats. Here, the influence of endogenous opioids on oxytocin release within the hypothalamic paraventricular and supraoptic nuclei was investigated using microdialysis in virgin and pregnant (day 19–21) rats. Rats fitted with a U-shaped microdialysis probe 3 days before testing were injected with naloxone (5 mg/kg body weight, s.c.) or vehicle (sterile saline) and, 3 min later, were forced to swim (10 min at 19°C). Within the paraventricular nucleus, basal and stimulated oxytocin release did not significantly differ between vehicle-treated virgin and pregnant rats. After naloxone, local oxytocin release in response to swimming was lowered in virgin rats ( P<0.01), whereas it was further increased in pregnant rats ( P<0.01). Within the supraoptic nucleus, basal oxytocin release was significantly lower in pregnant compared to virgin rats ( P<0.01). Forced swimming induced a similar rise in intranuclear oxytocin release in both vehicle-treated virgin and pregnant rats, but peak levels were still higher in the virgin controls. In contrast to the paraventricular nucleus, naloxone did not alter swim-induced oxytocin release within the supraoptic nucleus either in virgin or pregnant rats. Vasopressin release in the paraventricular nucleus was also increased by forced swimming but there was no effect of pregnancy or naloxone on it. In summary, in pregnancy, basal and stress-induced oxytocin release within the paraventricular nucleus was not changed, whereas it was blunted within the supraoptic nucleus. Further, within the paraventricular nucleus the excitatory effect of endogenous opioids on local oxytocin release seen in virgins was switched into an inhibitory action in pregnancy. In contrast, endogenous opioids were evidently not involved in the regulation of swim-induced oxytocin release within the supraoptic nucleus either in virgin or pregnant rats. Thus, pregnancy-related neuroendocrine plasticity also includes site-specific functional alterations in opioid receptor-mediated actions in the hypothalamus.

Histaminergic Control of Oxytocin Release in the Paraventricular Nucleus during Lactation in Rats

The central neurotransmitters regulating both systemic and central release of oxytocin (OT) during lactation are not completely defined. Although central histaminergic systems have been implicated in systemic release of OT, the role of this neurotransmitter in suckling-induced intranuclear OT secretion has not been investigated. Therefore, microdialysis of the paraventricular nucleus (PVN) was used to determine if suckling stimulates histamine release within the PVN and if nursing-induced intranuclear OT release is reduced by local blockade of either H1 or H2 histamine receptors. Female Holtzman rats were implanted with microdialysis probes adjacent to the PVN on lactation days 8–12. The next day, the pups and dam were separated for 4 h, reunited, and again separated. Histamine concentrations in dialysates were measured before, during, and following suckling. In separate animals, a similar separation/reunion paradigm was used, but the dialysate OT concentration was measured during PVN perfusion with vehicle or an H1 or H2 receptor antagonist. Suckling increased dialysate concentrations of both histamine and OT in the PVN. Furthermore, local pharmacological blockade of either H1 or H2 receptors prevented the increase in OT release in the PVN during suckling. These data demonstrate that activation of histamine receptors in the PVN is necessary for intranuclear release of OT induced by suckling and extend previous findings demonstrating a similar relationship between central histamine and systemic release of OT.

The oxytocin receptor system: structure, function, and regulation.

The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via G(q) proteins to phospholipase C-beta. The high-affinity receptor state requires both Mg(2+) and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has been characterized by mutagenesis and molecular modeling and is different from the antagonist binding site. The function and physiological regulation of the OT system is strongly steroid dependent. However, this is, unexpectedly, only partially reflected by the promoter sequences in the OT receptor gene. The classical actions of OT are stimulation of uterine smooth muscle contraction during labor and milk ejection during lactation. While the essential role of OT for the milk let-down reflex has been confirmed in OT-deficient mice, OT's role in parturition is obviously more complex. Before the onset of labor, uterine sensitivity to OT markedly increases concomitant with a strong upregulation of OT receptors in the myometrium and, to a lesser extent, in the decidua where OT stimulates the release of PGF(2 alpha). Experiments with transgenic mice suggest that OT acts as a luteotrophic hormone opposing the luteolytic action of PGF(2 alpha). Thus, to initiate labor, it might be essential to generate sufficient PGF(2 alpha) to overcome the luteotrophic action of OT in late gestation. OT also plays an important role in many other reproduction-related functions, such as control of the estrous cycle length, follicle luteinization in the ovary, and ovarian steroidogenesis. In the male, OT is a potent stimulator of spontaneous erections in rats and is involved in ejaculation. OT receptors have also been identified in other tissues, including the kidney, heart, thymus, pancreas, and adipocytes. For example, in the rat, OT is a cardiovascular hormone acting in concert with atrial natriuretic peptide to induce natriuresis and kaliuresis. The central actions of OT range from the modulation of the neuroendocrine reflexes to the establishment of complex social and bonding behaviors related to the reproduction and care of the offspring. OT exerts potent antistress effects that may facilitate pair bonds. Overall, the regulation by gonadal and adrenal steroids is one of the most remarkable features of the OT system and is, unfortunately, the least understood. One has to conclude that the physiological regulation of the OT system will remain puzzling as long as the molecular mechanisms of genomic and nongenomic actions of steroids have not been clarified.

Neurotransmitter interaction in release of intranuclear oxytocin in magnocellular nuclei of the hypothalamus.

Oxytocin (OT) is released within the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus in response to several stimuli. However, the neurotransmitters that control this intranuclear OT release are unknown. In vivo microdialysis was used to examine the roles of norepinephrine and histamine in intranuclear OT release in conscious, lactating female rats. Administration of alpha- or beta-noradrenergic agonists, or histamine, increased OT release in the PVN. In addition, the increase in PVN OT evoked by exogenous histamine was prevented by simultaneous blockade of either H1 or H2 receptors. Furthermore, histamine-induced release of intranuclear OT was also prevented by blockade of alpha-adrenergic receptors. Finally, the increase in magnocellular OT release induced by suckling was abolished by administration of alpha-adrenergic antagonists. These data demonstrate that norepinephrine and histamine are important neurotransmitters for release of intranuclear OT, and histamine releases intranuclear OT by stimulating norepinephrine release.

Stimulation of Central and Systemic Oxytocin Release by Histamine in the Paraventricular Hypothalamic Nucleus: Evidence for an Interaction with Norepinephrine

Central histaminergic neurons have been implicated in the control of oxytocin (OT) secretion in various physiological conditions, including parturition and lactation. The present studies investigated whether histamine also influences the central intranuclear release of OT, which is known to be important in the activation of OT neurons, and the possible interaction of histamine with norepinephrine in systemic and central OT release. Microdialysis probes were placed immediately adjacent to the hypothalamic paraventricular nucleus (PVN) and used for administration of artificial cerebrospinal fluid (ACSF) vehicle, ACSF containing histamine, ACSF containing histamine in combination with a specific H1 or H2 histamine receptor antagonist, or ACSF containing histamine and the α-adrenergic antagonist phentolamine. Dialysates and plasma were collected, and OT concentrations were determined using RIA. Dialysis of the PVN with ACSF containing histamine significantly increased the release of OT systemically and centrally within the PVN. Furthermore, the increases in OT concentration in dialysates and plasma were prevented by simultaneous administration of chlorpheniramine (an H1 receptor antagonist) or ranitidine (an H2 receptor antagonist) as well as by the adrenergic antagonist phentolamine. These data demonstrate that histamine acts within the PVN to increase both systemic and intranuclear release of OT. Furthermore, the increased OT release induced by histamine is dependent upon stimulation of both H1 and H2 histaminergic receptors and subsequent activation ofα -noradrenergic receptors. These findings suggest that histamine induces systemic and intranuclear OT release by stimulating the release of norepinephrine.

Stimulation of central and systemic oxytocin release by histamine in the paraventricular hypothalamic nucleus: evidence for an interaction with norepinephrine.

Central histaminergic neurons have been implicated in the control of oxytocin (OT) secretion in various physiological conditions, including parturition and lactation. The present studies investigated whether histamine also influences the central intranuclear release of OT, which is known to be important in the activation of OT neurons, and the possible interaction of histamine with norepinephrine in systemic and central OT release. Microdialysis probes were placed immediately adjacent to the hypothalamic paraventricular nucleus (PVN) and used for administration of artificial cerebrospinal fluid (ACSF) vehicle, ACSF containing histamine, ACSF containing histamine in combination with a specific H1 or H2 histamine receptor antagonist, or ACSF containing histamine and the alpha-adrenergic antagonist phentolamine. Dialysates and plasma were collected, and OT concentrations were determined using RIA. Dialysis of the PVN with ACSF containing histamine significantly increased the release of OT systemically and centrally within the PVN. Furthermore, the increases in OT concentration in dialysates and plasma were prevented by simultaneous administration of chlorpheniramine (an H1 receptor antagonist) or ranitidine (an H2 receptor antagonist) as well as by the adrenergic antagonist phentolamine. These data demonstrate that histamine acts within the PVN to increase both systemic and intranuclear release of OT. Furthermore, the increased OT release induced by histamine is dependent upon stimulation of both H1 and H2 histaminergic receptors and subsequent activation of alpha-noradrenergic receptors. These findings suggest that histamine induces systemic and intranuclear OT release by stimulating the release of norepinephrine.

Noradrenergic control of central oxytocin release during lactation in rats.

Noradrenergic systems regulate the systemic release of oxytocin (OT) in lactating rats. However, a role for norepinephrine (NE) in release of OT within the magnocellular nuclei during suckling has not been established. These studies were designed to determine 1) if suckling induces NE release in the supraoptic (SON) and paraventricular (PVN) nuclei of conscious rats and 2) the role of NE in the central, intranuclear release of OT within these nuclei. Female Holtzman rats were implanted with microdialysis probes adjacent to the PVN or SON on lactation days 8-12. The following day, the pups were isolated from the dams for 4 h. Microdialysis probes were perfused with artificial cerebrospinal fluid (ACSF) or with ACSF containing an alpha- or a beta-adrenergic receptor antagonist. Dialysate was collected before, during, and after suckling and analyzed for NE or OT. In an additional experiment, an alpha- or beta-adrenergic agonist was administered via the microdialysis probes into the PVN in nonsuckled, lactating rats. Extracellular NE increased in the PVN during suckling but was not detectable in the SON. OT concentrations in dialysates from the PVN and SON significantly increased during suckling. Blockade of either alpha-(in both PVN and SON) or beta- (PVN) adrenergic receptors prevented the suckling-induced increase in central OT release. OT release was increased in nonsuckled, lactating rats by central application of either an alpha- or beta-adrenergic agonist. These data demonstrate that intranuclear NE release is increased in the PVN by suckling and that subsequent stimulation of both alpha- and beta-noradrenergic receptors mediates intranuclear OT release.

Oxytocin neurone autoexcitation during morphine withdrawal in anaesthetized rats.

We investigated whether release of oxytocin into the supraoptic nucleus is involved in morphine-withdrawal excitation of oxytocin neurones. Retrodialysis of naloxone into the supraoptic nucleus of morphine-dependent rats increased intranuclear oxytocin release by 56.5p +/- 12.7% whereas no change was seen in vehicle-treated dependent rats. In another experiment, in morphine-dependent rats given intravenous (i.v.) naloxone, intracerebroventricular (i.c.v.) oxytocin receptor antagonist injection reduced the increase of plasma oxytocin concentration (to 28-fold) compared with i.c.v. vehicle (62-fold increase). Finally, the increase in oxytocin neurone firing rate following morphine-withdrawal in the presence of i.c.v. oxytocin antagonist infusion was 28% of the steady state firing rate (15-20 min later) and this was lower (p < 0.05) than the percentage increase in i.c.v. vehicle-infused rats (89%). Thus, central endogenous oxytocin may be involved in withdrawal excitation of oxytocin neurones.

Functional role of intrahypothalamic release of oxytocin and vasopressin: consequences and controversies

This brief review of vasopressin (VP) and oxytocin (OT) release into the extracellular space of the supraoptic (SON) and paraventricular (PVN) nuclei focuses on recent data illustrating the significance of their intranuclear release and the potential functional consequences. With the use of in vitro techniques, it has been demonstrated that administration of exogenous OT causes local peptide release and that, in vivo, this facilitates the milk ejection reflex. These findings lead to the idea that endogenous peptides are released into the hypothalamic nuclei. Microperfusion techniques have been used to monitor the dynamics of intranuclear OT and VP release in response to distinct stimuli. It is clear that intranuclear release of OT plays a role during reproductive states (parturition and lactation) and that intranuclear release of VP and OT is involved in osmoregulation. This review discusses 1) the origin of the intranuclearly released peptides, 2) stimuli which cause release into the hypothalamic nuclei, and 3) the function of intranuclear VP and OT, e.g., regulation of local morphology, feedback mechanisms and synchronization, and the possible role in regulating autonomic function and behavior.

Crosstalk in the magnocellular system during osmotic stimulation of one supraoptic nucleus

Neural connections linking the four magnocellular nuclei i.e.,. the paired supraoptic (SON) and paraventricular (PVN) nuclei, may contribute to the simultaneous and parallel changes in firing patterns of oxytocinergic neurons during reflex milk ejection. To investigate these neural connections in the absence of suckling, intranuclear release of oxytocin (OT) was stimulated by microdialysis of hypertonic CSF containing 1 M NaCl (HS-CSF) into the right SON area and glucose metabolism of both SONs and PVNs and the neural lobe of virgin and lactating (10–12 day) rats was mapped by the autoradiographic [ 14C]deoxyglucose (DG) method. OT in the microdialysates and in plasma, obtained before and after 80–90 min of dialysis with CSF or HS-CSF, was quantified by RIA. In both virgin and lactating rats, microdialysis of HS-CSF unilaterally into the SON area significantly ( p < 0.05) increased release of OT in the nucleus and into plasma, which was associated with enhanced ( p < 0.05) metabolic activity in the ipsilateral and contralateral SON and the neural lobe but not in either PVN. Compared with virgins, lactating rats were less active, had lower ( p < 0.05) glucose utilization in the hypothalamo-neurohypophysial system, and less ( p < 0.05) OT in plasma during microdialysis of HS-CSF into the SON area. The osmotic stimulus did not activate neural structures (suprachiasmatic and medial amygdaloid nuclei) near the SON in either hemisphere. Thus, neural connections or, less likely, transport of OT via the subarachnoid space, may function to recruit activation of cells in the contralateral SON following hypertonic stimulation of cells in the other SON.

Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: A microdialysis study

The release of the nonapeptides oxytocin and vasopressin within the hypothalamic supraoptic and paraventricular nuclei was measured in 30-min microdialysates in conscious female rats in the last three days of pregnancy, during parturition, immediately after parturition and during suckling, all in the same rats, and in virgin controls. Nonapeptide release within the supraoptic and paraventricular nuclei was unchanged during late pregnancy compared to virgin rats, but intranuclear oxytocin and not vasopressin release was elevated during parturition (relative to late pregnancy, supraoptic nucleus: to 254%, paraventricular nucleus: to 300%; P < 0.01) and during suckling also on days 8–10 of lactation (relative to pre-suckling, supraoptic nucleus: to 407%, paraventricular nucleus: to 275%; P < 0.02). Suckling-induced release of oxytocin was significantly reduced using Ca 2+-free, EDTA-containing (10 −4 M) microdialysis fluid and further stimulated by high K +-(56 mM), veratridine-containing (50 μM) microdialysis fluid. The opioid antagonist naloxone whether given by subcutaneous injection (5 mg/kg) or directly into the supraoptic nucleus by microdialysis (5 × 10 −6M) or microinjection (1.5 μl, 10 −6M) did not further enhance oxytocin release within either the supraoptic or paraventricular nuclei during parturition. In contrast to the selective release of oxytocin within the supraoptic and paraventricular nuclei during parturition and suckling, direct osmotic stimulation of the nuclei by microdialysing hypertonic medium (artificial cerebrospinal fluid; 1 M NaCl) increased intranuclear release of both oxytocin and vasopressin which was further enhanced after replacement of hypertonic with isotonic fluid. This rebound phenomenon served to confirm the precise location of the microdialysis probe ante mortem and the ability of the nuclei to adequately respond to the osmotic stimulus at the end of the experiment. The study has shown that oxytocin is released in the supraoptic and paraventricular nuclei during parturition as well as in lactation unrestrained by endogenous opioids during parturition. This intranuclear release of oxytocin may act by local positive feedback stimulation of oxytocin neurons to excite further oxytocin release in the brain and into blood during both parturition and lactation.