Effects of alpha-melanocyte-stimulating hormone on magnocellular oxytocin neurones and their activation at intromission in male rats

The peptides alpha-melanocyte-stimulating hormone (alpha-MSH) and oxytocin have very similar effects on several behaviours, including male sexual behaviour. Both induce penile erection and enhance copulatory behaviour when given centrally, suggesting that their central actions are not independent. Here, we used intromission as a physiological stimulus to investigate whether some central effects of alpha-MSH during male sexual behaviour are mediated by oxytocin neurones. We used the expression of the immediate-early gene product Fos to investigate oxytocin neurone activation at intromission and after intracerebroventricular (i.c.v.) administration of alpha-MSH (1 microg/5 microl) and studied the effects of i.c.v. administration of a MC4 receptor antagonist on Fos expression and on the latency of male rats to exhibit sexual behaviour in the presence of a receptive female. In rats that showed intromission, Fos was expressed in magnocellular oxytocin neurones in both the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but there was no significant activation of parvocellular oxytocin neurones of the PVN. Similarly, alpha-MSH increased Fos expression in magnocellular oxytocin neurones but had little or no effect in parvocellular oxytocin neurones. In male rats that achieved intromission, central injection of a MC4 receptor antagonist significantly attenuated the increase in Fos expression in magnocellular oxytocin neurones in both the PVN and the SON and increased mount and intromission latencies compared to vehicle-injected controls. Together, the results indicate that magnocellular oxytocin neurones are involved in the central regulation of male sexual behaviour, and that some of the central effects of alpha-MSH are likely to be mediated by magnocellular oxytocin neurones.     

Suppressed oxytocin neuron responses to immune challenge in late pregnant rats: a role for endogenous opioids

Cytokine challenge (mimicking infection) with systemic interleukin-1beta (IL-1beta) stimulates oxytocin neurons via a noradrenergic brainstem pathway similar to that involved in parturition. As the responses of oxytocin neurons to several stimuli are reduced in late pregnancy, we have investigated whether responses to IL-1beta are also suppressed. In virgin Sprague-Dawley rats, IL-1beta (500 ng/kg i.v.) rapidly increased oxytocin secretion (3.2-fold), via a central action as the firing rate of oxytocin neurons in the supraoptic nucleus was increased. In contrast, IL-1beta had no significant effect on the electrical or secretory activity of oxytocin neurons in late pregnant rats. In pregnancy activation of a central inhibitory opioid mechanism restrains oxytocin neuron responses to various stimuli. Accordingly, we tested the effects of the opioid antagonist, naloxone, on oxytocin neuron responses to IL-1beta in pregnancy. Naloxone (5 mg/kg i.v.) did not affect the oxytocin secretory response to IL-1beta in virgin rats, whereas in late pregnant rats naloxone revealed a greater oxytocin secretory response to IL-1beta (3.5-fold) than in virgin rats. In virgin rats, naloxone decreased oxytocin neuron firing rate after IL-1beta, however, in pregnant rats naloxone increased the firing rate response to IL-1beta to the level seen in virgin rats. Thus, systemic IL-1beta acts centrally to increase oxytocin secretion. In pregnancy this response is suppressed by endogenous opioids, thus preserving neurohypophysial oxytocin stores for parturition and minimizing the risk of preterm labour. The exaggerated oxytocin secretory response to IL-1beta in pregnancy after naloxone reflects increased oxytocin stores and/or increased efficiency of excitation-secretion coupling at the posterior pituitary.     

Late Pregnancy is a Critical Period for Changes in Phosphorylated Mitogen‐Activated Protein Kinase/Extracellular Signal‐Regulated Kinase 1/2 in Oxytocin Neurones

The physiological demands of parturition and lactation lead to the increased pulsatile release of oxytocin (OT) into the circulation from the neurohypophysial axons of OT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. These states of increased OT release are accompanied by a significant plasticity in magnocellular OT neurones and their synaptic connections, and many of these changes require activation of a central OT receptor. The mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway (MAPK/ERK) is assumed to be up‐regulated in the PVN during lactation, and many of the effects of OT in peripheral and brain tissue are mediated through a MAPK/ERK pathway. The present study investigated whether this pathway is altered in the SON and PVN during late pregnancy [embryonic day (E)20–21], which is a critical period for OT plasticity induction, and for lactation, when plastic changes are sustained. Based on immunoreactivity for phosphorylated ERK1/2 (pERK1/2), the results suggest an enhanced activation of MAPK/ERK pathway in OT neurones specifically during late pregnancy in both the SON and PVN. Although immunoblots from the SON confirm this pregnancy‐associated up‐regulation in late pregnancy, they also suggest enhancement into lactation as well. Together, the results suggest an important role for the MAPK/ERK pathway during reproductive changes in the SON and PVN.     

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague‐Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual‐label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin‐ or oxytocin‐labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post‐MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.     

Opioids and Coupling of the Anterior Peri-Third Ventricular Input to Oxytocin Neurones in Anaesthetized Pregnant Rats

In the pregnant rat the osmotic drive to oxytocin neurones is reduced and oxytocin secretion itself is inhibited by endogenous opioids. Coupling of the anterior peri-third ventricular input pathway, involved in osmoregulation, to magnocellular oxytocin neurones was studied in urethane-anaesthetized virgin and 21 day pregnant rats using electrical stimulation of the region anterior and ventral to the third cerebral ventricle (AV3V region) to drive the oxytocin neurones, and giving naloxone to prevent the action of any endogenous opioids on the system. Trains ofstimuli (0.5 mA, 1 ms pulses, 10 s on 10 s off, at either 10 Hz or 25 Hz for 10 or 2 min respectively) were given at 20 or 30 min intervals via an electrode stereotaxically-implanted in the AV3V region, and femoral arterial blood plasma samples collected immediately before and after each stimulation were radioimmunoassayed for oxytocin concentration. The first (control) AV3V stimulation increased plasma oxytocin concentration reproducibly and similarly in virgin and 21-day pregnant rats. Naloxone administered 10 min before the second stimulus increased basal plasma oxytocin concentration in virgin and pregnant rats and increased the oxytocin secretory response to 25 Hz AV3V stimulation in virgin but not pregnant rats, and the response was significantly greater in virgin rats. Naloxone reveals oxytocin secretion unrestrained by endogenous opioids, therefore it appears that there is an opioid-independent reduction in the excitatory coupling of the AV3V input to oxytocin neurones which may explain the reduced osmoresponsiveness of oxytocin neurones at the end of pregnancy. In other experiments, morphine (μ-opioid agonist; at 1 & 5 mg/kg 5 min before the second and third stimulation periods respectively) or U50,488 (κ-opioid agonist; 0.5 and 2.5 mg/kg respectively) was given to test the opioid sensitivity of the oxytocin neurone response to stimulation of the AV3V input. Morphine did not significantly affect the oxytocin secretory response to 25 Hz AV3V stimulation in either virgin or pregnant rats but there was a significant dose related inhibition of the oxytocin secretory response to 10 Hz stimulation in both groups. U50,488 also inhibited the oxytocin secretory response to 25 Hz AV3V stimulation in a dose-related manner in both virgin and pregnant rats. The inhibitory effects of morphine and U50,488 were not different between virgin and pregnant rats; this indicates that central opioid interactions with the AV3V input to oxytocin neurones are not changed in pregnancy. The initial similar oxytocin secretory response to AV3V stimulation in pregnant and virgin rats seems to be a product of a reduced effectiveness of the excitatory AV3V input to oxytocin neurones in late pregnancy and the previously established reduced posterior pituitary sensitivity to inhibitory endogenous opioids.     

Effect of Melanotan‐II on Brain Fos Immunoreactivity and Oxytocin Neuronal Activity and Secretion in Rats

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan‐II (MT‐II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v. , but not intranasal, administration of MT‐II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU‐9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT‐II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v. , but not intranasal, administration of MT‐II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT‐II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT‐II also increased Fos expression.     

Progesterone receptor expression in the pregnant and parturient rat hypothalamus and brainstem

Oxytocin is synthesized by magnocellular neurons in the supraoptic and paraventricular nuclei (SON and PVN) and during pregnancy progesterone prevents premature activation of oxytocin neurons. Progesterone receptors (PR) are not detectable in SON oxytocin neurons of non-pregnant rats, so we sought to determine whether they are expressed during pregnancy and parturition. In addition, we examined PR expression in brainstem and hypothalamic regions that have known direct projections to the SON. Neuronal immunoreactive PR (irPR)-labeled nuclei were counted in sections from proestrous virgin, late pregnant (day 21) and parturient rats (90 min from birth onset). IrPR nuclei were not evident in the SON at any stage but irPR expression in the medial preoptic nucleus (MPA) significantly increased in pregnancy and parturition (159% and 189% of proestrous controls, respectively). Other hypothalamic areas did not exhibit a significant change in irPR expression. In the nucleus tractus solitarius (NTS) in the brainstem, there was no significant change in irPR in late pregnancy, but there was a significant reduction in irPR expression at parturition (22% of proestrous controls). Very few NTS neurons immunoreactive for tyrosine hydroxylase (irTH), and thus putatively noradrenergic, contained irPR. These findings taken with evidence that brainstem irTH neurons projecting to the SON are stimulated at parturition, whereas MPA cells projecting to the SON are not, suggest that any direct actions of progesterone or progesterone withdrawal on NTS or SON neurons are not mediated through the classical PR. Upregulation of PR expression in the MPA during pregnancy and parturition may relate to the onset of maternal behavior and/or regulation of GnRH neuronal activity.     

Somatostatin Actions on Rat Supraoptic Nucleus Oxytocin and Vasopressin Neurones

Magnocellular neurones in the supraoptic nucleus (SON) receive major afferent inputs from the brainstem that have been implicated in the regulation of oxytocin and vasopressin secretion from the posterior pituitary. Notably, at parturition, some neurones that project from the nucleus tractus solitarii (NTS) in the brainstem directly to the SON are activated. Many of these are noradrenergic and regulate oxytocin secretion during parturition, whereas others contain somatostatin and their role is unclear. In the present study, we report that, at parturition, somatostatin mRNA expression in the NTS is significantly increased compared to pregnancy, suggesting an active role for these neurones at that time. Intracerebroventricular somatostatin infusion significantly increased plasma oxytocin secretion in both virgin female and pregnant rats. Intracerebroventricular somatostatin increased SON oxytocin and vasopressin neurone firing‐rates, and increased Fos expression in the SON and paraventricular nucleus and in the subfornical organ. Retrodialysis of somatostatin onto the ventrally exposed SON also increased vasopressin neurone firing rate but, unexpectedly, decreased oxytocin neurone firing rate. The experiments indicate that somatostatin neurones in the NTS are activated during parturition but, because the direct effects of somatostatin on oxytocin neurones are inhibitory, this direct pathway does not appear to contribute to enhanced oxytocin release at this time, although indirect somatostatin effects may do so.     

Modulation of oestrogen receptor-beta mRNA expression in rat paraventricular and supraoptic nucleus neurones following adrenal steroid manipulation and hyperosmotic stimulation

Magnocellular neurosecretory neurones in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei express oestrogen receptor beta (ERbeta) but not ERalpha. In the PVN, ERbeta is strongly expressed in the ventromedial parvocellular neurones projecting to the brainstem. We used quantitative in situ hybridization, with (35)S-labelled riboprobes, to study heterologous regulation by manipulating adrenal steroid hormones (72 h after adrenalectomy +/- corticosterone replacement; repeated stress: halothane inhalation, environmental cold, immobilization, each daily for 3 days) in male rats. Adrenalectomy increased ERbeta mRNA expression in the magnocellular PVN and SON, by 2.2 and 2.5-fold, respectively, with no effect in the ventromedial parvocellular PVN neurones. Corticosterone replacement partially prevented the increases in ERbeta mRNA expression in magnocellular PVN and SON neurones. Repeated stress over 72 h had no effect on ERbeta mRNA expression in the magnocellular PVN or SON, but increased expression 1.4-fold in the ventromedial parvocellular PVN neurones. Although consequences of hydromineral balance derangement after adrenalectomy may stimulate magnocellular neurones, strongly stimulating the neurones by giving intact male rats 2% saline to drink for 72 h decreased ERbeta mRNA expression in the magnocellular PVN and SON neurones by approximately 60%, and in the ventromedial parvocellular PVN neurones by 13%. Thus, ERbeta mRNA expression is negatively regulated by basal glucocorticoid secretion in magnocellular PVN and SON neurones, and positively regulated by stress in ventromedial parvocellular PVN neurones. However, ERbeta mRNA expression in magnocellular neurones is negatively linked to hyperosmotic stimulation of the neurones. The 6.25-fold variation in ERbeta mRNA expression in magnocellular neurones from salt-loading to adrenalectomy could alter their sensitivity to oestrogens. Consequently, regulation of oxytocin and vasopressin neurone activity via ERbeta is expected to vary according to their functional state and, in particular, on basal glucocorticoid actions.     

Possible Involvement of the Rat Hypothalamo‐Neurohypophysial/‐Spinal Oxytocinergic Pathways in Acute Nociceptive Responses

Oxytocin (OXT)‐containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo‐spinal pathway, as well as the hypothalamo‐neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT‐mRFP1 neurones that expressed Fos‐like immunoreactivity (‐IR) and OXT‐mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT‐mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos‐IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin‐releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT‐saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo‐neurohypophysial/‐spinal OXTergic pathways may be involved in acute nociceptive responses in rats.     

Increased Sensitivity of Monoamine Release in the Supraoptic Nucleus in Late Pregnancy: Region‐ and Stimulus‐Dependent Responses

Oxytocin neurone activation at birth depends upon noradrenaline‐mediated signals from the uterus via a brainstem pathway, as well as on factors within the supraoptic nucleus (SON), including oxytocin itself, and the system adapts during pregnancy to optimise the delivery process. We determined whether noradrenaline release in the SON in response to stimuli activating brainstem inputs or antidromically activating magnocellular neurones is enhanced at term pregnancy. Noradrenaline, serotonin and dopamine concentrations were measured in microdialysis samples collected from the dorsal and ventral SON before, during and after either i.v. cholecystokinin (CCK) or neural stalk stimulation in virgin and late pregnant rats. Each stimulus transiently increased noradrenaline and serotonin but not dopamine concentration in the dorsal SON, and responses were increased on days 21 and 22 of pregnancy compared to day 20 pregnant and virgin rats. Neural stalk stimulation induced sensitisation to subsequent stalk stimulation and so the responses in the dorsal SON were doubled; on day 22 of pregnancy, the area under the curve of monoamine concentration was 3.4‐fold greater than in virgins, suggesting that adaptations perinatally enhance responsiveness. In conclusion, there are enhanced responses of noradrenaline and serotonin release in the SON that can generate very high, transient extracellular concentrations at term. This may be a consequence of neuroendocrine adaptations in late pregnancy and probably contributes to optimal oxytocin neurone activation during parturition.     

Systemic Leptin Increases the Electrical Activity of Supraoptic Nucleus Oxytocin Neurones in Virgin and Late Pregnant Rats

In the rat hypothalamus, fasting attenuates the expression of oxytocin and this can be reversed by exogenous leptin administration. In the present study, we investigated the effects of systemically administered leptin on the electrical activity of magnocellular neurones in the supraoptic nucleus of urethane‐anaesthetised rats. In virgin female rats, systemic leptin significantly excited identified oxytocin neurones with no detected effects on the patterning of activity, as reflected by hazard function analyses. The lowest dose that was consistently effective was 100 μg/i.v., and this dose had no significant effect on vasopressin neurones. In virgin rats fasted overnight, the spontaneous firing rate of oxytocin neurones was significantly lower than in unfasted rats, although leptin had a similar excitatory effect as in unfasted rats. In late pregnant rats (days 19–21 of pregnancy), spontaneous firing rates of oxytocin neurones were higher than in virgins, and the initial response to leptin was similar to that in virgin rats, although the increase in activity was more persistent. In fasted pregnant rats, the mean spontaneous firing rate of oxytocin neurones was again lower than in unfasted rats, although leptin had no significant effect even at the higher dose of 1 mg/rat. Thus, fasting reduced the spontaneous firing rates of oxytocin neurones in nonpregnant rats, and this effect could be reversed by the excitatory effects of leptin. Pregnant rats showed some evidence of leptin resistance but only after an overnight fast.     

Oestradiol potentiates hormone secretion and neuronal activation in response to hypertonic extracellular volume expansion in ovariectomised rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 μg/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c‐Fos‐VP and c‐Fos‐OT double‐labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c‐Fos‐OT and c‐Fos‐VP double‐labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.     

Oxytocinase in the Female Rat Hypothalamus: A Novel Mechanism Controlling Oxytocin Neurones During Lactation

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase; P‐LAP) is expressed both peripherally and centrally. P‐LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, although its role in the hypothalamus is unclear. We investigated P‐LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, as well as its role in vivo during the milk‐ejection reflex. P‐LAP mRNA and protein were expressed in magnocellular neurones of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurones co‐expressed P‐LAP without strong subcellular co‐localisation of oxytocin and P‐LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P‐LAP showed a redistribution of P‐LAP to within 1 μm of the plasma membrane in the somata of oxytocin neurones during lactation. Both P‐LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c.v. injection of amastatin in anaesthetised suckling mothers increased the frequency of reflex milk ejections. Because hypothalamic P‐LAP expression and activity increase in lactation, and the prevention of its action mimics central oxytocin administration, we conclude that P‐LAP regulates auto‐excitatory oxytocin actions during the suckling‐induced milk‐ejection reflex.     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

Reproductive state changes NADPH-diaphorase staining in the paraventricular and supraoptic nuclei of female rats

It has been demonstrated in guinea pigs that nitric oxide synthase (NOS) activity is increased in late pregnancy in some peripheral tissues and in the cerebellum. To determine whether similar changes would be observed in areas of the brain known to play a role in parturition, staining for NADPH-diaphorase, a histochemical marker of NOS synthase, in the paraventricular (PVN) and supraoptic nuclei (SON) was compared among ovariectomized, virgin and late pregnant rats. The number of cells showing dense staining for NADPH-diaphorase increased in both the SON and PVN in late pregnancy compared to that observed in virgin and ovariectomized females. Thus, changes in reproductive state are associated with changes in NADPH-diaphorase staining in areas of the brain that are intimately involved in the control of reproductive function.     

Oestradiol acts through its beta receptor to increase vasopressin neuronal activation and secretion induced by dehydration

Vasopressinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei express oestrogen receptor (ER)β and receive afferent projections from osmosensitive neurones that express ERα. However, which subtype of these receptors mediates the effects of oestradiol on vasopressin (AVP) secretion induced by hydromineral challenge has not yet been demonstrated in vivo. Moreover, AVP secretion induced by hyperosmolality is known to involve activation of TRPV1 (transient receptor potential vanilloid, member 1) in magnocellular neurones, although whether oestradiol modulates expression of this receptor is unknown. Thus, the present study aimed to clarify the mechanisms involved in the modulation exerted by oestradiol on AVP secretion, specifically investigating the involvement of ERβ, ERα and TRPV1 receptors in response to water deprivation (WD). We observed that treatment with an ERβ agonist potentiated AVP secretion and vasopressinergic neuronal activation induced by WD. This increase in AVP secretion induced by WD was reversed by an ERβ antagonist. By contrast to ERβ, the ERα agonist did not alter plasma AVP concentrations or activation of AVP neurones in the SON and PVN. Additionally, Fos expression in the subfornical organ was not altered by the ERα agonist. TRPV1 mRNA expression was increased by WD in the SON, although this response was not altered by any treatment. The results of the present study suggest that ERβ mediates the effects of oestradiol on AVP secretion in response to WD, indicating that the effects of oestradiol occur directly in AVP neurones without affecting TRPV1.     

Interleukin-1beta release in the supraoptic nucleus area during osmotic stimulation requires neural function

Interleukin (IL)-1beta is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1beta is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0-180 min; basal), and after (180-240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1beta was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1beta+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1beta+ in the SON area was measurable and stable (pg/ml; mean +/- SEM) from 0-60 min (2.2 +/- 0.06), 60-120 min (2.32 +/- 0.05) and 120-180 min (2.33 +/- 0.06), likely originating locally from activated microglia (OX42+; IL-1beta+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1beta. In response to osmotic stimulation, IL-1beta increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1beta in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.