Endogenous Opioid Regulation of Oxytocin Secretion Through Pregnancy in the Rat

We have investigated the influence of endogenous opioids on oxytocin secretion during pregnancy. In blood-sampled consciousrats on days 18 and 21 of pregnancy plasma oxytocin concentration, measured by radioimmunoassay, was significantly increased compared to non-pregnant or post-partum rats. On days 15, 18 and 21 of pregnancy, but not in non-pregnant, early pregnant or post-partum rats, the opioid antagonist naloxone caused a significant increase in plasma oxytocin compared to vehicle injection, indicating activation of an endogenous opioid restraint over oxytocin secretion. Electrically stimulated neural lobes isolated from 16- and 21-day pregnant rats released more oxytocin than those from non-pregnant rats. However, naloxone (10^?5 M) was less effective at potentiating, and the k -opioid agonist U50,488 (10^?5 M) was less effective at inhibiting, stimulated release at the end of pregnancy than in non-pregnant rats suggesting desensitization of oxytocin nerve terminals to actions of endogenous opioids. Neural lobes from male rats drinking 2% saline for 4 days also showed desensitization of oxytocin nerve endings to naloxone. Neither neural lobe content of dynorphin A(_1–8), an endogenous k -opioid, nor prodynorphin mRNA expression, measured by in situ hybridization histochemistry in the supraoptic nucleus altered during pregnancy. However, neural lobe content of Met⁵enkephalin significantly decreased by day 21 of gestation suggesting enhanced release. We conclude that an endogenous opioid, possibly a product of proenkephalin A in oxytocin cells may be responsible for auto-inhibition of oxytocin release during gestation, and that this mechanism desensitizes in late pregnancy at a time when other opioid inputs to the oxytocin neurons become activated to provide an overall increase in opioid restraint of the system. The changes in opioid input through pregnancy may be involved in initiation and regulation of oxytocin secretion at parturition. A similar opioid mechanism, but possibly involving dynorphin, could explain desensitization in saline drinking rats and indicates that desensitization may be a consequence of chronic activation of secretion from the oxytocin nerve terminals rather than a phenomenon peculiar to pregnancy.     

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.     

Allopregnanolone and induction of endogenous opioid inhibition of oxytocin responses to immune stress in pregnant rats

In virgin rats, systemic administration of interleukin (IL)-1β (i.e. to mimic infection), increases oxytocin secretion and the firing rate of oxytocin neurones in the supraoptic nucleus (SON). However, in late pregnancy, stimulated oxytocin secretion is inhibited by an endogenous opioid mechanism, preserving the expanded neurohypophysial oxytocin stores for parturition and minimising the risk of preterm labour. Central levels of the neuroactive metabolite of progesterone, allopregnanolone, increase during pregnancy and allopregnanolone acting on GABA(A) receptors on oxytocin neurones enhances inhibitory transmission. In the present study, we tested whether allopregnanolone induces opioid inhibition of the oxytocin system in response to IL-1β in late pregnancy. Inhibition of 5α-reductase (an allopregnanolone-synthesising enzyme) with finasteride potentiated IL-1β-evoked oxytocin secretion in late pregnant rats, whereas allopregnanolone reduced the oxytocin response in virgin rats. IL-1β increased the number of magnocellular neurones in the SON and paraventricular nucleus (PVN) expressing Fos (an indicator of neuronal activation) in virgin but not pregnant rats. In immunoreactive oxytocin neurones in the SON and PVN, finasteride increased IL-1β-induced Fos expression in pregnant rats. Conversely, allopregnanolone reduced the number of magnocellular oxytocin neurones activated by IL-1β in virgin rats. Treatment with naloxone (an opioid antagonist) greatly enhanced the oxytocin response to IL-1β in pregnancy, and finasteride did not enhance this effect, indicating that allopregnanolone and the endogenous opioid mechanisms do not act independently. Indeed, allopregnanolone induced opioid inhibition over oxytocin responses to IL-1β in virgin rats. Thus, in late pregnancy, allopregnanolone induces opioid inhibition over magnocellular oxytocin neurones and hence on oxytocin secretion in response to immune challenge. This mechanism will minimise the risk of preterm labour and prevent the depletion of neurohypophysial oxytocin stores, which are required for 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.     

Sex-steroid induction of endogenous opioid inhibition on oxytocin secretory responses to stress

In pregnancy, endogenous opioids inhibit enhanced basal and stressor-stimulated oxytocin neurone activity and secretion. By contrast, stress responses of the hypothalamo-pituitary-adrenal (HPA) axis are reduced in pregnancy. We investigated whether the high levels of oestradiol and progesterone of pregnancy could induce these changes. Silastic capsules containing oestradiol or progesterone (or control capsules) were implanted s.c. in virgin female rats for 16 or 17 days, with or without progesterone removal on day 15 to mimic the progesterone withdrawal seen at the end of pregnancy. Plasma concentrations of oxytocin, adrenocorticotrophic hormone (ACTH) and corticosterone were measured in jugular vein blood samples from conscious rats. Under basal conditions, naloxone (5 mg/kg) increased oxytocin secretion in all groups, but had no greater effect in sex-steroid treated rats, and did not induce Fos expression in the supraoptic nucleus. Forced swimming, a stressor, increased oxytocin secretion at 5 min in vehicle-injected controls, and this response was slightly attenuated in the sex-steroid treated groups. Pretreatment with naloxone greatly enhanced the response in the sex-steroid treated rats, and was less effective in the controls. In rats treated with oestradiol alone, naloxone prolonged the response. Thus, the combined sex-steroid treatment enhanced the responsiveness of oxytocin neurones to the stressor, while simultaneously restraining oxytocin secretion via endogenous opioid inhibition. In the same rats, ACTH and corticosterone secretion was also stimulated by the stressor, but the hypothalamo-pituitary-adrenal (HPA) axis response was not attenuated in sex-steroid treated rats. Naloxone weakly reduced the HPA axis response in controls and was ineffective in the sex-steroid treated rats. We conclude that oestradiol and progesterone may be responsible for inducing the opioid restraint and enhanced oxytocin neurone responsiveness in pregnancy.     

The role of the AV3V region in the control of magnocellular oxytocin neurons

The AV3V region is important in the control of body fluid and Na⁺ regulation and projects to the supraoptic and paraventricular nuclei. Oxytocin from the neurohypophysis mediates milk ejection and is involved in parturition, but has also been recently implicated as a candidate natriuretic hormone. We have studied the role of the AV3V region in the control of magnocellular oxytocin neurons in rats. Electrical stimulation of the AV3V region increased the firing rate of supraoptic oxytocin neurons and evoked a concomitant release of oxytocin. Acute electrolytic AV3V lesions silenced supraoptic neurons and abolished their excitation by hyperosmotic stimulation. The lesions also abolished osmotically-induced release of oxytocin. Re-activation of supraoptic neurons by local glutamate restored their osmoresponsiveness to about 50% normal. Thus, while supraoptic neurons are directly osmosensitive, the AV3V region is essential for their normal osmoresponsiveness. Electrolytic AV3V lesions did not affect suckling-induced oxytocin secretion or, in conscious rats, the release of oxytocin secretion during parturition. Thus the AV3V region is not involved in the activation of oxytocin neurons during suckling or parturition.     

Convergent effects of oxytocin and a delta-opioid agonist in the bed nuclei of the stria terminalis of the peripartum rat

In the bed nuclei of the stria terminalis (BST), opioids may suppress the facilitatory effect of oxytocin on its own release pre-partum. In vitro electrophysiological recording showed that in virgin, late-pregnant, and lactating rats, a δ-opioid agonist inhibited a high proportion of BST neurons, many of which were also oxytocin responsive. Response magnitude did not differ significantly between groups, suggesting that the postulated pre-partum increase in opioid tone does not involve postsynaptic changes in neuronal sensitivity.     

Opposing effects of oxytocin and of a μ-receptor agonistic opioid peptide on the same class of non-pyramidal neurones in rat hippocampus

A study was made of the effects of opioid peptides on the spontaneous firing of oxytocin-responsive non-pyramidal neurones in hippocampal slices. D-Ala2-Gly-ol5-enkephalin (DAGO), a mu-opiate agonist, decreased or even suppressed the firing of these neurones, an effect reversed by naloxone. In contrast, U-50,488, a kappa-opiate agonist, had no effect. When the slices were synaptically uncoupled by elevating the concentration of external magnesium, oxytocin still excited non-pyramidal neurones and DAGO still inhibited them. Thus, opiates and oxytocin exerted direct, opposite effects on the same population of neurones, which apparently bear mu-type receptors. An indirect action of opioids on the excitability of pyramidal cells was apparent and is probably mediated by the same interneurones, since the amplitude of the depolarizing component of the synaptic potential elicited by stimulation of Schaffer's collaterals was increased in the presence of DAGO.     

Role of Anterior Peri-Third Ventricular Structures in the Regulation of Supraoptic Neuronal Activity and Neurohypophysical Hormone Secretion in the Rat

Neurohypophysical hormone release, and the electrical activity of single neurons of the supraoptic nucleus, were monitored in urethane-anaesthetized rats. Immediately after electrolytic lesions of the region anterior and ventral to the third ventricle (AV3V region), supraoptic neurons showed little spontaneous activity and their responses to ip injection of hypertonic saline were severely impaired; corresponding deficits were found in the secretion of both oxytocin and vasopressin. Similar deficits in oxytocin secretion were also found in rats following electrolytic lesions which destroyed all or part of the subfornical organ; however the effects of the lesions were not additive: rats with lesions of both the AV3V region and the subfornical organ region showed a similar degree of impairment of osmotically stimulated oxytocin secretion to rats with lesions of either site alone. Such deficits might occur either as a result of destruction of osmoresponsive projections to the magnocellular nuclei, or as a result of destruction of an afferent input which is essential for the full expression of the innate osmosensitivity of supraoptic neurons. To test the latter possibility, supraoptic neurons in AV3V-lesioned rats were activated by continuous application of glutamate, and then tested with ip injection of hypertonic saline. Five of seven cells tested responded significantly to the hyperosmotic stimulus, though the responses were significantly weaker than observed in sham-lesioned rats. We suggest that the innate osmosensitivity of supraoptic neurons does contribute to their responses to systemic osmotic stimulation, but that expression of this innate osmosensitivity requires inputs from the AV3V region and/or the subfornical organ, some of which may also be osmoresponsive. Electrical stimulus pulses applied to the AV3V region influenced the electrical activity of most supraoptic neurons strongly: the predominant response was a short-latency, short-duration inhibition followed by long-latency, long-duration excitation. Whereas intracerebroventricular administration of the angiotensin II antagonist saralasin reduced spontaneous or osmotically induced activity of supraoptic neurons, the neuronal responses to AV3V stimulation were impaired only with relatively high doses of saralasin. We conclude that angiotensin ll-sensitive neurons are an important component of the afferent pathways that sustain the excitability of supraoptic neurons, but that angiotensin is probably not the major transmitter of the projection from the AV3V region to the supraoptic nucleus.     

Permissive effect of centrally administered oxytocin on the excitatory response of oxytocin neurones to ventral tegmental stimulation in the suckled lactating rat

The mesencephalic ventral tegmentum has been implicated in the milk-ejection reflex and modulation of inputs from this region could provide a mechanism whereby central oxytocin facilitates synchronous bursting of oxytocin neurones during suckling. Experiments were therefore undertaken to investigate the effect of intracerebroventricular (i.c.v.) oxytocin on the response of oxytocin neurones to ventral tegmental stimulation. Oxytocin neurones were recorded in the supraoptic nucleus of urethane-anaesthetized lactating rats during suckling, and their response to single shock stimulation of the ventral tegmentum was monitored using peri-stimulus time-interval histograms. Before i.c.v. oxytocin, oxytocin neurones were either unresponsive to ventral tegmental stimulation, or displayed a small inhibition. However, after administration of oxytocin (2.2 ng i.c.v.), seven out of eight neurones tested displayed a pronounced excitatory response (onset latency 78.4 +/- 4.8 ms, duration 73.4 +/- 8.3 ms). This permissive effect on the excitatory response was only observed in the presence of suckling, and followed the same time course as facilitation of the milk-ejection reflex, being maximal immediately before each facilitated bursting response in the oxytocin neurones. The response to ventral tegmental stimulation remained unaltered after intraperitoneal administration of hypertonic saline to cause a generalized increase in the excitability of the oxytocin neurones. Moreover, i.c.v. oxytocin had no effect on the response of oxytocin neurones to stimulation of a descending input from the medial septum. In conclusion, administration of i.c.v. oxytocin has a selective permissive effect on the excitation of oxytocin neurones from the ventral tegmentum, and this supports previous in vitro studies suggesting that centrally released oxytocin may act as a modulator of afferent transmission to the magnocellular nuclei. This effect on the afferent excitation of oxytocin neurones may provide a mechanism whereby i.c.v. oxytocin facilitates suckling-evoked bursting activity.     

Neurohypophyseal hormone release and biosynthesis in rats with lesions of the anteroventral third ventricle (AV3V) region

Lesions of the periventricular tissue surrounding the anteroventral third ventricle (AV3V) have been shown to disrupt body fluid homeostasis. The acute post-lesion phase in rats is characterized by adipsia, the lack of an appropriate antidiuretic response, and plasma vasopressin levels which do not rise. Electron micrographs of the supraoptic nucleus and neural lobe of lesioned adipsic rats suggest no stimulation of biosynthetic activity, and large stores of neurosecretory material in the axon terminals. To directly investigate the status of these neurons, we determined neural lobe vasopressin and oxytocin content and the incorporation of [35S]cysteine into hypothalamic proteins in rats with sham-lesions or lesions of the AV3V after 3 days of adipsia or water deprivation, and in water replete sham-lesioned rats. The results demonstrate that adipsic rats with AV3V lesions have neural lobe vasopressin and oxytocin content equivalent to water-replete sham-lesioned rats. Neural lobe vasopressin and oxytocin levels of water-deprived sham-lesioned rats were significantly below those of all other groups. In addition, this group had a radioactivity incorporation rate into hypothalamic proteins which was two-fold greater than either of the other groups. The results indicate that 3-day adipsic AV3V-lesioned rats do not increase neurohypophyseal hormone release or biosynthesis as do 3-day water-deprived sham-lesioned rats. The periventricular tissue of the AV3V would therefore appear to be crucial in providing information to the hypothalamo-neurohypophyseal neurons on body fluid homeostasis.     

Effects of Different Opioid Receptor Antagonists on the Electrically-Evoked Release of Endogenous Dopamine from the Isolated Neural Lobe of the Rat Pituitary Gland in vitro

Isolated neural lobes of the rat pituitary gland were incubated in Krebs-HEPES solution which contained the dopamine uptake inhibitor GBR 12921 and in some experiments additionally pargyline. The release of endogenous dopamine evoked by electrical stimulation of the pituitary stalk was determined by high-performance liquid chromatography with electrochemical detection. (±)- Naloxone increased the evoked dopamine release maximally by 440% (EC₅₀ 209 nM). The (+)-enantiomer of naloxone (up to 10 μM) did not affect the release of dopamine. The preferential κ-opioid receptor antagonist MR 2266 increased the evoked dopamine release maximally by 135% (EC₅₀ 7 nM). MR 2267, the inactive (+)-enantiomer of MR 2266, had no effect on dopamine release. The δ-opioid receptor selective antagonist ICI 174864 increased the release of dopamine maximally by 120% (EC₅₀ 10 nM). The non-selective opioid receptor agonist etorphine up to 10 μM had no effect on the evoked dopamine release. In conclusion, endogenous opioids in the neurohypophysis strongly inhibit the release of endogenous dopamine from this gland. Activation of κ- and δ-opioid receptors appears to be involved in the inhibitory action of the endogenous opioids on the neurohypophysial release of dopamine.     

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.     

Hypothalamic opioid mechanisms controlling oxytocin neurones during parturition

The influences of opioids on oxytocin secretion and parturition were investigated in the rat. Morphine, administered centrally or peripherally, severely delays the course of established parturition. This delay is accompanied by reduced plasma oxytocin levels and is overcome by treatment either with the opioid antagonist naloxone, or by infusion of oxytocin. An endogenous opioid regulatory mechanism inhibiting oxytocin secretion becomes activated immediately prior to and during parturition. This mechanism does not operate earlier in pregnancy or during normal lactation and is not seen in nonpregnant animals. Naloxone acutely speeds up the course of established parturition, an effect accompanied by greatly elevated plasma oxytocin levels. The mechanisms underlying opioid regulation of oxytocin neurones were investigated at two sites. Precipitated withdrawal from chronic morphine treatment causes hypersecretion of oxytocin. This response is mediated by greatly enhanced electrical activity in the perikarya of oxytocin neurones indicating the presence of opioid receptors on oxytocin neurones and/or on their afferent input. Opioid receptors are also present in the neurohypophysis where they exert direct and noradrenaline mediated effects on secretion from oxytocin terminals in vitro.     

Changes in the active membrane properties of rat supraoptic neurones during pregnancy and lactation

To better understand the plasticity of intrinsic membrane properties of supraoptic magnocellular neuroendocrine cells associated with reproductive function, intracellular recordings were performed in oxytocin (OT) and vasopressin (VP) neurones from virgin, late pregnant (E19-22), and lactating (8-12 days of lactation) rats in vitro, using hypothalamic explants. OT neurones from virgin rats displayed a narrower spike width than neurones from pregnant and lactating rats, characterized by faster rise and decay times. Spike width changes in VP neurones were not as prominent as those observed in OT neurones. In OT neurones, the amplitude and the decay of the afterhyperpolarization following spike trains was significantly larger and faster, respectively, in pregnant and lactating rats compared to virgin rats. These properties did not change during pregnancy and lactation in VP neurones. The incidence of the depolarizing afterpotential following spikes significantly increased from approximately 20% in virgin rats to 40-50% during pregnancy and lactation in OT neurones, but was stable (80-90%) across states in VP neurones. Repetitive firing properties (frequency adaptation, the first interspike interval frequency and frequency-current (F-I) relationship) were altered during pregnancy and lactation in OT neurones, but not VP neurones. The increased incidence of depolarizing afterpotentials in OT neurones enhances excitability, while the increased afterhyperpolarization results in suppression of firing rate. Thus, the changes may favour the short bursting activity seen in OT neurones during lactation. These results confirmed reproductive state-dependent changes in intrinsic membrane properties of OT neurones during lactation, and suggest these changes are in place during late pregnancy. This argues that the plasticity in the electrical properties in OT neurones associated with lactation is not instigated by suckling.     

Activation of excitatory amino acid inputs to supraoptic neurons. I. Induced increases in dye-coupling in lactating, but not virgin or male rats

Mitral cells of the main and accessory olfactory bulbs have been shown to project monosynaptically to the supraoptic nucleus (SON) via the lateral olfactory tract (LOT) which uses excitatory amino acid transmitters. Data collected during characterization of these projections suggested that synaptic activation of SON neurons via LOT stimulation in slices influenced the incidence of dye-coupling. The present study pursued this suggestion using horizontally cut slices from male, virgin female and lactating rats. Neurons were confirmed to be excited by electrical stimulation of the tract, injected with Lucifer yellow, and synaptically activated for 10 min at 10 Hz (n = 92). Another 94 neurons were similarly confirmed and injected, but received no further stimulation. In an additional 8 slices, injected neurons were antidromically activated for 10 min at 10 Hz. Analyses done on 194 injected neurons from the 3 groups showed that synaptic activation resulted in a significant (P less than 0.01) increase in the incidence of coupling only in tissue from lactating rats. This increase was entirely due to larger numbers of cells being coupled dendrodendritically to the injected cells in the stimulated slices. Antidromic activation did not influence coupling. Increased coupling occurred among both oxytocin and vasopressin cell types. This is the first report of increased coupling resulting from synaptic activation in mammalian CNS. Changes seen only in lactating rats may be related to their altered SON ultrastructural morphology (i.e. dendritic bundling). Strong olfactory and vomeronasal input associated with some maternal behaviors may increase neuronal coupling and enhance hormone release in response to other incoming stimuli (e.g. suckling, dehydration).     

Beta-endorphin cells in the arcuate nucleus: projections to the supraoptic nucleus and changes in expression during pregnancy and parturition

Supraoptic nucleus oxytocin neurone activity and secretion are inhibited in late pregnancy and parturition by endogenous opioids. Here, we investigated alterations in the projections and gene expression of beta-endorphin/pro-opiomelanocortin neurones in the arcuate nucleus in the pregnant rat. All regions of the arcuate nucleus were found to contain cells immunoreactive for beta-endorphin fluorescent microbeads retrogradely transported from the supraoptic nucleus, and double-labelled neurones (beta-endorphin plus microbeads), showing that beta-endorphin neurones throughout the arcuate nucleus project to the supraoptic nucleus. There was an increase in the number of beta-endorphin-immunoreactive cells in the arcuate nucleus and an increase in the density of beta-endorphin fibres within the supraoptic nucleus and peri-supraoptic region in late pregnancy and parturition, suggesting enhanced expression of beta-endorphin and increased beta-endorphin innervation of the supraoptic nucleus. Pro-opiomelanocortin mRNA expression in the arcuate nucleus increased in late compared to early pregnancy: the number of positive neurones significantly increased in the caudal region. Fos expression (an indicator of neuronal activation) in the arcuate nucleus was colocalized in beta-endorphin neurones in both proestrus and parturient rats, but the number of positive cells did not increase during parturition, suggesting lack of activation of beta-endorphin neurones at birth. Thus, beta-endorphin cells in the arcuate nucleus project to the supraoptic nucleus and increased innervation during pregnancy may explain the enhanced endogenous opioid inhibition of oxytocin neurones.     

Effects of neurotensin on the organization of activity in supraoptic nucleus cells in virgin and lactating rats

Neurotensin increases the firing rate of supraoptic nucleus oxytocin and vasopressin neurones in vitro and induces Fos protein expression in the supraoptic nucleus in vivo. Here, we used extracellular single-unit electrophysiological recording combined with local microdialysis administration of neurotensin (1 mM at 2 micro l/min) to investigate the effects of locally applied neurotensin on the firing of oxytocin and vasopressin neurones in urethane-anaesthetized virgin and lactating rats. Neurotensin decreased the mean firing rate of oxytocin cells in virgin, but not lactating, rats. In addition, neurotensin increased the index of dispersion (a measure of the variability of firing) in virgin, but not lactating, rats. By contrast to oxytocin cells, neurotensin increased the mean firing rate of vasopressin cells in both virgin and lactating rats, but did not alter the index of dispersion. The increase in firing of phasic vasopressin cells was achieved through an increase in intraburst frequency (rather than an increase in burst duration or decrease in interburst interval), which resulted from a reduction of the spike-frequency adaptation that develops over the course of phasic bursts. Thus, neurotensin has differential effects on activity patterning in oxytocin and vasopressin cells and the effects on oxytocin cells, but not vasopressin cells, depend upon the physiological status of the animal. The increase in the variability of firing of oxytocin cells induced by neurotensin in virgin rats, but not in lactating rats, suggests that neurotensin (or other neurotransmitters/neuromodulators with similar actions) might establish conditions that predispose oxytocin cells to fire in milk-ejection bursts in lactating rats.     

Reduced activity of the noradrenergic system in the paraventricular nucleus at the end of pregnancy: implications for stress hyporesponsiveness

We investigated whether changes in noradrenaline neurotransmission in the hypothalamus could explain the hyporesponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis in late pregnancy. Noradrenaline release within the hypothalamic paraventricular nucleus in response to swim stress, as estimated by microdialysis and high-performance liquid chromatography, was lower in 20-day pregnant rats compared to virgin rats. Driving a central noradrenergic pathway using intravenous cholecystokinin increased adrenocorticotropic hormone (ACTH) secretion in virgin rats, but the response was significantly less in 16-day and 20-day pregnant rats. Thus, the activity of noradrenergic inputs to the paraventricular nucleus and the HPA axis is attenuated in late pregnancy. The sensitivity of the HPA axis to noradrenaline in pregnancy was investigated by intracerebroventricular administration of an alpha1-receptor antagonist, benoxathian, before and during exposure to swim stress. In virgin rats, benoxathian increased basal and stress-induced ACTH secretion, but in late pregnant rats the benoxathian effects were attenuated, indicating reduced sensitivity of the HPA axis to noradrenaline neurotransmission and/or the inability of the system to become disinhibited at this time. alpha1A-adrenoreceptor mRNA expression in the parvocellular and magnocellular paraventricular nucleus, measured by in situ hybridisation, was decreased in late pregnant compared to virgin rats. Additionally, blocking endogenous opioid inhibition with naloxone pretreatment restored the ACTH secretory response to cholecystokinin in pregnant rats. Thus, in late pregnancy, there is reduced noradrenergic input to the paraventricular nucleus and reduced alpha1A-receptor expression in the paraventricular nucleus, both of which may contribute to the reduced responsiveness of the HPA axis in pregnancy.     

Identification of brainstem projections mediating hemodynamic responses to stimulation of the anteroventral third ventricle (AV3V) region

The anteroventral third ventricle (AV3V) region is necessary for the development of several models of experimental hypertension in rats. Electrical stimulation of the AV3V region produces hindlimb vasodilation, mesenteric and renal vasoconstriction and bradycardia resulting in a depressor response. The effect of selective ablation of diencephalic and mesencephalic sites upon responses to AV3V stimulation was employed earlier to functionally identify descending tracts from the AV3V region through the ventromedial hypothalamus and rostral central gray. This study was performed in a similar manner to identify more caudal projections from the AV3V region to the level of pons and medulla. The vasodilator response to AV3V stimulation was dependent upon sites in and around the nucleus tractus solitarius, although the region receiving primary baroreceptor afferent input was not part of this projection. Visceral vasoconstrictor tracts were less clearly defined suggesting that these projections traverse diffusely through the ventrolateral tegmentum. These data suggest that a topographical differentiation of descending vasomotor tracts from the AV3V region exists in the brainstem of the rat.