Naloxone potentiates the release of oxytocin induced by systemic administration of cholecystokinin without enhancing the electrical activity of supraoptic oxytocin neurones

Summary Studies performed in conscious female rats confirmed that iv injection of cholecystokinin octapeptide (CCK; 20µ/kg) increased the circulating concentration of oxytocin but not that of vasopressin, and confirmed that the stimulation of oxytocin release was markedly facilitated after iv administration of naloxone (1mg/kg), indicating attenuation of oxytocin release by endogenous opioids. To investigate the site of action of the endogenous opioids, the electrical activity of putative oxytocin neurones in the supraoptic nucleus was recorded in urethaneanaesthetised female rats. Oxytocin neurones responded to CCK injection with an increase in firing rate lasting 5–15 min, but this response was not facilitated by prior injection of naloxone. The results suggest that the opioid influence upon CCK-induced oxytocin release operates at the level of the neurosecretory terminals in the neurohypophysis rather than centrally. Since CCK does not elevate vasopressin release, it appears unlikely that dynorphin, the opioid peptide co-existing with vasopressin, is responsible in these circumstances for the cross-inhibition of oxytocin release. It is suggested that products of proenkephalin A, the met-enkephalin precursor present in the supraoptic nucleus and in the neurohypophysis itself, may be active in the regulation of oxytocin release.     

Effects of naloxone and of intraperitoneal hypertonic saline upon oxytocin release and upon supraoptic neuronal activity

Urethane anaesthetized male rats were given an i.p. injection of hypertonic saline to increase plasma osmotic pressure. This injection resulted in significantly elevated plasma oxytocin levels and increased discharge activity of putative oxytocin cells in the supraoptic nucleus. Subsequent injection of naloxone (1 mg/kg) i.v. resulted in a similarly large increase in plasma oxytocin, but did not affect the discharge activity of putative oxytocin neurones. The results suggest that, following an i.p. injection of hypertonic saline, endogenous opioids act at the neurosecretory terminals to partially inhibit oxytocin release.     

Release of oxytocin in the hypothalamic paraventricular nucleus, but not central amygdala or lateral septum in lactating residents and virgin intruders during maternal defence

In lactating rats, the neuroendocrine responses of the oxytocinergic system and the hypothalamo-pituitary-adrenal axis to various kinds of stressors are attenuated. In this study, using intracerebral microdialysis in combination with a highly sensitive radioimmunoassay, we characterised oxytocin (OXT) release within the paraventricular nucleus (PVN), the central amygdala (CeA), and the medio-lateral septum (mS) before, during and after a psycho-social stressor (the maternal defence test) in both the virgin intruder and the lactating resident rat (day 3 of lactation). Within the PVN, local OXT release was found to increase significantly in virgin intruders during exposure to the resident (2.1-fold, P < 0.05), as well as in lactating residents when exposed to the virgin intruder, though to a lesser extent when compared with basal levels (1.7-fold, P < 0.05). In contrast, OXT release remained unchanged within the CeA and the mS of both virgin intruders and lactating residents. Release of OXT under basal conditions was clearly above the detection limit of the radioimmunoassay, and did not differ between lactating and virgin rats in any of the brain regions studied. Our study also demonstrates that recent surgery or ongoing intracerebral microdialysis does not affect the behavioural performance of the intruders or residents when comparing dialysed and non-dialysed rats. The results indicate that exposure to the maternal defence test is a relevant stressor for the brain OXT system which becomes activated in both intruder and resident rats, although to varying degrees depending upon their reproductive status and in a region-dependent manner. The behavioural and/or neuroendocrine functions of intra-PVN released OXT during this psycho-social challenge remain to be clarified.     

Brain oxytocin: differential inhibition of neuroendocrine stress responses and anxiety-related behaviour in virgin, pregnant and lactating rats

The involvement of brain oxytocin in the attenuated responsiveness of the hypothalamo-pituitary-adrenal axis and the oxytocin systems to external stressors found in pregnant and lactating rats has been studied, including both neuroendocrine and behavioural aspects. Intracerebroventricular infusion of an oxytocin receptor antagonist (0.75 microg/5 microl), but not of vehicle, elevated basal corticotropin and corticosterone secretion into blood of virgin female, but not of late pregnant or lactating rats. Oxytocin antagonist treatment further elevated the stress-induced (exposure to the elevated plus-maze or forced swimming) secretion of both corticotropin and corticosterone, but only in virgin and not in pregnant or lactating rats. Thus, corticotropin and corticosterone plasma concentrations remained attenuated in antagonist-treated pregnant and lactating animals. In contrast, infusion of the oxytocin antagonist significantly elevated the stress-induced secretion of oxytocin into blood in pregnant and lactating, but not in virgin, animals, indicating an autoinhibitory influence of intracerebral oxytocin on neurohypophysial oxytocin secretion induced by non-reproduction-related stimuli. Treatment with oxytocin antagonist 10 min prior to behavioural testing on the elevated plus-maze significantly reduced the anxiety-related behaviour in both pregnant and lactating rats, without exerting similar effects in virgin female rats. The results demonstrate a tonic inhibitory effect of endogenous oxytocin on corticotropin and, consequently, corticosterone secretion in virgin female rats, an effect which is absent in the peripartum period. In contrast, an anxiolytic action of endogenous oxytocin was detectable exclusively in pregnant and lactating rats. Therefore, we conclude that the actions of intracerebral oxytocin include independent effects on the responses of the hypothalamo-pituitary-adrenal axis and oxytocin systems to stressors and the anxiety-related behaviour which are modulated by the reproductive state of the animals.     

Brain oxytocin: differential inhibition of neuroendocrine stress responses and anxiety-related behaviour in virgin,pregnant and lactating rats

The involvement of brain oxytocin in the attenuated responsiveness of the hypothalamo-pituitary–adrenal axis and the oxytocin systems to external stressors found in pregnant and lactating rats has been studied, including both neuroendocrine and behavioural aspects. Intracerebroventricular infusion of an oxytocin receptor antagonist (0.75 μg/5 μl), but not of vehicle, elevated basal corticotropin and corticosterone secretion into blood of virgin female, but not of late pregnant or lactating rats. Oxytocin antagonist treatment further elevated the stress-induced (exposure to the elevated plus-maze or forced swimming) secretion of both corticotropin and corticosterone, but only in virgin and not in pregnant or lactating rats. Thus, corticotropin and corticosterone plasma concentrations remained attenuated in antagonist-treated pregnant and lactating animals. In contrast, infusion of the oxytocin antagonist significantly elevated the stress-induced secretion of oxytocin into blood in pregnant and lactating, but not in virgin, animals, indicating an autoinhibitory influence of intracerebral oxytocin on neurohypophysial oxytocin secretion induced by non-reproduction-related stimuli. Treatment with oxytocin antagonist 10 min prior to behavioural testing on the elevated plus-maze significantly reduced the anxiety-related behaviour in both pregnant and lactating rats, without exerting similar effects in virgin female rats.The results demonstrate a tonic inhibitory effect of endogenous oxytocin on corticotropin and, consequently, corticosterone secretion in virgin female rats, an effect which is absent in the peripartum period. In contrast, an anxiolytic action of endogenous oxytocin was detectable exclusively in pregnant and lactating rats. Therefore, we conclude that the actions of intracerebral oxytocin include independent effects on the responses of the hypothalamo-pituitary–adrenal axis and oxytocin systems to stressors and the anxiety-related behaviour which are modulated by the reproductive state of the animals.     

Release patterns of excitatory and inhibitory amino acids within the hypothalamic supraoptic nucleus in response to direct nitric oxide administration during forced swimming in rats

The effect of direct intrahypothalamic nitric oxide (NO) administration on the release of selected amino acids in the hypothalamic supraoptic nucleus (SON) with and without concomitant forced swimming was investigated. Adult male Wistar rats were chronically fitted with U-shaped microdialysis probes in the SON and forced to swim for 10-min in 20-degree C warm water. Thirty-min microdialysis samples were collected before, during and after the forced swimming period while NO was administered into the SON via microdialysis. The results show that NO administration in combination with forced swimming affects the release of aspartate, glutamate, taurine, and gamma aminobutyric acid (GABA) in different patterns. Whereas the release of the excitatory amino acids aspartate and glutamate was triggered only during NO administration and forced swimming, the inhibitory amino acids GABA and taurine were found in the extracellular fluid in increased concentrations also after the treatment. These data indicate that NO administration differently affects the release of excitatory and inhibitory amino acids within the SON. Thus, SON neurons which contain high concentrations of neuronal NO synthase, might contribute to the regulation of their own secretory activity by releasing NO that controls the orchestrated release of excitatory and inhibitory amino acids from axon terminals of afferences and interneurons as well as release from glial cells.     

Neuroactive steroids attenuate oxytocin stress responses in late pregnancy

In late pregnant rats neuroendocrine stress responses, expressed as increased oxytocin secretion and activation of the hypothalamo-pituitary-adrenal axis, are attenuated. These adaptations preserve the oxytocin store for parturition and prevent pre-term birth, and protect the fetuses from adverse programming by exposure to excess glucocorticoid. Mechanisms of adaptations for oxytocin neurones are reviewed, using challenge with systemic interleukin-1beta, simulating activation of immune signaling by infection, as a stressor of special relevance in pregnancy. In virgin rats, systemic interleukin-1beta stimulates the firing of oxytocin neurones, and hence oxytocin secretion, but interleukin-1beta has no effects in late pregnant rats. This lack of response is reversed by naloxone treatment just before interleukin-1beta administration, indicating endogenous opioid suppression of oxytocin responses in late pregnancy. This opioid presynaptically inhibits noradrenergic terminals impinging on oxytocin neurones. Finasteride pretreatment, inhibiting progesterone conversion to allopregnanolone, a positive GABA(A) receptor allosteric modifier, also restores an oxytocin response to interleukin-1beta. This finasteride effect is reversed by allopregnanolone treatment. In virgin rats allopregnanolone attenuates the oxytocin response to interleukin-1beta, which is exaggerated by naloxone. The effects of naloxone and finasteride in late pregnant rats in restoring an oxytocin response to interleukin-1beta are not additive. Accordingly, allopregnanolone may both enhance GABA inhibition of oxytocin neurone responses to interleukin-1beta, and induce opioid suppression of noradrenaline release onto oxytocin neurones.     

Pregnancy alters the density of opioid binding sites in the supraoptic nucleus and posterior pituitary gland of rats.

Opioid receptor binding was measured in cryostat sections of supraoptic nucleus (SON) and posterior pituitary of virgin and pregnant rats by quantitative receptor autoradiography after in vitro incubation with [3H]etorphine or [3H](-)-bremazocine in the presence of unlabelled sub-type-selective agonists. Mu-selective [3H]etorphine-binding in the SON was reduced on the last day (21) of pregnancy vs. virgin controls (9.9 +/- 2.2 vs. 31.7 +/- 6.5 fmol/mg). Kappa-selective [3H](-)-bremazocine binding to the SON was not altered by pregnancy. Kappa-selective [3H](-)-bremazocine-binding to the posterior pituitary was less on day 16 of pregnancy vs. virgin females (19.1 +2- 5.2 vs. 74.4 +/- 16.2 fmol/mg). The results suggest mechanisms for the changes in actions of opioids on oxytocin neurones in pregnancy.     

Facilitative role of prolactin-releasing peptide neurons in oxytocin cell activation after conditioned-fear stimuli

Emotional stress activates oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei and stimulates oxytocin release from the posterior pituitary. Oxytocin neurons in the hypothalamus have synaptic contact with prolactin-releasing peptide (PrRP) neurons. Intracerebroventricular administration of PrRP stimulates oxytocin release from the pituitary. These observations raise the possibility that PrRP neurons play a role in oxytocin response to emotional stress. To test this hypothesis, we first examined expression of Fos protein, an immediate early gene product, in the PrRP neurons in the medulla oblongata after conditioned-fear stimuli. Conditioned-fear stimuli increased the number of PrRP cells expressing Fos protein especially in the dorsomedial medulla. In order to determine whether PrRP cells projecting to the supraoptic nucleus are activated after conditioned-fear stimuli, we injected retrograde tracers into the supraoptic nucleus. Conditioned-fear stimuli induced expression of Fos protein in retrogradely labeled PrRP cells in the dorsomedial medulla. Finally we investigated whether immunoneutralization of endogenous PrRP impairs oxytocin release after emotional stimuli. An i.c.v. injection of a mouse monoclonal anti-PrRP antibody impaired release of oxytocin but not of adrenocorticotrophic hormone or prolactin and did not significantly change freezing behavior in response to conditioned-fear stimuli. From these data, we conclude that PrRP neurons in the dorsomedial medulla that project to the hypothalamus play a facilitative role in oxytocin release after emotional stimuli in rats.     

Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats

Summary To investigate the hypothesis that oxytocin may be released within the magnocellular nuclei in vivo, push-pull cannula perfusions were performed in anaesthetized lactating rats in one supraoptic nucleus of the hypothalamus while recording the intramammary pressure and/or the electrical activity of oxytocin cells in the contralateral supraoptic nucleus. Oxytocin content was measured in samples collected over 15 min, under various conditions: 1) with no stimulation; 2) during suckling and suckling-induced reflex milk ejections; 3) during electrical stimulation of the neurohypophysis by trains of pulses that mimicked oxytocin cell bursts; 4) under osmotic stimulation by i.p. injection of 2 ml of 1.5 M NaCl to evoke a tonic and sustained oxytocin release from the neurohypophysis. Oxytocin release within the supraoptic nucleus increased significantly during the milk ejection reflex and, to a lesser extent, during burst-like electrical stimulation of the neurohypophysis. In suckled rats, the increase started before the first reflex milk ejection occurred. There was no apparent correlation between the amount of oxytocin in the perfusates and the number of milk ejections and oxytocin cell bursts occurring during each perfusion period. The amount of oxytocin in the perfusates further increased during facilitation of the milk ejection reflex by intraventricular injections of oxytocin or its analogue, isotocin. When suckling failed to evoke the milk ejection reflex, there was no change in intra-supraoptic oxytocin release. There was also no change after osmotic stimulation. When the push-pull cannula was positioned outside the supraoptic nucleus, there was no increase in the amount of oxytocin during the three types of stimulation tested. These results provide evidence for an endogenous release of oxytocin within the magnocellular nuclei in lactating rats. It is suggested that the increase in such a release induced by suckling is likely to be a pre-requisite for the onset and the maintenance of the characteristic intermittent bursting electrical activity of oxytocin cells leading to milk ejections.     

Possible morphological bases for synchronisation of neuronal firing in the rat supraoptic nucleus during lactation

Oxytocin-containing neurones in the supraoptic and paraventricular nuclei of lactating rats display a periodic activation which results in a pulsatile release of hormone before each reflex milk ejection induced by suckling. This electrical activity occurs in an all-or-none fashion and is synchronised in the whole population of oxytocin neurones in both nuclei. The present report describes changes in the ultrastructure of the supraoptic nucleus of lactating animals which may serve as morphological bases for such a functional synchronisation.In the supraoptic nuclei of normal rats, neurosecretory neurones are usually separated by elements of the neuropil, particularly glial processes. At rare intervals, adjacent neurosecretory somata, and dendrites, are seen to be in direct apposition. The only specialisations apparent between the contiguous membranes are occasional attachment plates. In nuclei of lactating rats, quantitative analysis indicated that 34% of profiles of the sectioned neurosecretory cell bodies were in direct contact with each other and 22% with profiles of dendrites, a 5-fold increase over the corresponding frequencies observed in normal male and virgin female animals. Such contacts involved 10% of the total measured soma surface membrane (compared to 1.5% in the controls). The number of attachment plates supporting the apposing membranes also increased significantly as did the mean size of the individual appositions. There was also a higher incidence of presynaptic terminals contacting more than one post-synaptic element (soma or dendrite) in the same plane of section, a rare phenomenon in the normal nucleus. No further increases were evident in these appositional relations in virgin female and lactating rats deprived of water for one day, a stimulus which enhances vasopressin release.It is postulated that the structural reorganisation observed in the nuclei of lactating animals may lead to electrical interactions between the neurosecretory cells and may thus be one of the factors supporting the synchronisation of neuronal activity during the episodic release of oxytocin.     

Septal projections to nuclei functioning in oxytocin release

Terminal degeneration was observed in the paraventricular and supraoptic nuclei of the hypothalamus following lesions in the septum of the squirrel monkey. The degeneration was usually greater in the supraoptic nucleus. A lesion in the posterior dorsal septum resulted in more degeneration in the paraventricular nucleus. Lesions in the dorsal gyrus rectus, genu of the corpus callosum and extreme rostral septum resulted in no identifiable degeneration in the two nuclei. These nuclei have been shown by various investigators to facilitate milk ejection (oxytocin release). Stimulation of the mesencephalic central gray has been shown to influence oxytocin release. Following a lesion in the mid-septal area, degenerating terminals were seen in the mesencephalic central gray. Our results show that the septum is directly connected with the supraoptic and paraventricular nuclei and mesencephalic tectum. Possibly the septum functions in modulating mechanisms having to do not only with lower autonomic responses such as blood pressure, and bladder emptying, but oxytocin release as well.     

Electrical activity in the supraoptic and paraventricular nuclei associated with neurohypophysial hormone release

1. Unit recordings were made from the hypothalamus in anaesthetized male rats using steel or glass micro-electrodes.2. Stimuli which are known to release vasopressin and oxytocin (electrical stimulation of the central end of the severed right vagus nerve and intracarotid injection of CaCl(2) solution) also excite units in the supraoptic and paraventricular nuclei. In addition, these units are excited by intracarotid injections of carbachol, acetylcholine and NaCl (5%) which are less effective stimuli for vasopressin release.3. These stimuli also excite units from which potentials can be evoked by stimulation of the pituitary stalk and which are likely to be neurosecretory neurones. Neurosecretory neurones can conduct electrical impulses like other less specialized nerve cells. Estimations of conduction velocity range between 0.4 and 1.3 m/sec.4. The proportion of units in the supraoptic and paraventricular nuclei which are excited and the degree of excitation corresponds approximately to the amount of vasopressin released by the stimuli. In addition, excitation of the supraoptic nucleus seems to be more directly associated with vasopressin release and excitation of the paraventricular nucleus with oxytocin release.     

Neurohypophysial opioids and oxytocin secretion: source of inhibitory opioids

Summary When electrical stimuli are applied to the neural stalk of the pituitary, oxytocin, vasopressin, and probably several opioid peptides also contained in nerve terminals in the gland are released: one action of the released opioids appears to be to inhibit oxytocin release by an action that has been likened to pre-synaptic inhibition. Thus, when Clarke et al. (1979) stimulated the neural stalk following intravenous injection of the opioid antagonist naloxone, they observed that the evoked oxytocin release was potentiated. In the present study we confirm this result and show that oxytocin release evoked by stimulation of the supraoptic nucleus is similarly potentiated by naloxone. This finding is consistent with the hypothesis that the opioid responsible for inhibition of oxytocin release coexists with either oxytocin or vasopressin. We further report that the specific -receptor antagonist ICI 174864 does not potentiate oxytocin release either in vivo or in vitro. Thus, it seems unlikely that the enkephalins, putative -receptor agonists present in neurohypophysial fibres, are the opioids responsible for the observed inhibition of oxytocin release.     

Oxytocin and ADH secretion in relation to electrical activity in antidromically identified supraoptic and paraventricular units

1. Electrical recordings were made from antidromically identified supraoptic and paraventricular units during intracarotid injections of hypertonic and isotonic sodium chloride solutions in rats.2. The blood concentrations of vasopressin and oxytocin were estimated by bio-assay before and at different intervals after similar injections.3. Although a significant change in the action potential activity of the supraoptic nucleus was associated with hormone release, the results were not entirely consistent with a simple relationship between action potential activity and hormone secretion. Firstly, although some units were excited by the stimulus a substantial number were inhibited. Secondly, the blood concentration of the hormones, particularly ADH, remained elevated for longer than might have been expected if additional hormone had ceased to be secreted as soon as firing rates had returned to control values.4. There were substantial differences between the initial blood concentrations of vasopressin and oxytocin but the firing rates of units in the supraoptic and paraventricular nuclei appeared to be the same.5. Although significantly less paraventricular than supraoptic units were affected by hypertonic injections the blood concentration of oxytocin was increased by a factor of 8 whereas that of vasopressin was increased by a factor of 2.7.     

Opioid modulation of magnocellular neurosecretory cell activity

Magnocellular neurosecretory cells of the hypothalamic supraoptic and paraventricular nuclei secrete the hormones, oxytocin and vasopressin, into the systemic circulation from the posterior pituitary gland. Oxytocin is important for parturition and is essential for lactation. Vasopressin regulates body fluid homeostasis. The secretion of these hormones is altered in response to peripheral stimuli that are conveyed via projections from other parts of the brain. Endogenous opioid peptide systems interact with the magnocellular neurosecretory system at several levels to restrain the basal secretion of these hormones as well as their secretory responses to various physiological stimuli. The inhibition of basal secretion can occur at the level of the neurosecretory terminals where endogenous opioids inhibit the release of oxytocin, and at the cell bodies of magnocellular cells to modulate the activity pattern of vasopressin cells. The responses of the magnocellular neurosecretory system to physiological stimuli are also regulated by these mechanisms but in addition probably also by pre-synaptic inhibition of afferent inputs to magnocellular cells as well as direct effects on the cell bodies of afferent input cells to modulate their activity. Here, we review the mechanisms and functional consequences of opioid interactions with oxytocin and vasopressin cells.     

Electrophysiological evidence for a projection from the arcuate nucleus to the supraoptic nucleus

The electrical activity of single neurones in the hypothalamic arcuate and supraoptic nuclei was recorded in urethane-anaesthetized rats. Stimulus pulses applied to the supraoptic nucleus antidromically activated 3 out of 41 cells recorded in the ipsilateral arcuate nucleus, confirming that there is a projection from the arcuate nucleus to the region of the supraoptic nucleus. Stimulation of the arcuate nucleus inhibited 17 out of 19 continuously firing (putative oxytocin) supraoptic neurones. Inhibition was followed by a marked post-stimulus excitation in 12 cells. The responses were not abolished by i.v. injection of the opioid antagonist naloxone. Thus at least part of the input to the magnocellular oxytocin system that arises from or passes through the arcuate nucleus, is not mediated by opioid peptides.     

Impaired central stress-induced release of noradrenaline in rats with heart failure: a microdialysis study

Sympathetic hyperactivity in rats with heart failure is associated with increased extracellular noradrenaline in the hypothalamic paraventricular nucleus at rest. However, it is unknown how this nucleus responds to stressful stimuli. In the present study we therefore examined the basal and stress-induced release of noradrenaline in the paraventricular nucleus of conscious Sprague-Dawley rats with heart failure measured by in vivo microdialysis. Basal noradrenaline concentration in the paraventricular nucleus of rats with heart failure was more than double that in sham-operated controls. Immobilization stress decreases noradrenaline levels in the paraventricular nucleus of rats with heart failure to 57% of baseline, while it increased in sham-operated controls to 228%. However, serum corticosterone was similarly elevated at 30 and 90 min post-stress in both experimental groups. We have shown that heart failure causes an impairment of the central noradrenergic system's response to acute sympatho-excitation but does not affect the hypothalamo-pituitary-adrenocortical response.     

Involvement of oxytocin and its receptor in nociceptive modulation in the central nucleus of amygdala of rats

Studies have demonstrated that oxytocin plays important roles in pain modulation in the central nervous system. Oxytocin-ergic neurons are found in paraventricular nucleus and supraoptic nucleus of the hypothalamus. The oxytocin-ergic neurons send fibers from hypothalamus to amygdala and high density of oxytocin receptors are found in the central nucleus of amygdala (CeA). The present study was performed to investigate the influences of oxytocin and its receptors on nociceptive responses in the CeA of rats. Intra-CeA injection of 0.1, 0.5 or 1 nmol of oxytocin induced dose-dependent increases in the handpaw withdrawal latency induced by noxious thermal and mechanical stimulation in rats. The oxytocin-induced anti-nociception could be blocked by the selective oxytocin antagonist 1-deamino-2-d-Tyr-(Oet)-4-Thr-8-Orn-oxytocin. The present study demonstrated that oxytocin and its receptors are involved in nociceptive modulation in the CeA of rats.