Gareth Leng

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.

Central Actions of Peptide and Non-Peptide Growth Hormone Secretagogues in the Rat

Evidence for a central site of action of growth-hormone-releasing peptide (GHRP-6) was sought by (1) counting the number of Fos-positive nuclei within the brain following intracerebroventricular or intravenous injection of peptide and non-peptide GH secretagogues and (2) characterizing the electrophysiological responses of neuroendocrine arcuate neurones (recorded in vivo) following intravenous injection of GHRP-6. Conscious male rates were chronically implanted with intracerebroventricular or intravenous catheters. Dense nuclear Fos staining was induced throughout the ventral arcuate nucleus of rats injected intracerebroventricularly with low doses of GHRP-6 but not in rats injected with the endogenous GH-releasing hormone GHRH or in vehicle-treated controls. The non-peptidyl GH secretagogues L-692,585 and L-692,429 also induced Fos expression in the arcuate nucleus, and the pattern of distribution was similar to that described for GHRP-6. No increase in Fos expression was observed in rats given a systemic injection of a high dose of GHRH. In pentobarbitone-anaesthetized male rats, the effects of intravenous injection of GHRP-6 on the electrical activity of arcuate neurones was predominantly excitatory for putative neuroendocrine cells and inhibitory for the remaining unidentified cells. These results suggest that (1) GHRP-6 and non-peptidyl GH secretagogues have a central site of action involving the activation of a subpopulation of arcuate neurones and (2) this action is not mimicked by the central or peripheral effects of GHRH.

Presynaptic actions of morphine: blockade of cholecystokinin-induced noradrenaline release in the rat supraoptic nucleus.

1. This study aimed to establish the site at which morphine acts to inhibit oxytocin release in response to peripheral administration of cholecystokinin (CCK). 2. Conscious rats were given morphine or vehicle followed by CCK or vehicle (I.V.). Fos immunoreactivity was apparent 90 min after CCK injection in the supraoptic nucleus of vehicle‐ but not morphine‐pretreated animals. 3. In the dorsomedial (C2/A2) and the ventrolateral (C1/A1) regions of the brainstem, about half of the cells immunoreactive for tyrosine hydroxylase (TH) expressed Fos‐like protein after CCK injection. In the C2/A2 region, 20% of the Fos‐positive cells also showed TH immunoreactivity, whereas in the C1/A1 region 68% did so. Morphine treatment did not significantly change the number of cells expressing Fos immunoreactivity, or the percentage of TH‐positive cells expressing Fos‐like protein. 4. Amine release was measured in the supraoptic nucleus of urethane‐anaesthetized rats using a microdialysis probe. An I.V. injection of CCK increased the concentrations in the dialysate of noradrenaline and serotonin, but not of either adrenaline or dopamine. Pretreatment with morphine (I.V.) blocked the effects of CCK in a naloxone‐reversible manner. 5. Inclusion of morphine in the dialysate also blocked the increase in noradrenaline and serotonin in response to CCK in a naloxone‐reversible manner. 6. These observations indicate that morphine acts near or within the supraoptic nucleus to block CCK‐evoked noradrenaline release presynaptically. This presynaptic action of morphine may be a cause of the blockade of oxytocin release after CCK.

Effects of Opioid Agonists and Antagonists on Oxytocin and Vasopressin Release in vitro

The rat neurohypophysis contains both opioid receptors and substantial amounts of endogenous opioid peptides. Inhibitory influences of opioids on the secretion of both oxytocin and vasopressin have been described. We have examined the effects of a range of opioid agonists and antagonists with differing relative selectivities towards opioid receptor subclasses on the secretion of oxytocin and vasopressin from the isolated neurohypophysis. Oxytocin and vasopressin release evoked by brief periods of electrical stimulation in control experiments was compared to evoked release in the presence of test compounds. Oxytocin release was depressed approximately 25% by the delta-agonist (D-Ala2, D-Leu5)-enkephalin but not affected by putative kappa-agonists or by beta-endorphin. The use of opioid antagonists revealed a strong inhibition of oxytocin secretion by endogenous opioids released during electrical stimulation. Naloxone, relatively mu-selective, enhanced oxytocin secretion by up to 90% with a half-maximal effect at approximately 10(-6) M. MR2266, a relatively kappa-selective antagonist also enhanced oxytocin secretion but displayed agonist-like activity at high concentrations. ICI 154129, a delta-selective antagonist, was without effect on oxytocin secretion. Vasopressin release was unaffected by any of the agonists tested and not potentiated by antagonists at a range of stimulation frequencies. The data do not support the suggestion of an inhibitory endogenous opioid influence over vasopressin secretion within the neurohypophysis but indicate that an endogenous opioid peptide, possibly acting via mu- or kappa rather than delta-receptors, strongly suppresses the secretion of oxytocin.

Electrical activation and c-fos mRNA expression in rat neurosecretory neurones after systemic administration of cholecystokinin.

1. The expression of c‐fos mRNA in the rat hypothalamus was examined by in situ hybridization following systemic administration of cholecystokinin (CCK), a procedure known to activate magnocellular oxytocin neurons but not magnocellular vasopressin neurones. 2. Conscious male rats were given a single I.P. injection of 50 micrograms/kg CCK, c‐fos mRNA signal was apparent in the supraoptic and paraventricular nuclei in rats killed 10 min after injection but not in uninjected or saline‐(vehicle) injected rats. The density of c‐fos mRNA at both sites was further elevated in rats killed 30 min or 60 min following injection, and was absent in rats killed 4 h after injection. 3. In the paraventricular nucleus the most dense expression of c‐fos mRNA following CCK administration was in the medial, mainly parvocellular portion of the nucleus, in an area corresponding to the distribution of corticotrophin‐releasing factor mRNA determined by in situ hybridization in adjacent sections. 4. The I.P. injection of CCK increased plasma oxytocin concentrations, measured by specific radioimmunoassay from 13 +/‐ 5 pg/ml in control rats to 107 +/‐ 9 pg/ml in the rats killed 10 min after injection, a similar response to that observed previously in urethane‐anaesthetized rats. 5. In each of six urethane‐anaesthetized rats, recordings were made from single neurones in the supraoptic nucleus, identified antidronomically as projecting to the posterior pituitary and identified electrophysiologically as putative oxytocin neurones. Following I.P. injection of 50 micrograms/kg CCK, the neurones increased their firing rate by a mean of 1.3 +/‐ 0.2 spikes/s averaged over the 10 min following injection. 6. From the appearance of c‐fos mRNA in supraoptic neurones following CCK administration we conclude that this message is expressed in magnocellular oxytocin neurones, since vasopressin neuronal activity and vasopressin release is known to be unaffected by this stimulus, and since the supraoptic nucleus contains essentially only oxytocin neurones and vasopressin neurones. 7. We conclude that c‐fos mRNA expression can be induced in supraoptic oxytocin neurones following brief and modest episodes of electrical activation, suggesting that c‐fos may be involved in the gene regulation of these neurones under physiological conditions.

Naloxone excites oxytocin neurones in the supraoptic nucleus of lactating rats after chronic morphine treatment

1. Lactating rats were implanted with a cannula in a lateral cerebral ventricle to deliver morphine (up to 50 micrograms/h) chronically from a subcutaneous osmotically driven mini‐pump. After infusion of morphine for 5 days the rats were anaesthetized with urethane and prepared with ventral surgery for recording the electrical activity of single, antidromically identified neurones in the supraoptic nucleus. 2. A single I.V. injection of naloxone (5 mg/kg) in these rats provoked a long‐lasting, large increase in intramammary pressure, but in control rats had negligible effects. Concentrations in plasma of oxytocin, measured by radioimmunoassay in samples of femoral arterial blood, rose from 44.7 +/‐ 2.5 to 1072.1 +/‐ 89.5 pg/ml (means +/‐ S.E.M.) 6 min after naloxone in the morphine‐treated rats. In control rats, the concentration of oxytocin in plasma rose only from 42.1 +/‐ 2.9 to 125.1 +/‐ 28.2 pg/ml after naloxone. 3. Naloxone produced a transient increase in arterial blood pressure in morphine‐treated but not control rats. Concentrations in plasma of vasopressin, measured by radioimmunoassay in samples of femoral arterial blood, rose in morphine‐treated rats from 7.4 +/‐ 2.4 to 29.2 +/‐ 3.7 pg/ml after naloxone, but did not rise significantly in control rats. 4. Naloxone (1‐5 mg/kg) produced a prompt and prolonged increase in the discharge rate of each of ten continuously active (putative oxytocin) cells recorded from ten morphine‐treated rats. The discharge rate of the six cells tested at the highest dose (5 mg/kg) increased by an average of 6.3 Hz (360%) within 5 min, and the firing rate remained elevated for at least 30 min; the discharge rate of six continuously active supraoptic neurones recorded in control rats was not affected by naloxone. 5. The firing activity of five phasic (putative vasopressin) supraoptic neurones in morphine‐treated rats was increased for at least 30 min by the injection of naloxone; these increases were the result of a raised intraburst firing rate with no change in burst duration or frequency. One phasic neurone was inhibited for 15 min, and one phasic neurone was unaffected. 6. The excitatory effects of naloxone on neurones in the supraoptic nucleus of morphine‐treated rats were not explained by changes in blood pressure or osmolarity and did not depend on suckling or a cholinergic pathway. 7. The concentrations of oxytocin in plasma and the operation of the milk‐ejection reflex were similar in the controls and morphine‐treated rats, prior to naloxone. These findings indicate tolerance to initial inhibitory effects of morphine on oxytocin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversible fatigue of stimulus‐secretion coupling in the rat neurohypophysis.

Single rat neurointermediate lobes were impaled on a stimulating electrode and continuously perifused with oxygenated medium. The secretion of oxytocin and vasopressin into the medium was measured by specific radio‐immunoassays. The temporal profile of vasopressin release during a 20 min period of 13 Hz stimulation was compared with that of oxytocin. The results indicate that although the rate of secretion of both oxytocin and vasopressin declines over 20 min, the extent and time course of this fatigue is different for the two hormones. This difference could not be accounted for by differences in the rate of diffusion of released hormone from the tissue which was similar to the rate of wash‐out of [14C]sucrose from the extracellular space in pre‐labelled glands. In separate experiments glands were exposed to a prolonged period (60‐70 min) of 13 Hz stimulation interrupted by brief silent periods (30 s‐2 min duration). Some recovery from the fatigue of vasopressin secretion was evident after even the shortest of these silent periods. In further experiments glands were stimulated electrically for 18, 36, 54 and 72 s at 13 Hz: the order of presentation of the periods of stimulation was randomized between experiments. The vasopressin release rate declined markedly and progressively between 18 and 72 s. In contrast, the oxytocin release rate was relatively uniform throughout 72 s of stimulation. Thus vasopressin secretion is subject to a relatively rapid and dramatic fatigue. The results support the hypothesis that the phasic discharge patterns characteristic of vasopressin secreting neurones optimize the efficiency of vasopressin release from the nerve terminals in the neurohypophysis by avoiding the fatigue of stimulus‐secretion coupling that accompanies continual stimulation.

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.

Uterine Contractile Activity Stimulates Supraoptic Neurons in Term Pregnant Rats Via a Noradrenergic Pathway1

Oxytocin secretion is important for the normal progress of parturition in the rat. We tested the hypotheses that contractions of the uterus before pup delivery activate oxytocin neurons, and that they do so via a noradrenergic projection. In anesthetized 22-day (term) pregnant rats, iv oxytocin pulses enhanced both uterine contractile activity and the firing rate of oxytocin and vasopressin neurons in the supraoptic nucleus, and these were significantly correlated. The same oxytocin treatment also increased the expression of Fos in both the supraoptic nucleus and the nucleus of the tractus solitarius, but not in 21-day pregnant or virgin rats. In five of eight rats on the day of expected parturition, noradrenaline release in the supraoptic nucleus (sampled by microdialysis) exhibited sudden peaks during oxytocin administration, seen in only one of nine rats given vehicle pulses. Noradrenaline release was significantly greater in rats that went into labor or gave birth to a pup than in rats not in labor. In rats infused with the a1-noradrenergic receptor antagonist, benoxathian, into the supraoptic nucleus before and during iv oxytocin administration, Fos expression in supraoptic neurons was significantly less than that in vehicle controls. Thus, at term pregnancy, uterine contractions activate both oxytocin and vasopressin neurons in the SON, and this activation involves a noradrenergic pathway. (Endocrinology 142: 633–644, 2001) P CAN occur in the absence of oxytocin in the mouse, as is apparent from oxytocin gene knockout studies (1, 2). Nevertheless, oxytocin antagonists administered before or during parturition in the rat delay the onset and progress of delivery of young, respectively (3), suggesting an important role for oxytocin in the control of normal parturition, at least in this species. During parturition in the rat, the firing rate of magnocellular oxytocin neurons in the rat supraoptic nucleus (SON) is increased, and superimposed upon this elevated electrical activity are intermittent high frequency bursts of action potentials, which occur at about the time of delivery of each pup (4) and lead to pulsatile oxytocin secretion from the posterior pituitary (5, 6). The response of the rat uterus to systemic oxytocin administration increases on the day of delivery, in the last few hours before labor starts (7), and the pulsatile pattern of oxytocin appears to be particularly efficacious, as the onset of labor and optimal progress of parturition (8, 9) can be promoted by pulsatile administration of oxytocin at doses that are less effective when given continuously (9). Uterine contractions and the passage of fetuses through the birth canal further enhance oxytocin secretion; thus, oxytocin release from the pituitary during parturition is driven by a positive feedback loop (the Ferguson reflex) (10). This positive feedback is relayed via a neuronal pathway through the brainstem (11) including the A2 neurons of the nucleus of the tractus solitarius (NTS). Neurons in both the SON and the NTS express Fos during parturition, reflecting their activation at this time, and pulsatile administration of oxytocin to term pregnant rats results in the induction of Fos expression at both sites even before birth is induced (8, 9). Triple labeling has shown that some of these NTS neurons are both retrogradely labeled from the SON and contain tyrosine hydroxylase, the rate-limiting enzyme in noradrenaline synthesis (12). The above pattern of Fos expression in the SON and NTS during birth superficially resembles that after iv cholecystokinin (CCK). CCK increases the firing rate of oxytocin neurons, accompanied by increased Fos expression in the SON and increased oxytocin secretion (13, 14); CCK also induces Fos expression (8, 14) in noradrenergic neurons in the NTS and increases noradrenaline release within the SON (15), and local destruction of the noradrenergic innervation of the SON by selective neurotoxins prevents CCK-induced Fos expression in the SON, but not expression in the NTS or in the intact SON contralateral to the lesion (14). Thus, noradrenaline appears to mediate the activation of oxytocin neurons in the SON after systemic CCK. As noradrenaline release within the SON increases immediately before and during parturition (16), we hypothesized that this noradrenergic pathway may also mediate positive feedback from the contracting uterus to the SON. Received July 25, 2000. Address all correspondence and requests for reprints to: Dr. A. J. Douglas, Laboratory of Neuroendocrinology, Department of Biomedical Sciences, University Medical School, Edinburgh, United Kingdom EH8 9XD. E-mail: alison.j.douglas@ed.ac.uk. * This work was supported by The Wellcome Trust (Project Grant 047318/Z/96/Z) and the Biotechnology and Biological Sciences Research Council. † Current address: Max Planck Institute of Psychiatry, Kraepelinstrasse, 80804 Munich, Germany. 0013-7227/01/

Involvement of cholecystokinin receptor types in pathways controlling oxytocin secretion

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