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M. J. O. Clarke and G. E. Gillies

ABSTRACT

Primary cultures of rat hypothalamic neurones were maintained either in a serum-supplemented medium or in a serum-free chemically defined medium for up to 6 weeks. The release of the 41 amino acid-containing peptide, corticotrophin-releasing factor (CRF-41), vasopressin (AVP) and somatostatin (SRIF) were followed using immunoassays. In response to K+ (56 mmol/l) depolarization both the quantities of peptides released and the magnitude of responses were significantly greater from cultures maintained in the fully supplemented defined medium. As a consequence, release of CRF-41 and AVP could be measured directly, without requiring the concentration step necessary for cultures grown in serum. The response to K+ depolarization increased with the age of the culture, suggesting neuronal maturation.

Responses to K+ depolarization were Ca2+-dependent, and the addition of corticosterone (100 nmol/l) to the defined medium caused a significant reduction in the response of neurones secreting CRF-41 and AVP, but not those secreting SRIF, to depolarization. This suggests the retention in vitro of the responsiveness of stress-associated neuropeptides to the negative feedback effects of corticosterone. Neurones producing CRF-41 and AVP responded significantly in a dose-dependent manner to acetylcholine stimulation, whereas those producing SRIF did not. As cultures matured, the CRF-41- and AVP-producing neurones became more sensitive to acetylcholine with the maximal response at 1 nmol acetylcholine/1.

In conclusion, the culture of rat hypothalamic neurones is improved in terms of peptide output when the cultures are maintained in a defined medium. Differential responses of the peptidergic neurones may be seen in the presence of corticosterone and neurotransmitters, illustrating the retention in vitro of specific receptor-mediated responses which have been observed in vivo. This model should prove useful in the further study of the physiological, pharmacological and biochemical maturation and control of peptidergic neurones.

J. Endocr. (1988) 116, 349–356

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G. CLARKE, D. W. LINCOLN, and LYNDA P. MERRICK

During suckling, anaesthetized lactating rats release regular (about every 7 min) but brief )ulses of oxytocin (0·5–1·0 mu.) which produce single transient increases in intramammary [unk]ressure. Drugs which selectively impair synaptic transmission were used to determine the [unk]ole of dopamine and noradrenaline in regulating this natural reflex. Diethyldithiocarbamate 100–200 mg/kg, i.v.) and α-methylparatyrosine (100–400 mg/kg, i.v.) which inhibit the [unk]ynthesis of catecholamines both blocked the suckling-induced release of oxytocin. The milk[unk]jection reflex was also inhibited in a dose-dependent manner by the intravenous administraion of the dopamine antagonists, fluphenazine (0·7 mg/kg), pimozide (1·4 mg/kg), cis-lupenthixol (4·5 mg/kg) and metoclopramide (6·0 mg/kg), and caused a significant inhibition P < 0·01) of the reflex in 50% of the rats tested. The α-adrenoceptor antagonist phenoxy[unk]enzamine (1·4 mg/kg) was similarly effective. Dopamine (40 μg), bromocriptine (10 μg), [unk]pomorphine (100 μg), noradrenaline (10 μg) and phenylephrine (2 μg) injected into the;erebral ventricles evoked a sustained release of oxytocin which produced multiple increases n intramammary pressure; isoprenaline (4 μg) was ineffective. The release of oxytocin evoked [unk]y dopamine and noradrenaline was prevented by cis-flupenthixol and phenoxybenzamine respectively. None of the drugs used affected the mammary sensitivity to exogenous oxytocin lor were their actions modified by pretreatment with propranolol (1 mg/kg). The results [unk]uggest that the neural pathway for the reflex release of oxytocin during suckling in the rat [unk]ontains both dopaminergic and noradrenergic synapses, the latter acting through α-[unk]drenoceptors and being distal in the pathway to the dopaminergic component.

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T P Fletcher, G B Thomas, F R Dunshea, L G Moore, and I J Clarke

Abstract

The putative negative feedback effects of IGF-I and IGF-II on GH secretion were tested by intracerebroventricular (icv) and intrapituitary administration to sheep. Over two consecutive days, serial jugular blood samples were taken at 10 min intervals for 6 h from ewes (n=3/group) fitted with indwelling stainless steel cannulae into the lateral or third cerebral ventricles. The sheep were injected (icv) with either vehicle or purified ovine IGF-I (2, 4 or 8 μg). IGF-I injection had no effect on plasma GH secretion. Serial blood samples were taken from a second group of nine ewes in which ovine or recombinant human (rh) IGF-I was infused (2·5 μg/h for 2 h) into the third ventricle; once again, IGF-I failed to affect the episodic pattern of GH secretion. Three ewes fitted with indwelling stainless steel cannulae placed in the anterior pituitary gland were consecutively infused with either ovine or rhIGF-I (2·5 μg/h for 2 h) or vehicle. Plasma GH concentrations were suppressed in 3/3 sheep from 1–1·5 h after the commencement of infusion and GH levels remained low for the remainder of the sampling period. In another group of five ewes synergistic effects of IGF-I and IGF-II on GH secretion were tested by icv infusion of rhIGF-I, rhIGF-II, or rhIGF-I+rhIGF-II (5 μg/h for 2 h) or vehicle (sterile 10 mm HCl/saline). Each sheep received each treatment in a randomised design. Infusion (icv) of IGF-I and IGF-II alone or in combination failed to alter GH secretion.

These observations suggest that IGF-I derived from peripheral tissues may modulate GH release at the pituitary level but that IGF-I acts neither alone nor in conjunction with IGF-II as a negative feedback regulator of GH secretion via the hypothalamus in the ewe.

Journal of Endocrinology (1995) 144, 323–331

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G. B. Thomas, J. T. Cummins, G. A. Smythe, R. M. Gleeson, R. C. Dow, G. Fink, and I. J. Clarke

ABSTRACT

The concentrations of dopamine, noradrenaline and their respective primary neuronal metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethyleneglycol (DHPG) were measured in the hypophysial portal and peripheral plasma of sheep and rats by combined gas chromatography–mass spectrometry. Hypophysial portal and jugular blood samples were taken at 5- to 10-min intervals for 3–7 h from six conscious ovariectomized ewes. Blood was also collected for 30 min under urethane anaesthesia from the cut pituitary stalk from 16 pro-oestrous female and five intact male rats.

In ovariectomized ewes, noradrenaline concentrations were higher in hypophysial portal plasma than in peripheral plasma (6·6 ± 0·8 vs 2·2 ± 0·4 nmol/l). In contrast, dopamine was undetectable (<1 nmol/l) in the portal and peripheral plasma of all ewes. Plasma levels of DOPAC and DHPG in portal and jugular samples were similar. In all pro-oestrous female rats, plasma concentrations of dopamine were higher in portal blood than in jugular blood (8·0±1·4 vs 4·8± 0·6 nmol/l). Detectable concentrations of dopamine were measured in the portal plasma of two out of five male rats. Noradrenaline concentrations were higher in portal plasma than in peripheral plasma of both female (8·3 ± 1·7 vs 3·7 ± 0·6 nmol/l) and male (14·8± 2·7 vs 6·1± 1·2 nmol/l) rats.

These data show that noradrenaline, but not dopamine, is secreted into the long portal vessels in sheep. The results suggest that there are species differences in the secretion of hypothalamic dopamine into hypophysial portal blood.

Journal of Endocrinology (1989) 121, 141–147

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G. B. Thomas, J. T. Cummins, L. Cavanagh, and I. J. Clarke

ABSTRACT

Hypothalamic control of prolactin secretion was studied in ovariectomized ewes by comparing the effects of hypothalamo-pituitary disconnection (HPD) and sham-operation (sham-HPD). HPD caused a two-fold increase in plasma prolactin concentrations on days 1 and 7 following surgery during anoestrus and a tenfold increase during the breeding season. Thereafter, concentrations gradually declined to be similar to those in sham-HPD ewes by day 43 (breeding season) and day 145 (anoestrus). The maximum plasma prolactin response to HPD was similar during the two seasons (anoestrus: 128 ± 15 vs breeding season: 118 ± 13 μg/l). Sham-HPD had no effect on plasma prolactin concentrations. Prolactin pulse frequency was not affected by HPD, but increases in plasma prolactin concentrations were associated with increases in pulse amplitude. At the time of the normal anoestrus, plasma prolactin concentrations rose in both the HPD and sham-HPD ewes, raising the question of extra-hypothalamic regulation of seasonal changes in prolactin secretion. Plasma LH and FSH concentrations became undetectable in HPD ewes but were unaltered in sham-HPD ewes. We conclude that hypothalamic inhibition of pituitary prolactin secretion in the sheep can be demonstrated by HPD but that this effect is not sustained. This transience may indicate the additional requirement of hypothalamic-releasing factors in the control of prolactin release. In addition, the surgically isolated ovine pituitary of the HPD animal has an inherent pulsatile secretion of prolactin.

J. Endocr. (1986) 111, 425–431

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P J Shen, A I Smith, R G Evans, and I J Clarke

Abstract

The negative feedback regulation by ovarian steroids of luteinizing hormone secretion may be partially mediated by a hypothalamic endogenous opioid mechanism. This could be affected by ovarian steroid-regulated changes in hypothalamic opioid receptor binding mechanisms. In this report we show that in the presence of blocking concentrations of site-selective opioid analogues, [3H]diprenorphin homogeneously labelled μ, δ or κ receptor subtypes respectively. Using this receptor binding model, we characterized each opioid receptor subtype in the hypothalamic preoptic area and medio-basal hypothalamus of ovariectomized (OVX) and OVX plus progesterone- or oestradiol-17β (OE2)-treated ewes. In the preoptic area, progesterone treatment did not influence the affinity or capacity of δ or κ receptor binding sites, but significantly reduced μ receptor subtype content (20% less than control) with no statistically significant change in affinity. There was no effect of OE2 on either the affinity or capacity of each opioid receptor subtype in this area. In the mediobasal hypothalamus, progesterone treatment significantly decreased δ subtype receptor affinity (22 ± 11 nm vs control 7 ± 2 nm) and increased binding capacity (78 ± 9 fmol/mg protein vs control 37 ± 16 fmol/mg protein). OE2 treatment had a similar, though more profound effect on affinity (51 ± 17 nm) and binding capacity (139 ± 26 fmol/mg protein) at the δ receptor binding site. There were no significant changes in the affinity or capacity of μ or κ binding sites in the medio-basal hypothalamus. These results indicate that steroid hormones modulate hypothalamic opioid receptors in the OVX ewe in a receptor subtype- and region-dependent manner. The precise role of these steroid-induced changes in opioid receptor characteristics remains to be determined.

Journal of Endocrinology (1995) 145, 559–567

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G. B. Thomas, J. T. Cummins, B. Yao, K. Gordon, and I. J. Clarke

ABSTRACT

In order to determine whether pituitary prolactin release was directly related to the secretion of TRH into hypophysial portal blood, serial portal and jugular venous blood samples were collected from seven lactating and three non-lactating ewes. In another experiment, samples were collected from five ovariectomized ewes while being exposed to an audio-visual stress and then later administered with chlorpromazine. Secretion of TRH was pulsatile in all ewes and independent of prolactin secretion; TRH pulses coincided with significant increases in prolactin secretion in only 15% of cases and only 29% of prolactin pulses were associated with TRH pulses. Sixty-seven per cent of suckling bouts were associated with increases in prolactin secretion, but only 22% of these were associated with significant increases in TRH secretion. Chlorpromazine increased prolactin levels fourfold but did not affect portal concentrations of TRH. Audio-visual stress was not a reliable method of causing prolactin release in this model. Mean portal concentrations of TRH and jugular concentrations of prolactin were not significantly correlated. These results show that hypothalamic TRH and pituitary prolactin are secreted independently in the sheep, implying that increases in prolactin release caused by suckling or chlorpromazine are not the direct result of increased TRH secretion.

J. Endocr. (1988) 117, 115–122

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K. T. O'Byrne, L. Eltringham, G. Clarke, and A. J. S. Summerlee

ABSTRACT

The effect of relaxin on electrically evoked release of oxytocin from the posterior pituitary was examined by monitoring changes in intramammary pressure in the anaesthetized lactating rat.

The amount of oxytocin released by electrical stimulation of the neurohypophysis in vivo was dramatically reduced following i.v. injection of highly purified porcine relaxin (2·5–10 μg/rat). Relaxin inhibited oxytocin release in a dose-dependent manner and the onset of inhibition occurred within 6–10 min and lasted for 10–60 min. No effect on the sensitivity of the mammary gland to exogenous oxytocin was observed after relaxin treatment. During the period of inhibition, i.v. injection of the opioid antagonist naloxone chloride (1 mg/kg) completely and immediately restored electrically evoked oxytocin release. The neurohypophysis is known to contain endogenous opioid peptides, therefore the effect of relaxin on electrically stimulated release of oxytocin from the rat isolated neural lobe in vitro was examined. Relaxin (500–2000 ng/ml) failed to inhibit oxytocin release in vitro.

The results suggest that relaxin can inhibit the release of oxytocin from terminals in the neurohypophysis, but by an indirect mechanism. This action appears to be mediated through endogenous opioid peptides whose source is not clear. They are unlikely to be of neurohypophysial origin and may probably come from the adrenal medulla, since acute adrenalectomy negated the inhibitory effect of relaxin on oxytocin release.

J. Endocr. (1986) 109, 393–397

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R. G. GOSDEN, J. A. RUSSELL, J. CLARKE, I. PIPER, and A. S. McNEILLY

Treatment of rats with bromocriptine between days 5 and 8 after the post-partum mating resulted in suppression of serum prolactin levels and caused luteal regression. Although this treatment led to embryonic resorption when suckling was prevented by removing litters soon after birth, the diapausing embryos of animals nursing a litter of eight pups were unaffected by the treatment. These results suggest that the high levels of prolactin and progesterone in the circulation during lactation are not responsible for maintenance of the diapausing state.

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G. B. Thomas, J. T. Cummins, J. M. Hammond, R. J. E. Horton, and I. J. Clarke

ABSTRACT

Surgical disconnection of the ovine hypothalamus from the pituitary gland (hypothalamo-pituitary disconnection; HPD) has provided a useful experimental model for studying the control of gonadotrophin secretion. The objective of the present study was to define the characteristics of prolactin secretion using stimuli acting through the hypothalamus or directly on the pituitary gland in HPD ewes. Prolactin responses to either a stressful stimulus or the dopaminergic antagonists metoclopramide (20 mg i.v.) or chlorpromazine (50 mg i.v.) seen in intact animals (sham-HPD) were completely abolished by HPD. Injection of TRH (100 μg i.v.) caused an immediate release of prolactin in both groups of ewes. In the HPD ewes plasma prolactin concentrations remained raised for at least 3 h after TRH injection, whereas in sham-HPD ewes prolactin concentrations began to decline after 20 min. Administration of bromocriptine (1 mg i.v.) 10 min after TRH inhibited the prolonged response to TRH in HPD ewes. The results support the hypothesis that prolactin exerts a short-loop feedback effect on its own secretion at the hypothalamic level.

J. Endocr. (1986) 111, 433–438