Brattleboro rats, homozygous for diabetes insipidus, and Long–Evans rats were anaesthetized with urethane, and antidromically identified neurones were recorded from the supraoptic nucleus. Phasically firing neurones were studied during repeated electrical stimulation of the neural stalk, whereby most supraoptic neurones, but not the recorded neurone, were activated antidromically. Such stimulation consistently modified the discharge pattern of phasic neurones in Long–Evans rats, but was relatively ineffective in Brattleboro rats. These results suggest that the effects of neural stalk stimulation on discharge patterns in Long–Evans rats may be substantially mediated by the evoked release of vasopressin or neurophysin.
JA Russell and G Leng
S. A. Way and G. Leng
In urethane-anaesthetized ovariectomized rats, injection of porcine relaxin (7·5 and 15 μg/kg, i.v.) caused a sustained increase in circulating plasma oxytocin and vasopressin concentrations; 10 μg relaxin/rat i.v. produced a smaller but significant increase in plasma oxytocin concentration in conscious ovariectomized rats. A significant increase in oxytocin concentration and inhibition of the spontaneous milk-ejection reflex was also seen in anaesthetized (ovary intact) lactating rats following injection of relaxin (7·5 μg/kg, i.v.). To investigate whether relaxin acts by increasing the electrical activity of oxytocin neurones or by facilitating stimulus-secretion coupling in the pituitary, the electrical activity of neurones in the supraoptic nucleus was recorded in urethane-anaesthetized lactating rats and in ovariectomized rats. Porcine relaxin (10 μg/rat, i.v.) increased the firing rate of both oxytocin and vasopressin neurones in the supraoptic nucleus in lactating rats. The response to relaxin was unaffected by subsequent injection of naloxone (1 mg/kg, i.v.). Oxytocin neurones were also activated by injection of relaxin (10 μg/rat) into ovariectomized rats. Combining the electrophysiological data, the neuronal activation following relaxin was significantly correlated with the level of spontaneous activity prior to relaxin injection. The results show that relaxin acts centrally to increase circulating plasma oxytocin and vasopressin concentrations by an opioid-independent mechanism.
Journal of Endocrinology (1992) 132, 149–158
R. E. J. Dyball and G. Leng
Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI rats) were anaesthetized with urethane. Extracellular recordings were made from antidromically identified neurones of the supraoptic nucleus. About half (77 out of 153) of the neurones recorded in DI rats showed phasic patterns of discharge activity similar to those which are characteristic of vasopressin-secreting neurones in normal rats during hyperosmotic stimulation. Significantly fewer neurones showed phasic activity in DI rats which had been pretreated with vasopressin tannate at a dose which significantly reduced urine volume, water intake and plasma osmolality. Acute systemic hyperosmotic stimulation, induced by an i.p. injection of 1 ml 1·5 m-NaCl, increased the discharge rate of each of 14 neurones from DI rats by 1–5 spikes/s. Hypo-osmotic stimulation, induced by an intragastric injection of 10 ml tap water, reduced the discharge rate of each of four neurones from DI rats by 50% or more.
We conclude that supraoptic neurones in DI rats respond normally to acute systemic osmotic stimuli despite the total absence of vasopressin in these rats and despite their chronically disturbed water balance. This implies that the osmoreceptor mechanism which drives the supraoptic nucleus does not adapt substantially during prolonged disturbance of water balance and functions outside the normal physiological range of plasma osmolality, and that the reported alteration of noradrenergic innervation of the neurones in DI rats does not affect their osmotic responsiveness.
J. Endocr. (1985) 105, 87–90
S L Dickson, O Doutrelant-Viltart and G Leng
In the rat, the synthetic GH secretagogue GH-releasing peptide (GHRP-6) acts centrally to activate a subpopulation of arcuate neurones as reflected by increased electrical activation and by the detection of Fos protein in cell nuclei. Since GHRP-6 also induces GH secretion via a direct action on the pituitary, we set out to determine whether the central actions of GHRP-6 are mediated by GH itself. First, we demonstrated that peripherally administered GHRP-6 induces Fos expression in the arcuate nucleus of GH-deficient animals (dw/dw rats and lit/lit mice). Secondly, in dw/dw rats, neither intracerebro-ventricular injection of 15 μg recombinant bovine GH nor 1 μg recombinant human IGF-I resulted in an increase in the number of cells expressing Fos protein in the arcuate nucleus (or in any other hypothalamic structure studied). These results support our hypothesis that GHRP-6 has a central site and mechanism of action and provide evidence to suggest that the activation of arcuate neurones by GHRP-6 is not mediated by a central action of GH or IGF-I. Furthermore, since the lit/lit mouse pituitary does not release GH following GHRP-6 administration, our finding that the central actions of GHRP-6 remain intact in these animals suggests the possible existence of two subpopulations of putative GHRP-6 receptors.
Journal of Endocrinology (1995) 146, 519–526
J. A. Russell, R. E. Blackburn and G. Leng
The region anterior and ventral to the third ventricle (AV3V) region is a major source of excitatory afferents to the magnocellular neuroendocrine system, and is essential for the osmotically regulated release of oxytocin. We investigated whether this input has a similarly essential role in parturition. Rats were implanted with a guide cannula in the AV3V region on days 9–18 of pregnancy. Following the birth of the third pup, rats were anaesthetized briefly with ether and either given an electrolytic AV3V lesion or a sham procedure was carried out. In eight AV3V-lesioned rats the mean (± s.e.m.) median interbirth interval following the lesion was 6·3 ± 1·2 min compared with 5·2 ± 0·6 min in 11 sham-lesioned rats. All rats completed delivery of their litters. The mean plasma concentration of oxytocin was unchanged following the sham procedure (pre-sham 17·1±2·8 pmol/l, n = 8; 15 min post-sham 18·1±2·7 pmol/l, n = 8; 30 min post-sham 19·2 ± 3·5 pmol/l, n = 8). In AV3V-lesioned rats, plasma oxytocin was significantly raised following the lesion (pre-lesion 14·6±1·3 pmol/l, n = 7; post-lesion 58·3 ± 9·8 pmol/l, n = 7) and was still higher than the sham-treated group after 30 min (55·8 ± 9·9 pmol/l). Thus there was no significant difference in the time-course of parturition between AV3V-lesioned rats and sham-lesioned rats, and no evidence that the lesion impaired the release of oxytocin. Furthermore, in rats given an AV3V lesion on the morning of the expected day of delivery, parturition was neither delayed nor disrupted, suggesting that the AV3V region does not contribute to the mechanisms controlling the onset of parturition.
Journal of Endocrinology (1989) 121, 109–115
R. E. Blackburn, G. Leng and J. A. Russell
The involvement of the region anterior and ventral to the third ventricle (AV3V region) in the control of oxytocin release was investigated using electrical stimulation and electrolytic lesioning techniques in the rat. Electrical stimulation (0·5 mA, 50 Hz, 15–25 s) of the AV3V region of lactating rats evoked a reproducible rise in intramammary pressure equivalent to that induced by 0·25–0·5 mu. oxytocin (i.v.). Increases in circulating concentrations of oxytocin, as determined by specific radioimmunoassay, confirmed that AV3V stimulation released oxytocin in both lactating and non-lactating rats. The increases in plasma oxytocin concentration evoked by electrical stimulation of the AV3V region were dependent upon intensity and frequency of stimulation, and electrode position. A significant (P<0·05) increase followed stimulation at 25 Hz and 0·1 mA, and a maximal response was obtained with 50 Hz and 1·0 mA. Stimulation of the area in and around the nucleus medianus produced the greatest rise in oxytocin secretion.
The milk-ejection reflex was not abolished after acute electrolytic ablation of the AV3V region in urethane-anaesthetized lactating rats, but electrolytic lesion of the AV3V region prevented the increase in plasma oxytocin concentration which normally followed an osmotic stimulus (1 ml 1·5 mol NaCl/l, i.p.),
These studies provide evidence that the AV3V region is a major source of excitatory afferents to oxytocin neurones; this input is essential for the osmoresponsiveness of these neurones but plays little role in the control of such neurones during reflex milk ejection.
J. Endocr. (1987) 114, 253–261
S L Dickson, O Doutrelant-Viltart, R E J Dyball and G Leng
Previously, we demonstrated that the synthetic hexapeptide GH-releasing peptide (GHRP-6) activates a subpopulation of arcuate neurones, as reflected by increased electrical activation and by the detection of Fos protein in cell nuclei. Here we set out to determine (1) what proportion of the cells activated by GHRP-6 are neurosecretory neurones and (2) whether the cells activated by GHRP-6 contain tyrosine hydroxylase (TH; a marker of dopaminergic cells in this region) or β-endorphin. In the first study, adult male rats were injected i.v. with the retrograde tracer, Fluorogold, to detect cells which project outside the blood–brain barrier (and are therefore likely to be neurosecretory neurones). Three days later the conscious rats were injected i.v. with 50 μg GHRP-6 and the brains processed for the immunocytochemical detection of Fos protein. Between 68% and 82% of the arcuate neurones expressing Fos protein following GHRP-6 injection were retrogradely labelled with Fluorogold. In the second study, conscious male rats, bearing a chronically implanted jugular catheter, were killed 90 min following an i.v. injection of 50 μg GHRP-6 and the brains were processed for the double immunocytochemical detection of Fos protein and either TH or β-endorphin. Less than 7% (mean ± s.e.m.= 6·7 ± 2·6% nuclei/section per rat) of the arcuate neurones expressing Fos protein following GHRP-6 injection were TH-containing cells. Of 143β-endorphin-containing arcuate cells detected only four cells were identified as containing Fos protein. Thus, the majority of arcuate neurones activated by GHRP-6 (1) project outside the blood–brain barrier (and are therefore likely to be neurosecretory neurones) and (2) were not identified as TH- or β-endorphin-containing cells.
Journal of Endocrinology (1996) 151, 323–331
I A Antonijevic, A J Douglas, S Dye, R J Bicknell, G Leng and J A Russell
The physiological importance of oxytocin for the initiation and maintenance of labour and delivery is controversial. We investigated the effects of two novel peptide oxytocin antagonists on the onset and the progress of delivery in rats implanted with a jugular vein cannula one day before term. During delivery rats were given either an oxytocin antagonist (OVT16, n=10, or F382, n=7, 30 μg/kg) or vehicle (n=10, 9) after the birth of the second pup and the time to deliver five more pups was recorded. Other rats were given an injection of F382 (30 μg/kg, n=7) or vehicle (n=9) after the birth of the fourth pup and the time to deliver three more pups was recorded. In another experiment rats were given repeated injections of F382 (30 or 60 μg/kg, n=13, 11) or vehicle (n=32) prepartum on the day of expected term and the time of onset and the progress of delivery was recorded.
Rats given an antagonist after the second pup delivered the next five pups in 100 ± 8 min (F382) and 83 ± 12 min (OVT16), significantly slower than the respective controls (51 ± 6 and 49 ± 6 min, U-test, P<0·05). Four of the 7 rats given F382 after the fourth pup showed no prolongation of delivery (time between pups 4–7: 24·7±2·9 vs 27·5±3·1 min in controls), while in the other three rats delivery was prolonged (time between pups 4–7: 86 ± 4·3 min). The onset of delivery was significantly delayed in rats given repeated injections of F382 at 60 μg/kg on the afternoon of expected delivery but before labour had begun (median shift 14·5 h vs controls, U-test, P<0·05). These results suggest that in rats oxytocin is involved in both the initiation and the maintenance of parturition and that it is more important during the early rather than the later stages of delivery.
Journal of Endocrinology (1995) 145, 97–103
S. N. Thornton, G. Leng, R. J. Bicknell, C. Chapman and T. Purdew
Plasma samples obtained at 4-h intervals from goats for at least 24 h before and then during 24 h of deprivation of water were analysed by radioimmunoassay for vasopressin and oxytocin concentrations. The samples were also analysed for osmolality and sodium concentration. The differential effect of night/day versus day/night deprivation was also studied. During the two periods before the two deprivations osmolality varied in a regular manner, with low values occurring at 08.00 h. Sodium concentration followed osmolality, whereas vasopressin did not vary during the period before deprivation. During deprivation vasopressin increased along with osmolality and sodium concentration, with the beginning of the increase occurring after the morning feed. Oxytocin levels did not increase during the period of deprivation.
These results do not support the hypothesis of general release of neurohypophysial hormones in response to osmotic stimuli but instead indicate there are species variations with respect to hormonal response to water deprivation.
J. Endocr. (1986) 110, 335–340