Sandra Dye

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.

Acute Action of Oestrogen on Medial Preoptic Gamma-Aminobutyric Acid Neurons: Correlation with Oestrogen Negative Feedback on Luteinizing Hormone Secretion

The acute effects of oestrogen on the medial preoptic area (MPOA) γ‐aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17β‐oestradiol (5 μg/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen‐min blood samples were taken to determine the time‐course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17β‐oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle‐treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17β‐ oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17β‐oestradiol compared with controls (P<0.05). To determine the effects of the 17β‐oestradiol treatment on pituitary responsiveness to LH‐releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17β‐oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17β‐oestradiol‐treated rats was approximately 50% that of control‐treated values. This study demonstrates the acute and potent action of 17β‐oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.

Involvement of cholecystokinin receptor types in pathways controlling oxytocin secretion

1 Intravenous administration of cholecystokinin (CCK) results in a transient activation of oxytocin neurones in the rat, and hence to oxytocin secretion: this activation is followed by expression of c‐fos mRNA and of Fos‐like immunoreactivity (Fos‐LI) in magnocellular oxytocin neurones. Fos‐like immunoreactivity is also induced in the regions of the brainstem that are thought to relay information from the periphery to the hypothalamus. 2 Administration of the selective CCKA receptor antagonist MK‐329, but not the CCKB receptor antagonist L‐365,260, prior to CCK injection, prevented oxytocin release as measured by radioimmunoassay and oxytocin neuronal activation as measured by electrophysiology and by the lack of induction of c‐fos mRNA. 3 MK‐329 abolished the release of adrenocorticotrophic hormone (ACTH) following injection of CCK. 4 MK‐329 prevented the expression of Fos‐LI in the hypothalamic magnocellular nuclei and in the area postrema and dorsal vagal complex of the brainstem. 5 L‐365,260 had no effect on the expression of Fos‐LI in the brainstem, but attenuated that seen in the hypothalamic magnocellular nuclei. 6 We conclude that CCK acts on CCKA receptors, either in the area postrema or on peripheral endings of the vagus nerve, to cause the release of hypothalamic oxytocin and ACTH. Information may be carried to the hypothalamus in part by CCK acting at CCKB receptors.

Induction of members of the Fos/Jun family of immediate-early genes in identified hypothalamic neurons: in vivo evidence for differential regulation

In situ hybridization was used to measure the expression of members of the Fos/Jun family of immediate-early genes in hypothalamic neurons in vivo following defined stimuli that utilize different afferent pathways. Only c-jun messenger RNA was expressed in the hypothalamic supraoptic and paraventricular nuclei of control animals. Intravenous infusions of sodium chloride solutions of different tonicity produced a range of plasma osmolalities within physiological limits. While the induction of c-fos and jun B messenger RNAs followed the stimulus intensity, the expression of c-jun was repressed at low levels of stimulation. A higher level of osmotic stimulation was able to co-induce c-jun with the c-fos, jun B and fos B genes, suggesting that other signalling pathways may then be activated. Parturition or systemic administration of cholecystokinin, that activate supraoptic and paraventricular neurons via ascending afferent pathways from the brainstem, both induced c-fos, but not the other genes, in the magnocellular nuclei. Use of double in situ hybridization confirmed that, unlike with osmotic stimulation, induction of c-fos only occurred in oxytocin neurons. These two stimuli did not cause a concomitant repression of c-jun messenger RNA expression in magnocellular oxytocin neurons. These patterns of induction provide evidence for the differential regulation of members of this family of genes in a physiological context.

Kappa-Opioid Restraint of Oxytocin Secretion: Plasticity through Pregnancy

Oxytocin neurone terminals display a powerful opioid-mediated auto-inhibitory mechanism which restrains secretory activity. In pregnancy, upregulation of this mechanism may underlie the accumulation of large stores of oxytocin in the pituitary. In the present study, conscious pregnant rats were cannulated to allow blood samples to be taken for measurement of oxytocin secretion in response to intravenous administration of cholecystokinin (CCK). In each rat, we measured the secretory response to CCK before and after administration of norbinaltorphine (norBNI), a selective kappa-opioid antagonist, or of naloxone, a relatively mu-selective antagonist. Throughout pregnancy, the stimulatory effect of CCK was enhanced by prior administration of norBNI. In pregnant rats norBNI also consistently increased basal oxytocin secretion, while naloxone had no effect, suggesting that in pregnancy there is active restraint of oxytocin secretion by endogenous opioids acting at kappa-receptors. However, in late pregnancy, between days 17 and 20, there was a significant reduction in the efficacy of norBNI in potentiating the oxytocin release in response to CCK, compared to its efficacy either in non-pregnant rats or in rats between days 8 and 13 of pregnancy. This suggests that in late pregnancy, endogenous kappa-opioid restraint is downregulated. In addition, the present results demonstrate an attenuation in the potency with which CCK evoked oxytocin release in late pregnancy. The attenuation was unrelated to opioid restraint at the level of the pituitary since it coincided with apparent desensitization of this auto-inhibitory mechanism.

Perinatal and adult factors responsible for the sexually dimorphic calcitonin gene-related peptide-containing cell population in the rat preoptic area.

Neurons containing calcitonin gene-related peptide in the medial preoptic nucleus exhibit the largest neurochemically defined sex difference in the rat preoptic area with a 20-fold difference in cell numbers. The gonadal steroid hormones responsible for this sexual dimorphism have been investigated by examining calcitonin gene-related peptide immunoreactivity in the preoptic area of adult rats receiving a variety of perinatal and adult gonadal steroid manipulations. Cells immunoreactive for calcitonin gene-related peptide were examined in two populations within the preoptic area, one in its ventrolateral aspect and the other located in the lateral division of the medial preoptic nucleus. Cell profile counts estimate numbers of calcitonin gene-related peptide-containing cells in the medial preoptic nucleus of the female to be 22.2 +/- 3.0 cells/section compared with 1.0 +/- 0.2 in the male (P < 0.01). No sex differences existed in the preoptic ventrolateral population of calcitonin gene-related peptide cells (males 4.3 +/- 0.2, females 4.4 +/- 0.6 cells/section). Gonadectomy of male rats on postnatal day 2 resulted in the appearance of a calcitonin gene-related peptide-containing cell population in the medial preoptic nucleus which was indistinguishable from intact female rats (19.3 +/- 2.2 cells/section). Gonadectomy of adult male rats resulted in a modest increase in calcitonin gene-related peptide cell numbers within the medial preoptic nucleus (8.8 +/- 0.4 cells/section) and this was fully reversed by replacement of testosterone (0.7 +/- 0.2 cells/section).(ABSTRACT TRUNCATED AT 250 WORDS)

INCREASED FOS EXPRESSION IN PREOPTIC CALCITONIN GENE-RELATED PEPTIDE (CGRP) NEURONES FOLLOWING MATING BUT NOT THE LUTEINIZING HORMONE SURGE IN FEMALE RATS

The functional relationship between sexually dimorphic neural populations and sex differences in reproductive functioning is unclear. The present study has investigated the function of the sexually dimorphic, estrogen‐receptive, calcitonin gene‐related peptide (CGRP) neurones in the female preoptic area by examining patterns of Fos immunoreactivity within these cells in relation to the luteinizing hormone surge and lordosis behaviour. In the first experiment, ovariectomized rats were treated with estradiol alone or estradiol plus progesterone to induce the luteinizing hormone surge. The percentage of CGRP neurones with Fos‐positive nuclei was not different in estradiol alone (18 ± 4%) and estradiol/progesterone‐treated (24 ± 3%) rats although the number of Fos‐immunoreactive cells in the medial preoptic nucleus was increased 2‐fold (P<0.01) in estrogen/progesterone‐treated rats and 40 ± 5% of luteinizing hormone‐releasing hormone neurones were found to express Fos in this group. In the second experiment, ovariectomized rats were treated with estradiol and progesterone and either, mated with a single male or placed in an empty cage, for 30 min. The number of Fos‐immunoreactive cells in the medial preoptic nucleus was increased 4‐fold in mated rats (P<0.01) and the percentage of CGRP neurones with Fos‐positive nuclei increased from 24 ± 3% to 38 ± 2% (P<0.01) in mated animals. No differences were detected in the number of luteinizing hormone‐releasing hormone neurones with Fos‐positive nuclei in mated and non‐mated animals.