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Oxytocin is secreted by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons to induce uterine contractions during parturition. Increased activation of oxytocin neurons at parturition involves a network of afferent inputs that increase oxytocin neuron excitability. Kisspeptin fibre density increases around oxytocin neurons during pregnancy, and central kisspeptin administration excites oxytocin neurons only in late pregnancy. Kisspeptin signals via extracellular regulated kinase 1/2 (ERK1/2) and p38. Therefore, to determine whether kisspeptin excites oxytocin neurons via ERK1/2-p38 signalling in late-pregnant rats, we performed immunohistochemistry for phosphorylated ERK1/2 (pERK1/2) and phosphorylated p38 (p-p38) in oxytocin neurons of non-pregnant and late-pregnant rats. Intracerebroventricular (ICV) kisspeptin administration (2 µg) did not affect pERK1/2 or p-p38 expression in SON and PVN oxytocin neurons of non-pregnant or late-pregnant rats. Furthermore, ICV kisspeptin did not affect pERK1/2 or p-p38 expression in brain areas with major projections to the SON and PVN: the nucleus tractus solitarius, rostral ventrolateral medulla, locus coeruleus, dorsal raphe nucleus, organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, anteroventral periventricular nucleus, periventricular nucleus and arcuate nucleus. Hence, kisspeptin-induced excitation of oxytocin neurons in late pregnancy does not appear to involve ERK1/2 or p38 activation in oxytocin neurons or their afferent inputs.

Increased cardiac sympathetic nerve activity in type 2 diabetes mellitus (DM) suggests impaired autonomic control of the heart. However, the central regions that contribute to the autonomic cardiac pathologies in type 2 DM are unknown. Therefore, we tested the hypothesis that neuronal activation would be increased in central sympathoregulatory areas in a pre-clinical type 2 DM animal model. Immunohistochemistry in 20-week-old male Zucker diabetic fatty (ZDF) rats revealed an increased number of neurones expressing ΔFosB (a marker of chronic neuronal activation) in the intermediolateral column (IML) of the spinal cord in DM compared to non-diabetic (non-DM) rats (P < 0.05). Rostral ventrolateral medulla (RVLM) neurones activate IML neurones and receive inputs from the hypothalamic paraventricular nucleus (PVN), as well as the nucleus tractus solitarius (NTS) and area postrema (AP), in the brainstem. We observed more ΔFosB-positive noradrenergic RVLM neurones (P < 0.001) and corticotrophin-releasing hormone PVN neurones (P < 0.05) in DM compared to non-DM rats. More ΔFosB-positive neurones were also observed in the NTS (P < 0.05) and AP (P < 0.01) of DM rats compared to non-DM rats. Finally, because DM ZDF rats are obese, we also expected increased activation of pro-opiomelanocortin (POMC) arcuate nucleus (ARC) neurones in DM rats; however, fewer ΔFosB-positive POMC ARC neurones were observed in DM compared to non-DM rats (P < 0.01). In conclusion, increased neuronal activation in the IML of type 2 DM ZDF rats might be driven by RVLM neurones that are possibly activated by PVN, NTS and AP inputs. Elucidating the contribution of central sympathoexcitatory drive in type 2 DM might improve the effectiveness of pharmacotherapies for diabetic heart disease.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nucleus. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that enhanced activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single-point blood measurements of basal prolactin, thyroid-stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt, Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up-regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS.

Key points During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin-secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation. Abstract Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane-anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s−1, respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s−1 in lactating rats but excited none in virgin or pregnant rats (χ22 = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 μg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s−1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect phosphorylation of extracellular regulated kinase 1 or 2, or of Akt in the supraoptic or paraventricular nuclei of virgin or lactating rats. Hence, prolactin inhibition of oxytocin neurones is lost in lactation, which might allow concurrent elevation of prolactin secretion from the pituitary gland and activation of oxytocin neurones for synthesis and delivery of milk to the newborn.

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague-Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual-label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin- or oxytocin-labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post-MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.

Pregnancy in rodents is associated with hyperphagia, increased fat deposition, elevated leptin concentrations and insensitivity to the satiety action of leptin. To investigate the hormonal mechanisms involved in the development of this state of pregnancy-induced leptin resistance, we have used a pseudopregnancy rat model. We have previously demonstrated that pseudopregnant rats have a normal feeding response to leptin, although, if pseudopregnancy is extended using chronic i.c.v. ovine prolactin infusion along with progesterone implants, then leptin no longer suppresses food intake. The present study aimed to investigate the effect of chronically high lactogen levels, as seen in mid-pregnancy, on leptin-induced activation of hypothalamic Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction and mRNA expression of leptin (LepR-B) and prolactin (Prlr-L) receptors, using pseudopregnant rats chronically infused with ovine prolactin. Groups of virgin (dioestrous) and pseudopregnant rats were treated with chronic i.c.v. infusion of either prolactin (2.5 μg μL-1 h-1 for 5 days) or vehicle (artificial cerebrospinal fluid [aCSF]) via a minipump connected to a cannula surgically implanted into the lateral ventricle. Rats were fasted overnight and then received an i.c.v. injection of leptin (400 ng) or vehicle (aCSF) and were perfused 30 minutes later. In chronic vehicle-infused pseudopregnant rats, i.c.v. leptin increased the number of phosphorylated STAT3 positive cells in the arcuate nucleus and ventromedial nucleus (VMH) of the hypothalamus, similar to all acute-leptin treated virgin groups. This effect of leptin, however, was not observed in the pseudopregnant rats that were chronically infused with prolactin. A quantitative polymerase chain reaction analysis also showed decreased expression of LepR-B in the arcuate and VMH nuclei, as well as decreased Prlr-L in the arcuate nucleus of prolactin-infused "extended pseudopregnancy" rats. These data suggest that the attenuation of the leptin-induced suppression of food intake caused by chronically high lactogen levels in pseudopregnant rats is associated with impaired leptin-induced activation of the JAK/STAT pathway in specific hypothalamic nuclei.

Key points Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized. We found that that kisspeptin fibre density around oxytocin neurones increases in late-pregnant rats. These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy. Oxytocin neurones were excited by central kisspeptin administration in late-pregnant rats but not in non-pregnant rats or early- to mid-pregnant rats. Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth. Abstract The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day 21 of gestation) had a higher density of kisspeptin-positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non-pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT-PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone–supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late-pregnant rats (days 18–21 of gestation) but not in non-pregnant rats, or in early- or mid-pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition.

Key pointsIncreased appetite and weight gain occurs during pregnancy, associated with development of leptin resistance, and satiety responses to the anorectic peptide α-melanocyte stimulating hormone (α-MSH) are suppressed.This study investigated hypothalamic responses to α-MSH during pregnancy, usi

Oxytocin is required for normal birth and lactation. Oxytocin is synthesised by hypothalamic supraoptic and paraventricular nuclei neurons and is released into the circulation from the posterior pituitary gland. Under basal conditions, circulating oxytocin levels are relatively constant but during birth and lactation, pulsatile oxytocin release triggers rhythmic contraction of the uterus during birth and milk ejection during suckling. Oxytocin levels are principally determined by the pattern of action potential firing that is, in turn, determined by the interplay between the intrinsic properties of the oxytocin neurons, regulation of their excitability by surrounding glia as well as by synaptic drive from their afferent inputs. During birth and suckling, oxytocin neurons fire high-frequency bursts of action potentials that are coordinated across the population of neurons and these bursts underpin the pulsatile secretion of oxytocin required for normal birth and lactation. Neuroglial regulation of oxytocin neurons changes during pregnancy to favour burst firing. However, these changes still require afferent input activity to drive activity. While it has long been known that noradrenergic inputs to oxytocin neurons are activated during birth and lactation, the involvement of other afferent inputs is less clear. Here, we provide a brief overview of the current understanding of the mechanisms that regulate oxytocin neuron activity during pregnancy and lactation, and focus on recent evidence from our laboratory identifying an input that increases kisspeptin production to excite oxytocin neurons in late pregnancy. This article is protected by copyright. All rights reserved.

Myocardial infarction (MI) initiates an increase in cardiac sympathetic nerve activity (SNA) that facilitates potentially fatal arrhythmias. The mechanism(s) underpinning sympathetic activation remain unclear. Some neuronal populations within the hypothalamic paraventricular nucleus (PVN) have been implicated in SNA. This study elucidated the role of the PVN in triggering cardiac SNA following MI (left anterior descending coronary artery ligation). By means of c-Fos, oxytocin, and vasopressin immunohistochemistry accompanied by retrograde tracing we showed that MI activates parvocellular oxytocin neurons projecting to the rostral ventral lateral medulla. Central inhibition of oxytocin receptors using atosiban (4.5 µg in 5 µl, i.c.v.), or retosiban (3 mg/kg, i.v.), prevented the MI-induced increase in SNA and reduced the incidence of ventricular arrhythmias and mortality. In conclusion, pre-autonomic oxytocin neurons can drive the increase in cardiac SNA following MI and peripheral administration of an oxytocin receptor blocker could be a plausible therapeutic strategy to improve outcomes for MI patients., Roy et al. showed that activation of parvocellular pre-autonomic oxytocin neurons increased sympathetic nerve activity following myocardial infarction. This and other aberrant physiological changes induced by acute myocardial infarction were decreased by oxytocin receptor antagonists, hinting to their potential therapeutic role.

During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin-secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation.Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane-anaesthetised virgin, pregnant and lactating rats. Prolactin (2 μg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s

Oxytocin secretion is required for successful reproduction. Oxytocin is synthesised by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei and the physiological demand for oxytocin synthesis and secretion is increased for birth and lactation. Therefore, we used a polymerase chain reaction (PCR) array screen to determine whether genes that might be important for synthesis and/or secretion of oxytocin are up- or down-regulated in the supraoptic and paraventricular nuclei of late-pregnant and lactating rats, compared to virgin rats. We then validated the genes that were most highly regulated using real time-quantitative PCR. Among the most highly regulated genes were those that encode for suppressors of cytokine signalling, which are intracellular inhibitors of prolactin signalling. Prolactin receptor activation changes gene expression via phosphorylation of signal transducer and activator of transcription 5 (STAT5). Using double-label immunohistochemistry, we found that phosphorylated STAT5 was expressed in almost all oxytocin neurones of late-pregnant and lactating rats but was almost absent from oxytocin neurones of virgin rats. We conclude that increased prolactin activation of oxytocin neurones might contribute to the changes in gene expression by oxytocin neurones required for normal birth and lactation.

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

Pregnancy is associated with hyperphagia, increased fat mass, hyperleptinaemia and hyperprolactinaemia. The neuroendocrine control of bodyweight involves appetite-regulating centres in the hypothalamus, containing both orexigenic and anorexigenic neurons that express leptin receptors (LepR). In the rat, central leptin resistance develops during mid pregnancy, well after hyperphagia becomes apparent, to negate the appetite suppressing effects of leptin. We have investigated the hypothalamic response to leptin during pregnancy and examined the role of pregnancy hormones in inducing these changes. We have shown that there are multiple levels of leptin resistance during pregnancy. Despite elevated serum leptin, neuropeptide Y and agouti related peptide mRNA in the arcuate nucleus are not suppressed and may even be increased during pregnancy. LepR mRNA and leptin-induced pSTAT3 expression, however, are relatively normal in the arcuate nucleus. In contrast, both LepR and leptin-induced pSTAT3 are reduced in the ventromedial hypothalamic nucleus. Injecting α-melanocyte-stimulating hormone (α-MSH) into the brain, to bypass the first-order leptin-responsive neurons in the arcuate nucleus, also fails to suppress food intake during pregnancy, suggesting that pregnancy is also a melanocortin-resistant state. Using a pseudopregnant rat model, we have demonstrated that in addition to the changes in maternal ovarian steroid secretion, placental lactogen production is essential for the induction of leptin resistance in pregnancy. Thus, hormonal changes associated with pregnancy induce adaptive changes in the maternal hypothalamus, stimulating food intake and then allowing elevated food intake to be maintained in the face of elevated leptin levels, resulting in fat deposition to provide energy stores in preparation for the high metabolic demands of late pregnancy and lactation.

Pregnancy in rats is associated with hyperphagia, increased fat deposition, and elevated plasma leptin concentrations. Elevated leptin would be expected to inhibit food intake, but hypothalamic leptin resistance develops around midpregnancy, allowing hyperphagia to be maintained and excess energy to be stored as fat in preparation for future metabolic demands of lactation. To investigate the hormonal mechanisms inducing leptin resistance during pregnancy, the anorectic response to leptin was examined during pseudopregnancy. Pseudopregnant rats have identical hormonal profiles to early pregnancy, but no placenta formation, allowing differentiation of maternal and placental hormone effects on appetite. To investigate the effect of leptin on food intake, d-9 pseudopregnant rats were injected with leptin (4 μg) via an intracerebroventricular (icv) cannula, and then food intake was measured 24 h later. Pseudopregnant rats were hyperphagic but had normal anorectic responses to leptin. We therefore hypothesized that a longer exposure time to high concentrations of progesterone might be required to mimic the leptin resistance that occurs on d 14 of pregnancy. Pseudopregnant rats were given progesterone to prolong pseudopregnancy beyond the time that leptin resistance develops during pregnancy. However, rats remained responsive to icv leptin. To model the placental lactogen secretion that occurs during pregnancy, pseudopregnant rats were given progesterone and chronic icv ovine prolactin infusion. Central icv injection of leptin had no effect on food intake in pseudopregnant rats receiving chronic ovine prolactin. These results suggest that chronically high lactogen levels, secreted by the placenta during the second half of pregnancy, induce central leptin resistance.

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9–16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

The hormone leptin modulates a diverse range of biological functions, including energy homeostasis and reproduction. Leptin promotes GnRH function via an indirect action on forebrain neurons. We tested whether leptin deficiency or leptin resistance due to a high-fat diet (HFD) can regulate the potent reproductive neuropeptide kisspeptin. In mice with normalized levels of estradiol, leptin deficiency markedly reduced kisspeptin gene expression, particularly in the arcuate nucleus (ARC), and kisspeptin immunoreactive cell numbers in the rostral periventricular region of the third ventricle (RP3V). The HFD model was used to determine the effects of diet-induced obesity and central leptin resistance on kisspeptin cell number and gene expression. DBA/2J mice, which are prone to HFD-induced infertility, showed a marked decrease in kisspeptin expression in both the RP3V and ARC and cell numbers in the RP3V after HFD. This is the first evidence that kisspeptin can be regulated by HFD and/or increased body weight. Next we demonstrated that leptin does not signal (via signal transducer and activator of transcription 3 or 5, or mammalian target of rapamycin) directly on kisspeptin-expressing neurons in the RP3V. Lastly, in leptin receptor-deficient mice, neither GnRH nor kisspeptin neurons were activated during a preovulatory-like GnRH/LH surge induction regime, indicating that leptin's actions on GnRH may be upstream of kisspeptin neurons. These data provide evidence that leptin's effects on reproductive function are regulated by kisspeptin neurons in both the ARC and RP3V, although in the latter site the effects are likely to be indirect.

Obesity is associated with resistance to the actions of both leptin and insulin via mechanisms that remain incompletely understood. To investigate whether leptin resistance per se contributes to insulin resistance and impaired glucose homeostasis, we investigated the effect of acute leptin administration on glucose homeostasis in normal as well as leptin- or leptin receptor-deficient mice. In hyperglycemic, leptin-deficient Lepob/obmice, leptin acutely and potently improved glucose metabolism, before any change of body fat mass, via a mechanism involving the p110α and β isoforms of phosphatidylinositol-3-kinase (PI3K). Unlike insulin, however, the anti-diabetic effect of leptin occurred independently of phospho-AKT, a major downstream target of PI3K, and instead involved enhanced sensitivity of the hypothalamus to insulin action upstream of PI3K, through modulation of IRS1 (insulin receptor substrate 1) phosphorylation. These data suggest that leptin resistance, as occurs in obesity, reduces the hypothalamic response to insulin and thereby impairs peripheral glucose homeostasis, contributing to the development of type 2 diabetes.

Appetite and food intake are increased during pregnancy, comprising an adaptive response that facilitates energy storage in preparation for the high metabolic demands of pregnancy and subsequent lactation. To maintain the increased energy intake in the face of increased adiposity and rising leptin levels, pregnant females become resistant to the central anorectic actions of leptin. In rats, pregnancy-induced leptin resistance is characterised by elevated neuropeptide Y and reduced pro-opiomelanocortin expression in the arcuate nucleus, reduced leptin receptor mRNA levels and suppression of leptin-induced phosphorylated signal transducer and activator of transcription-3 protein in the ventromedial hypothalamic nucleus, as well as a loss of anorectic responses to both leptin and alpha-melantocyte-stimulating hormone. Our recent data suggest that this leptin-resistance may also cause central insulin resistance and an altered peripheral glucose homeostasis. The specific hormone changes during pregnancy that might mediate these effects on leptin signalling are a current focus of investigation. In pseudopregnant rats, chronic i.c.v. infusion of ovine prolactin to mimic patterns of placental lactogen secretion that occur during pregnancy completely blocked the ability of leptin to suppress food intake. These data suggest that placental lactogen secretion may mediate the hormone-induced loss of response to leptin during pregnancy. This action of prolactin/placental lactogen appears to be mediated downstream of the primary leptin-responsive neurones in the mediobasal hypothalamus, possibly in the paraventricular nucleus. Our studies show complex hormone-induced adaptations in the normal hypothalamic pathways regulating body weight homeostasis during pregnancy.

Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro-opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti-related gene protein (AgRP) neurones are first-order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double-label immunohistochemistry was used to quantify leptin-induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using α-melanocyte-stimulating hormone (MSH) as a marker. The percentage of α-MSH neurones containing leptin-induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with α-MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of α-MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first-order leptin-responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system.

Pregnancy is associated with hyperphagia, increased fat mass, hyperleptinaemia and hyperprolactinaemia. The neuroendocrine control of bodyweight involves appetite-regulating centres in the hypothalamus, containing both orexigenic and anorexigenic neurons that express leptin receptors (LepR). In the rat, central leptin resistance develops during mid pregnancy, well after hyperphagia becomes apparent, to negate the appetite suppressing effects of leptin. We have investigated the hypothalamic response to leptin during pregnancy and examined the role of pregnancy hormones in inducing these changes. We have shown that there are multiple levels of leptin resistance during pregnancy. Despite elevated serum leptin, neuropeptide Y and agouti related peptide mRNA in the arcuate nucleus are not suppressed and may even be increased during pregnancy. LepR mRNA and leptin-induced pSTAT3 expression, however, are relatively normal in the arcuate nucleus. In contrast, both LepR and leptin-induced pSTAT3 are reduced in the ventromedial hypothalamic nucleus. Injecting alpha-melanocyte-stimulating hormone (alpha-MSH) into the brain, to bypass the first-order leptin-responsive neurons in the arcuate nucleus, also fails to suppress food intake during pregnancy, suggesting that pregnancy is also a melanocortin-resistant state. Using a pseudopregnant rat model, we have demonstrated that in addition to the changes in maternal ovarian steroid secretion, placental lactogen production is essential for the induction of leptin resistance in pregnancy. Thus, hormonal changes associated with pregnancy induce adaptive changes in the maternal hypothalamus, stimulating food intake and then allowing elevated food intake to be maintained in the face of elevated leptin levels, resulting in fat deposition to provide energy stores in preparation for the high metabolic demands of late pregnancy and lactation.

Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.

Numerous studies have documented prolactin regulation of a variety of brain functions, including maternal behavior, regulation of oxytocin neurons, regulation of feeding and appetite, suppression of ACTH secretion in response to stress, and suppression of fertility. We have observed marked changes in expression of prolactin receptors in specific hypothalamic nuclei during pregnancy and lactation. This has important implications for neuronal functions regulated by prolactin. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these functions may be enhanced or exaggerated in the maternal brain. The adaptations of the maternal brain allow the female to exhibit the appropriate behavior to feed and nurture her offspring, to adjust to the nutritional and metabolic demands of milk production, and to maintain appropriate hormone secretion to allow milk synthesis, secretion, and ejection. This review aims to summarize the evidence that prolactin plays a key role in regulating hypothalamic function during lactation and to discuss the hypothesis that the overall role of prolactin is to organize and coordinate this wide range of behavioral and neuroendocrine adaptations during pregnancy and lactation.