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The present series of studies aimed to further our understanding of the role of melanin-concentrating hormone (MCH) neurones in the central regulation of luteinising hormone (LH) release in the female rat. LH release was stimulated when MCH was injected bilaterally into the rostral preoptic area (rPOA) or medial preoptic area (mPOA), but not when injected into the zona incerta (ZI), of oestrogen-primed ovariectomised rats. In rats that were steroid-primed to generate a surge-like release of LH, MCH administration into the ZI blocked this rise in LH release: no such effect occurred when MCH was injected into the rPOA or mPOA. In vitro, MCH stimulated gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants. Double-label immunohistochemistry showed GnRH-immunoreactive neurones in the vicinity of and intermingled with immunoreactive MCH processes. MCH is the endogenous ligand of the MCH type 1 receptor (MCH1-R). Previously, we have shown a role for melanocortin-5 receptors (MC5-R) in the stimulatory action of MCH, so we next investigated the involvement of both MCH1-R and/or MC5-R in mediating the actions of MCH on GnRH and hence LH release. The stimulatory action of MCH in the rPOA was inhibited by administration of antagonists for either MCH1-R or MC5-R. However, in the mPOA, the action of MCH was blocked only by the MC5-R antagonist. LH release was stimulated by an agonist for MC5-R injected into the rPOA or mPOA; this was blocked by the MC5-R antagonist but not the MCH1-R antagonist. These results indicate that both MCH1-R and MC5-R are involved in the central control of LH release by MCH.

A number of neuropeptides implicated in the hypothalamic regulation of appetite are synthesized in the arcuate nucleus (Arc). Neuropeptide Y (NPY) and agouti-related protein (Agrp) are orexigenic. The pro-opiomelanocortin (POMC) product alpha-melanocyte-stimulating hormone (alpha-MSH) is anorectic. Intracerebroventricular administration of cocaine- and amphetamine-regulated transcript (CART) decreases food intake. However, recent results show that CART is orexigenic when injected into discrete hypothalamic nuclei. There is almost complete coexpression of NPY and Agrp mRNA in Arc neurones, and the majority of CART-containing neurones in the Arc also contain POMC mRNA. We investigated possible interactions between these neuropeptides in vitro using a rat hypothalamic explant system. Administration of 1, 10 and 100 nm of NPY to hypothalamic explants significantly increased release of Agrp(83-132)-immunoreactivity (IR). NPY (10 and 100 nm) significantly increased the release of CART(55-102)-IR and alpha-MSH-IR from hypothalamic explants. Agrp(83-132) (10 nm) administered to hypothalamic explants significantly increased the release of NPY-IR. Agrp(83-132) (10 and 100 nm) significantly decreased the release of CART(55-102)-IR from hypothalamic explants. Administration of 1, 10 and 100 nm CART(55-102) to hypothalamic explants resulted in a significant increase in NPY-IR release. Administration of 10 nm CART(55-102) to hypothalamic explants significantly increased the release of Agrp(83-132)-IR. NDP-MSH (10 nm) administered to hypothalamic explants significantly increased the release of NPY-IR. NDP-MSH (10 and 100 nm) significantly increased the release of Agrp(83-132)-IR from hypothalamic explants. These data suggest that orexigenic neuropeptides in the arcuate nucleus stimulate the release of each other, perhaps reinforcing orexigenic behaviour via a positive-feedback loop. Our results are also in keeping with the possibility that the melanocortin-3 receptor in the arcuate nucleus may influence the release of arcuate neuropeptides.

The hypothalamic melanocortin system is important in the central regulation of food intake and body weight. We have previously demonstrated that intracerebroventricular administration of alpha-melanocyte stimulating hormone (alpha-MSH), a nonselective MC3 and MC4 receptor agonist, stimulated plasma thyroid-stimulating hormone, and agouti-related protein (AgRP), an MC3 and MC4 receptor antagonist, suppressed it. In this study, we investigated the effects of MC3 and MC4 receptor (MC3-R and MC4-R) selective agonists and antagonists on the release of thyrotropin-releasing hormone (TRH) from hypothalamic explants in vitro. alpha-MSH stimulated TRH release from the rat hypothalamic explants (alpha-MSH 100 nm 230 +/- 22.9% basal, P < 0.005). In contrast, gamma 2-MSH, a selective MC3-R agonist, suppressed TRH release (gamma 2-MSH 10 microns 76.2 +/- 7.4% basal, P < 0.05). AgRP (83-132), a nonselective MC3/4-R antagonist, induced no change in TRH release whilst JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH 11-22), a selective MC4-R antagonist, suppressed it (JKC-363 10 nm 57.2 +/- 11.5% basal, P < 0.05). Both AgRP (83-132) and JKC-363 blocked alpha-MSH stimulated TRH release but only AgRP (83-132) blocked the inhibitory effect of gamma 2-MSH on TRH release. These data suggest differential roles for the MC3 and MC4 receptors in TRH release; MC3-R agonism inhibiting and MC4-R agonism stimulating TRH release.

Orexin-A is synthesized in the posterolateral hypothalamus and immunoreactive fibres project to many central nervous system structures, including the paraventricular nucleus, which is rich in corticotropin releasing factor (CRF) neurones and neuropeptide Y (NPY) innervation. We investigated the central effects of orexin-A on the hypothalamic-pituitary-adrenal (HPA) axis by measuring plasma concentrations of corticosterone and adrenocorticotropic hormone (ACTH) in vivo. We explored the potential neuropeptide pathways involved by investigating the effects of orexin-A on CRF, NPY, arginine vasopressin (AVP) and noradrenaline release from hypothalamic explants in vitro. Intracerebroventricular (i.c.v.) injection of orexin-A (3 nmol) in male rats stimulated increases in plasma concentrations of corticosterone between 10 and 40 min after injection, and of plasma ACTH at 20 and 90 min after injection. Orexin-A significantly stimulated CRF and NPY release from hypothalamic explants in vitro. Orexin-A did not stimulate CRF release in the presence of the selective NPY Y1 receptor antagonist, BIBP3226. BIBP3226 alone did not alter CRF release from hypothalamic explants. Orexin-A had no effect in vitro on the release of other neuropeptides, AVP and noradrenaline, involved in the central regulation of the HPA axis. These results suggest that orexin-A is involved in activation of the HPA axis, and that these effects could be mediated via the release of NPY.

The adipocyte derived hormone leptin has been implicated as an important nutritional signal to the reproductive system, but the role of other adipocyte related cytokines is not clear. Tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 are present in adipose tissue and released into the circulation where plasma levels correlate positively with body mass index and body fat mass. These cytokines could play a role in signalling nutritional status to the hypothalamic-pituitary-gonadal axis. We investigated the effects of TNF-alpha and IL-6 on basal and luteinizing hormone releasing hormone (LHRH) stimulated luteineizing hormone (LH) release from cultured anterior pituitary cells, harvested from either proestrus female or male Wistar rats. We examined the effects of TNF-alpha and IL-6 on LHRH release from hypothalamic explants harvested from proestrus female and male rats in vitro. IL-6 significantly suppressed LHRH stimulated LH release from male dispersed pituitaries throughout the dose range, but did not influence basal LH release. IL-6 had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from proestrus females. By contrast, TNF-alpha significantly suppressed LHRH stimulated LH release in dispersed pituitaries from proestrus female rats in a dose responsive manner, but did not influence basal LH release. TNF-alpha had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from male rats. TNF-alpha and IL-6 had no effect on LHRH release from male hypothalamic explants in vitro. TNF-alpha and IL-6 had no effect on LHRH release from proestrus female hypothalamic explants in vitro. TNF-alpha and IL-6 have differential effects in dispersed pituitaries harvested from males and proestrus female rats. TNF-alpha and IL-6 may be important in mediating some of the nutritional effects on the reproductive axis by acting at the level of the anterior pituitary rather than the hypothalamus.

Orexin immunoreactive fibres are abundant in the hypothalamus suggesting a neuroendocrine regulatory role. Intracerebroventricular (ICV) administration of orexin A suppressed plasma prolactin in male rats by 71% at 20 min post-injection and 83% at 90 min post-injection (P < 0.005 vs saline at both time points). To investigate whether this effect was through the tuberoinfundibular dopaminergic (TIDA) system, a supra-maximal dose of domperidone, a dopamine receptor antagonist, was injected intraperitoneally (i.p.) prior to ICV injection of orexin A. ICV orexin A significantly suppressed domperidone (9 mg/kg)-stimulated plasma prolactin levels, by up to 40% (i.p. domperidone + ICV orexin A 3 nmol 34.5 +/- 7.4 ng/ml and i.p. domperidone + ICV orexin A 20 nmol 43.5 +/- 4.3 ng/ml, both P < 0.005 vs i.p. domperidone + ICV saline 57.9 +/- 2.7 ng/ml). Orexin A, 100 nM, significantly stimulated release of neurotensin, vasoactive intestinal polypeptide, somatostatin, corticotropin releasing factor and luteinizing hormone releasing hormone, but had no effect on release of dopamine, thyrotropin releasing hormone (TRH), vasopressin or melanin-concentrating hormone from hypothalamic explants in vitro. Orexin A did not alter basal or TRH stimulated prolactin release in dispersed pituitary cells harvested from male rats. The data suggest that ICV administration of orexin A suppresses plasma prolactin in part through a pathway independent of the dopaminergic system.

Ciliary neurotrophic factor (CNTF) is a member of the neuropoietic family of cytokines. CNTF exerts its actions through activation of a receptor complex, which shows similarity of sequence, second messenger systems and distribution to the leptin receptor. Leptin has been demonstrated to exert profound effects on the hypothalamo-pituitary gonadal axis. This study examines the in vitro effects of CNTF on hypothalamic luteinizing hormone releasing hormone release (LHRH) and pituitary luteinizing hormone (LH) release compared to those of leptin in the female. We report that CNTF stimulates LHRH release from medial basal hypothalamic explants harvested from proestrous female rats and this effect is of similar magnitude to that seen with leptin. In contrast, CNTF suppresses LHRH-stimulated LH release from dispersed anterior pituitary cells harvested from proestrous female rats but has no effect on basal LH release. Leptin stimulates basal LH release but has no effect on LHRH-stimulated LH release. The suppressive effect of CNTF on LHRH-stimulated LH release has been confirmed in perifused anterior hemipituitaries. These results suggest a differential effect of CNTF on the hypothalamo-pituitary gonadal axis and a possible role in the modulation of pituitary gonadal function.