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Aims: The aim of the present study is to examine the orexin A (OXA) signaling can leave any impact on the hypothalamic-pituitary-gonadal (HPG) axis and this impact can be relayed through the pathway of RF amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibitory hormone)/G-protein coupled receptor (GPR)-147 (RFRP-3 receptor) as a novel target for controlling of HPG axis in the male rats., Materials and Methods: Male rats were categorized randomly into experimental groups including control vehicle, OXA, and its antagonists' group and went through to surgical cannulation into the third ventricle. After the intracerebroventricular injection of each solution, blood samples were collected for measurements of the LH and testosterone using radioimmunoassay method. Hypothalamus of the animals were isolated for analysis of the relative expression of Rfrp-3 / Gpr -147 along with Gnrh gene by Real time-PCR. Also, in the different cohort of animal sexual behavior test was done., Results: It was shown that OXA significantly decreases the mean serum level of the LH and testosterone and, at the same time, its antagonists neutralize this impact. Moreover, we demonstrated that OXA has reduced the hypothalamic gene expression of Gnrh and increased the expression of Rfrp-3 and Gpr-147 genes . While OXA antagonists neutralize this impact., Conclusions: The results of this study are related to the impact of orexin on the HPG axis. It is recommended that RFRP-3/GPR-147 system as the interneural pathway relay the data of orexin to the neurons of GnRH., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Phoenixin (Pnx), a recently discovered neuropeptide, has been implicated in reproduction. Pnx mainly exists in two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleosts. To determine the roles of Pnx in the regulation of reproduction in Scatophagus argus, the physiological characterization of the Pnx was analyzed. During ovary development, the expression of pnx in phase IV was higher than in phase II and III in the hypothalamus. In the pituitary, pnx expression was highest in phase IV, moderate in phase III, and lowest in phase II. When hypothalamus and pituitary fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of GnRHR (gonadotropin releasing hormone receptor), lh (luteinizing hormone) and fsh (follicular stimulating hormone) in the pituitary increased significantly, except GnRH (gonadotropin releasing hormone) in the hypothalamus. Similarly, the expression of GnRHR, lh and fsh in the pituitary increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight (bw)), except GnRHR and fsh treated with 10 ng/gbw Pnx-20 in the pituitary and GnRHs in the hypothalamus. These results indicate that Pnx may not only stimulate the reproduction of the S. argus through the hypothalamic-pituitary-gonadal (HPG) axis, but also directly through the pituitary., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Study Objectives: Experimental studies over the last 15 years established a role in sleep of the tuberal hypothalamic neurons that express melanin-concentrating hormone (MCH). Controversies still remain regarding their actual contribution to both slow-wave sleep (SWS) and paradoxical sleep (PS also known as REM sleep) or PS alone., Methods: To address this point, we compared effects of chemogenetic activation and inhibition of MCH neurons on SWS and PS amounts and EEG rhythmic activities in transgenic Pmch-cre mice., Results: In agreement with recently reported optogenetic data, the activation of MCH neurons invariably facilitates PS onset and maintenance. Our chemogenetic experiments further disclose that the ultradian rhythm of SWS is also notably related to the activity of MCH neurons. We observed that the mean duration of SWS episodes is significantly extended when MCH neurons are inhibited. Conversely, when they were excited, SWS bouts were drastically shortened and depicted substantial changes in δ rhythmic activities in electroencephalographic recording likely reflecting a deeper SWS., Conclusions: According to these original findings, we propose that when MCH neurons are physiologically recruited, SWS depth is increased and the extinction of SWS episodes is accelerated, two joint physiological processes strengthening the probability for natural SWS to PS transition and likely facilitating PS onset.

Gonadotropin-inhibitory hormone (GnIH) has been characterized by its ability to inhibit either basal or gonadotropin-releasing hormone (GnRH)-induced gonadotropin synthesis and release in birds and mammals. However, the physiological role of GnIH on the reproductive axis in fish remains inconclusive, with most studies focusing on the orders Cypriniformes and Perciformes. To gain insight into the role of GnIH in the regulation of reproduction in the order Pleuronectiformes, we first cloned the LPXRFa gene, the piscine ortholog of GnIH, in the half-smooth tongue sole. The full-length cDNA of LPXRFa was 918bp in size with an open reading frame (ORF) of 585bp that encoded a 194 amino acids preprohormone with a calculated molecular mass and isoelectric point of 21.73kDa and 6.52, respectively. The LPXRFa precursor encoded two putative peptide sequences that included -MPMRF or -MPQRF motifs at the C-terminal. Tissue distribution analysis showed that LPXRFa transcripts could be detected at high levels in the brains of both sexes and to a lesser extent in the ovary, heart and stomach of females, while a noteworthy expression was observed in the kidney and muscle of males. Furthermore, the expression patterns of LPXRFa mRNA during ovarian maturation were also investigated. In the brain, the mRNA expression of LPXRFa increased significantly at stage III, declined at stage V and reached a maximum at stage VI. In the pituitary, the levels of LPXRFa mRNA remained stable during ovarian maturation and increased significantly to the top level at stage V and then declined back to basal levels. In contrast, the ovarian LPXRFa mRNA levels declined sharply at stage III and remained depressed over the course of ovarian maturation. Taken together, our results provide further evidence for the existence of LPXRFa in the order Pleuronectiformes and suggest its possible involvement in the regulation of reproduction in the female tongue sole., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The neurobiological mechanism of puberty onset in primates is currently only partly understood. A recent study reported an important role of Dmx-like 2 (DMXL2), a gene encoding rabconnectin-3α vesicular protein, in human subjects with mental retardation and neuroendocrine impairment of reproduction. To further characterize the potential role of DMXL2 in the regulation of reproduction, we analyzed the expression of DMXL2 in hypothalami of newborn, infantile, juvenile, pubertal, and postpubertal female and male common marmoset monkeys. Additionally, as the relative hypothalamic levels of gonadotropin-inhibitory hormone (GnIH) transcript during postnatal development are unknown in primates, we also quantified messenger RNA (mRNA) levels of RFRP, a gene encoding GnIH. Moreover, the transcript levels of kisspeptin, a well-known regulator of the hypothalamic neurohormonal axis controlling reproduction, were also checked. Transcript and protein levels of DMXL2 and Kiss1 transcript levels increase from the newborn to the infantile and from the juvenile (prepubertal) to the pubertal and the postpubertal period. We also noted a clear upsurge in RFRP transcript levels in the prepubertal period. In conclusion, the hypothalamic expressions of Kiss1 and DMXL2 mRNA increase during infantile, pubertal, and adult stages compared to newborn and juvenile stages in common marmoset monkeys. In contrast, the expression of RFRP mRNA upsurges in juvenile monkeys. Further mechanistic studies are needed to characterize the potential inhibitory role of the GnIH-GPR147 signaling in the prepubertal period and the role of DMXL2 in the molecular cascade regulating the neuroendocrine reproductive axis in primates.

Acupuncture has shown the therapeutic effect on various neurodegenerative disorders including Parkinson's disease (PD). While investigating the neuroprotective mechanism of acupuncture, we firstly found the novel function of melanin-concentrating hormone (MCH) as a potent neuroprotective candidate. Here, we explored whether hypothalamic MCH mediates the neuroprotective action of acupuncture. In addition, we aimed at evaluating the neuroprotective effects of MCH and elucidating underlying mechanism in vitro and in vivo PD models. First, we tested whether hypothalamic MCH mediates the neuroprotective effects of acupuncture by challenging MCH-R1 antagonist (i.p.) in mice PD model. We also investigated whether MCH has a beneficial role in dopaminergic neuronal protection in vitro primary midbrain and human neuronal cultures and in vivo MPTP-induced, Pitx3 -/- , and A53T mutant mice PD models. Transcriptomics followed by quantitative PCR and western blot analyses were performed to reveal the neuroprotective mechanism of MCH. We first found that hypothalamic MCH biosynthesis was directly activated by acupuncture treatment and that administration of an MCH-R1 antagonist reverses the neuroprotective effects of acupuncture. A novel finding is that MCH showed a beneficial role in dopaminergic neuron protection via downstream pathways related to neuronal survival. This is the first study to suggest the novel neuroprotective action of MCH as well as the involvement of hypothalamic MCH in the acupuncture effects in PD, which holds great promise for the application of MCH in the therapy of neurodegenerative diseases.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Reward and energy homeostasis are both regulated by a network of hypothalamic neuropeptide systems. The melanin-concentrating hormone (MCH) and its MCH-1 receptor (MCH1-R) modulate alcohol intake, but it remains unknown to what extent this reflects actions on energy balance or reward. Here, we evaluated the MCH1-R in regulation of caloric intake and motivation to consume alcohol in states of escalated consumption., Methods: Rats had intermittent access (IA) to alcohol and were divided into high- and low-drinking groups. Food and alcohol consumption was assessed after administration of an MCH1-R antagonist, GW803430. Next, GW803430 was evaluated on alcohol self-administration in protracted abstinence induced by IA in high-drinking rats. Finally, the effect of GW803430 was assessed on alcohol self-administration in acute withdrawal in rats exposed to alcohol vapor. Gene expression of MCH and MCH1-R was measured in the hypothalamus and nucleus accumbens (NAc) in both acute and protracted abstinence., Results: High-drinking IA rats consumed more calories from alcohol than chow and GW803430 decreased both chow and alcohol intake. In low-drinking rats, only food intake was affected. In protracted abstinence from IA, alcohol self-administration was significantly reduced by pretreatment with GW803430 and gene expression of both MCH and the MCH1-R were dysregulated in hypothalamus and NAc. In contrast, during acute withdrawal from vapor exposure, treatment with GW803430 did not affect alcohol self-administration, and no changes in MCH or MCH1-R gene expression were observed., Conclusions: Our data suggest a dual role of MCH and the MCH1-R in regulation of alcohol intake, possibly through mechanisms involving caloric intake and reward motivation. A selective suppression of alcohol self-administration during protracted abstinence by GW803430 was observed and accompanied by adaptations in gene expression of MCH and MCH1-R. Selective suppression of escalated consumption renders the MCH1-R an attractive target for treatment of alcohol use disorders., (Copyright © 2016 by the Research Society on Alcoholism.)

Aims: Melanin-concentrating hormone (MCH) is implicated in the control of food intake, body weight regulation and energy homeostasis. Lactation is an important physiological model to study the hypothalamic integration of peripheral sensory signals, such as suckling stimuli and those related to energy balance. MCH can be detected in the medial preoptic area (MPOA), especially around the 19th day of lactation, when this hormone is described as displaying a peak synthesis followed by a decrease after weaning. The physiological significance of this phenomenon is unclear. Therefore, we aimed to investigate hypothalamic changes associated to sensory stimulation by the litter, in special its influence over MCH synthesis., Main Methods: Female Wistar rats (n=56) were euthanized everyday from lactation days 15-21, with or without suckling stimulus (WS and NS groups, respectively). MCH and Fos immunoreactivity were evaluated in the MPOA and lateral and incerto-hypothalamic areas (LHA and IHy)., Key Findings: Suckling stimulus induced Fos synthesis in all regions studied. An increase on the number of suckling-induced Fos-ir neurons could be detected in the LHA after the 18th day. Conversely, the amount of MCH decreased in the MPOA from days 15-21, independent of suckling stimulation. No colocalization between MCH and Fos could be detected in any region analyzed., Significance: Suckling stimulus is capable of stimulating hypothalamic regions not linked to maternal behavior, possibly to mediate energy balance aspects of lactation. Although dams are hyperphagic before weaning, this behavioral change does not appear to be mediated by MCH., (Copyright © 2016. Published by Elsevier Inc.)

Exposure to short days (SD) induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD) phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE) cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response.

Neurons synthesizing melanin-concentrating hormone (MCH) are described in the posterior hypothalamus of all vertebrates investigated so far. However, their anatomy is very different according to species: they are small and periventricular in lampreys, cartilaginous fishes or anurans, large and neuroendocrine in bony fishes, or distributed over large regions of the lateral hypothalamus in many mammals. An analysis of their comparative anatomy alongside recent data about the development of the forebrain, suggests that although very different, MCH neurons of the caudal hypothalamus are homologous. We further hypothesize that their divergent anatomy is linked to divergence in the forebrain - in particular telencephalic evolution., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glucocorticoid deficiency is associated with a decrease of food intake. Orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP), and the anorexigenic peptide proopiomelanocortin (POMC), expressed in the arcuate nucleus of the hypothalamus (ARC), are regulated by meal-induced signals. Orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin, expressed in the lateral hypothalamic area (LHA), also control food intake. Thus, the present study was designed to test the hypothesis that glucocorticoids are required for changes in the expression of hypothalamic neuropeptides induced by feeding. Male Wistar rats (230-280 g) were subjected to ADX or sham surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received corticosterone in the drinking water (B: 25 mg/L, ADX+B). Six days after surgery, animals were fasted for 16 h and they were decapitated before or 2 h after refeeding for brain tissue and blood collections. Adrenalectomy decreased NPY/AgRP and POMC expression in the ARC in fasted and refed animals, respectively. Refeeding decreased NPY/AgRP and increased POMC mRNA expression in the ARC of sham and ADX+B groups, with no effects in ADX animals. The expression of MCH and orexin mRNA expression in the LHA was increased in ADX and ADX+B groups in fasted condition, however there was no effect of refeeding on the expression of MCH and orexin in the LHA in the three experimental groups. Refeeding increased plasma leptin and insulin levels in sham and ADX+B animals, with no changes in leptin concentrations in ADX group, and insulin response to feeding was lower in this group. Taken together, these data demonstrated that circulating glucocorticoids are required for meal-induced changes in NPY, AgRP and POMC mRNA expression in the ARC. The lower leptin and insulin responses to feeding may contribute to the altered hypothalamic neuropeptide expression after adrenalectomy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is classically defined as a motor disorder resulting from decreased dopamine production in the basal ganglia circuit. In an attempt to better diagnose and treat PD before the onset of severe motor dysfunction, recent attention has focused on the early, non-motor symptoms, which include but are not limited to sleep disorders such as excessive daytime sleepiness (EDS) and REM behavioral disorder (RBD). However, few animal models have been able to replicate both the motor and non-motor symptoms of PD. Here, we present a progressive rat model of parkinsonism that displays disturbances in sleep/wake patterns. Epidemiological studies elucidated a link between the Guamanian variant of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) and the consumption of flour made from the washed seeds of the plant Cycas micronesica (cycad). Our study examined the effects of prolonged cycad consumption on sleep/wake activity in male, Sprague-Dawley rats. Cycad-fed rats exhibited an increase in length and/or number of bouts of rapid eye movement (REM) sleep and Non-REM (NREM) sleep at the expense of wakefulness during the active period when compared to control rats. This hypersomnolent behavior suggests an inability to maintain arousal. In addition, cycad-fed rats had significantly fewer orexin cells in the hypothalamus. Our results reveal a novel rodent model of parkinsonism that includes an EDS-like syndrome that may be associated with a dysregulation of orexin neurons. Further characterization of this early, non-motor symptom, may provide potential therapeutic interventions in the treatment of PD., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The distribution of hypothalamic neurons expressing the peptides melanin-concentrating hormone (MCH; 'MCH neurons') or hypocretin/orexin (H/O; 'H/O neurons') was assessed with immunocytochemistry in male rats at high spatial resolution. Data were plotted on a rat brain atlas that includes a recently revised parcellation scheme for the lateral hypothalamic zone. Quantitative analysis revealed approximately three times more MCH neurons than H/O neurons in the hypothalamus, and approximately twice as many within the parcellations of the lateral hypothalamic area (LHA). The LHA contained 60% of MCH neurons and 81% of H/O neurons, and the same five LHA regions contained the vast majority of MCH (87%) or H/O (93%) neurons present within the LHA: namely the LHA dorsal region (LHAd: 31% of H/O; 38% of MCH), suprafornical region (LHAs: 28% of H/O; 11% of MCH), ventral region medial zone (LHAvm: 15% of H/O; 16% of MCH), juxtadorsomedial region (LHAjd: 14% of H/O and MCH) and magnocellular nucleus (LHAm: 5% of H/O; 7% of MCH). The zona incerta (ZI) contained 18% of MCH neurons. A high co-abundance of MCH and H/O neurons outside of the LHA was present in the posterior hypothalamic nucleus (PH: 11% of H/O; 9% of MCH). Morphological analysis revealed MCH and H/O neurons as typically tri-polar with irregularly shaped somata. These data provide a quantitative analysis of neurons expressing either MCH or H/O peptides within the rat hypothalamus, and they clarify differences in the distribution pattern for different subsets of these neuron types, especially within the LHA.

Background: There is growing evidence suggesting that hypothalamic galanin (GAL), which is known to stimulate intake of a fat-rich diet, has a role in promoting the consumption of ethanol. The present study further examined this possibility in GAL knockout (GALKO) mice., Methods: Two groups of female and male GALKO mice, compared to wild-type (WT) controls, were trained to voluntarily drink increasing concentrations of ethanol, while maintained on lab chow and water. They were examined in terms of their daily ethanol intake and preference, acute consumption of a high-fat diet, preference for flavored solutions, and expression of different peptides shown to stimulate ethanol intake., Results: In the GALKO mice compared to WT, the results revealed: (i) a 35 to 45% decrease in ethanol intake and preference, which was evident only at the highest (15%) ethanol concentration, was stronger in female than in male mice, and was seen with comparisons to littermate as well as nonlittermate WT mice; (ii) a 48% decrease in acute intake of a fat-rich diet, again stronger in female than male mice; (iii) no difference in consumption of sucrose or quinine solutions in preference tests; (iv) a total loss of GAL mRNA in the hypothalamic paraventricular nucleus (PVN) of female and male mice; and (v) a gender-specific change in mRNA levels of peptides in the perifornical lateral hypothalamus (PFLH), orexin and melanin-concentrating hormone, which are known to stimulate ethanol and food intake and were markedly decreased in females while increased in males., Conclusions: These results provide strong support for a physiological role of PVN GAL in stimulating the consumption of ethanol, as well as a fat-rich diet. Ablation of the GAL gene produced a behavioral phenotype, particularly in females, which may reflect the functional relationship of galanin to ovarian steroids. It also altered the peptides in the PFLH, with their reduced expression contributing to the larger behavioral effects observed in females and their increased expression attenuating these effects in males.

Regulation of energy homeostasis in animals involves adaptation of energy intake to its loss, through a perfect regulation of feeding behavior and energy storage/expenditure. Factors from the periphery modulate brain activity in order to adjust food intake as needed. Particularly, "first order" neurons from arcuate nucleus are able to detect modifications in homeostatic parameters and to transmit information to "second order" neurons, partly located in the lateral hypothalamic area. These "second order" neurons have widespread projections throughout the brain and their proper activation leads them to a coordinated response associated to an adapted behavior. Among these neurons, melanin-concentrating hormone (MCH) expressing neurons play an integrative role of the various factors arising from periphery, first order neurons and extra-hypothalamic arousal systems neurons and modulate regulation of feeding, drinking and seeking behaviors. As regulation of MCH release is correlated to regulation of MCH neuronal activity, we focused this review on the electrophysiological properties of MCH neurons from the lateral hypothalamic area. We first reviewed the knowledge on the endogenous electrical properties of MCH neurons identified according to various criteria which are described. Then, we dealt with the modulations of the electrical activity of MCH neurons by different factors such as glucose, glutamate and GABA, peptides and hormones regulating feeding and transmitters of extra-hypothalamic arousal systems. Finally, we described the current knowledge on the modulation of MCH neuronal activity by cytokines and chemokines. Because of such regulation, MCH neurons are some of the best candidate to account for infection-induced anorexia, but also obesity.

Background: Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours., Methods: Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice., Results: In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments., Conclusion: This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.

A study was undertaken in Corriedale ewes to test the lipostat theory using data obtained from a model of seasonal change in food intake and body composition. The theory predicts adipose-derived factors signal to the brain and vice versa, to maintain homeostasis. It is held that leptin acts on cells in the brain to regulate food intake and energy expenditure, through "first order" neurons in the arcuate nucleus (ARC). These cells are thought to receive information that is relayed to "second order" neurons, to regulate food intake and other functions. In this study, groups (n=4-5) of ovariectomized ewes were maintained under natural conditions and sampled at various points across the year. Food intake, body composition and indices of metabolic function were measured prior to collection of brains for in situ hybridization analysis. Expression of genes encoding for neuropeptide Y (NPY), pro-opiomelanocortin (POMC), orexin (ORX), melanin concentrating hormone (MCH) and leptin receptor (ObRb) was quantified. NPY gene expression was high when food intake was also high but, across the year, changes in NPY and POMC gene expression did not correspond predictably to plasma leptin levels or leptin receptor gene expression. Negative correlation was found between adiposity (omental and whole body fat) and gene expression of MCH and ORX, suggesting that changes in expression of genes for "second order" orexigenic peptides are closely linked to changes of metabolic state, even when similar relationships cannot be shown for expression of genes in "first order" neurons. These data provide support for the lipostat theory.

PRL and PrRP cDNAs have been isolated from euryhaline silver sea bream (Sparus sarba). The PRL cDNA consists of 1360bp encoding 212 amino acids whereas the PrRP cDNA contains 631bp encoding preproPrRP with 122 amino acids. The mature PrRP sequence within the preprohormone is identical to the PrRPs isolated from other fish species. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of organs and tissues including the intestines, olfactory rosette and various brain regions such as hypothalamus and pituitary. Expression levels of PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50ppt). In the pituitary, both PRL and PrRP mRNA were significantly higher in fish adapted to low salinities (0 and 6ppt) and the expression profiles of both hormones closely paralleled each other. However, expression of hypothalamic PrRP was significantly higher in fish adapted to iso-osmotic salinity (12ppt) when pituitary PRL expression was low. The present study demonstrates, for the first time, a synchronized mRNA expression pattern between PRL and PrRP in fish pituitary but a disparity of mRNA expression levels between hypothalamic PrRP and pituitary PRL during salinity adaptation. These data suggest that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulation of pituitary PRL expression in silver sea bream.

Prolactin-releasing peptide (PrRP) was recently isolated from bovine hypothalamus. PrRP is the natural ligand for an orphan G-protein-coupled receptor, hGR3, and directly stimulates prolactin secretion from the anterior pituitary in vitro and in vivo. It has also been reported that PrRP plays an important role as a neurotransmitter and/or neuromodulator in the brain. Although much knowledge has been gained concerning PrRP in the adult rat brain, little attention has been paid to the fetal and postnatal stages. We therefore examined the development of PrRP neurons in the rat brain. In immunocytochemical and in situ hybridization experiments, we observed the transient appearance of PrRP-producing cells in the area postrema (AP), in which PrRP-producing cells do not exist in the normal adult rat. PrRP-producing cells in the AP were detected at P14, and many PrRP-producing cells were observed at P17, though none were detected at P19. This is the first report of the appearance of PrRP-producing cells in the postnatal AP. Our findings suggest that PrRP may play a previously unknown role in the AP of postnatal rats.

Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo-pituitary-adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland.

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.

Neurons producing melanin-concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the 'cocaine- and amphetamine-regulated transcript' (CART) peptide, while another expresses neither one of these two molecules. MCH+/CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/CART- axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART- projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions.

Glucagon-like peptide 1 (GLP-1) is produced by neurons in the caudal brainstem that receive sensory information from the gut and project to several hypothalamic regions involved in arousal, interoceptive stress, and energy homeostasis. GLP-1 axons and receptors have been detected in the lateral hypothalamus, where hypocretin neurons are found. The electrophysiological actions of GLP-1 in the CNS have not been studied. Here, we explored the GLP-1 effects on GFP (green fluorescent protein)-expressing hypocretin neurons in mouse hypothalamic slices. GLP-1 receptor agonists depolarized hypocretin neurons and increased their spike frequency; the antagonist exendin (9-39) blocked this depolarization. Direct GLP-1 agonist actions on membrane potential were abolished by choline substitution for extracellular Na+, and dependent on intracellular GDP, suggesting that they were mediated by sodium-dependent conductances in a G-protein-dependent manner. In voltage clamp, the GLP-1 agonist Exn4 (exendin-4) induced an inward current that reversed near -28 mV and persisted in nominally Ca2+-free extracellular solution, consistent with a nonselective cationic conductance. GLP-1 decreased afterhyperpolarization currents. GLP-1 agonists enhanced the frequency of miniature and spontaneous EPSCs with no effect on their amplitude, suggesting presynaptic modulation of glutamate axons innervating hypocretin neurons. Paraventricular hypothalamic neurons were also directly excited by GLP-1 agonists. In contrast, GLP-1 agonists had no detectable effect on neurons that synthesize melanin-concentrating hormone (MCH). Together, our results show that GLP-1 agonists modulate the activity of hypocretin, but not MCH, neurons in the lateral hypothalamus, suggesting a role for GLP-1 in the excitation of the hypothalamic arousal system possibly initiated by activation by viscera sensory input.

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms., (Copyright 2004 Elsevier Inc.)

Neurons from the lateral hypothalamus that synthesize melanin-concentrating hormone (MCH) play an important role in the regulation of energy homeostasis. Relatively little is known of the cellular physiology and transmitter responses of these neurons, in part because of the difficulty in identifying live MCH cells. Here we use a novel approach of transfection of specific gene constructs with the MCH promoter driving green fluorescent protein (GFP) or red fluorescent protein (dsRed2) in CNS cultures to identify live rat MCH neurons; all neurons expressing the reporter gene showed MCH immunoreactivity, indicating selective expression. MCH neurons had a resting membrane potential of -57.5 +/- 0.6 mV, a linear current-voltage relation and a mean input resistance of 1,013 MOmega. Long depolarizing pulses revealed significant spike frequency adaptation. Functional glutamate and GABA receptors were expressed by MCH neurons. MCH neurons were hyperpolarized by norepinephrine in the presence or absence of tetrodotoxin, suggesting direct inhibition. Orexigenic peptides neuropeptide Y (NPY) and MCH showed no direct effect on membrane potential, input resistance, action potential width, or afterhyperpolarization potential, but inhibited voltage-dependent calcium channels, indicating that MCH neurons expressed both MCH and NPY receptors.

Background: Peptidergic neurons containing the melanin-concentrating hormone (MCH) and the hypocretins (or orexins) are intermingled in the zona incerta, perifornical nucleus and lateral hypothalamic area. Both types of neurons have been implicated in the integrated regulation of energy homeostasis and body weight. Hypocretin neurons have also been involved in sleep-wake regulation and narcolepsy. We therefore sought to determine whether hypocretin and MCH neurons express Fos in association with enhanced paradoxical sleep (PS or REM sleep) during the rebound following PS deprivation. Next, we compared the effect of MCH and NaCl intracerebroventricular (ICV) administrations on sleep stage quantities to further determine whether MCH neurons play an active role in PS regulation., Results: Here we show that the MCH but not the hypocretin neurons are strongly active during PS, evidenced through combined hypocretin, MCH, and Fos immunostainings in three groups of rats (PS Control, PS Deprived and PS Recovery rats). Further, we show that ICV administration of MCH induces a dose-dependent increase in PS (up to 200%) and slow wave sleep (up to 70%) quantities., Conclusion: These results indicate that MCH is a powerful hypnogenic factor. MCH neurons might play a key role in the state of PS via their widespread projections in the central nervous system.

Pregnancy and lactation provide excellent models of physiological hyperphagia and hyperprolactinemia. To identify possible factors associated with the increased feeding in these situations, we measured hypothalamic mRNA levels of three orexigenic neuropeptides--NPY, MCH, and orexins--in nonpregnant, pregnant, and lactating rats by in situ hybridization. NPY mRNA content in the arcuate nucleus was significantly increased during pregnancy and lactation. However, MCH and prepro-orexin expression was decreased in both states. 48 or 72 h of fasting in pregnant and lactating rats further elevated NPY mRNA levels and increased the low MCH mRNA content. Surprisingly, no effect was observed in prepro-orexin mRNA levels. Finally, we investigated the possible effect of high PRL levels on these orexigenic signals using a model of hyperprolactinemia induced by pituitary graft. NPY mRNA content was unchanged, but MCH and prepro-orexin mRNA levels were significantly decreased. Our results suggest that the increased NPY expression might be partly responsible for the hyperphagia observed during pregnancy and lactation. MCH and prepro-orexin may be involved in the adaptation of other homeostatic mechanisms and their decreased levels in these physiological settings could be mediated by the elevated circulating PRL levels.

In recent years, the key role of melanin-concentrating hormone (MCH) in regulating mammalian energy balance has been confirmed through several lines of evidence. When administered exogenously, MCH leads to a rapid and robust feeding response and chronic infusions result in the development of mild obesity. At the physiological level, it is known that MCH expression changes in states of altered energy balance, such as fasting and obesity. Genetic studies with mice have shown that ablation of either the gene for prepro-MCH or the gene encoding the MCH receptor leads to a lean phenotype. Finally, the administration of MCH antagonists appears to inhibit both feeding and the development of diet-induced obesity. The aim of this article is to review the recent data on MCH and MCH receptors in light of their emerging roles in energy homeostasis.

The hypothalamus has been long considered important in feeding and other motivated behaviors. The identification of neuropeptides expressed in the hypothalamus has initiated efforts to better elucidate the underlying molecular mechanisms involved. The neuropeptides orexin and melanin-concentrating hormone (MCH) are expressed in the lateral hypothalamus (LH) and have been implicated in regulation of feeding behavior. Neurons expressing these neuropeptides have extensive projections to regions of the brain important for behavioral responses to drugs of abuse, raising the possibility that the pathways may also be important in addiction. Regulation of LH intracellular signaling pathways in response to drugs of abuse supports a role for the LH neuropeptides in addiction.

Real-time Taqman RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P<0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P<0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the codon reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin secretion.

Many hypothalamic neuropeptides are involved in the regulation of energy homeostasis and feeding behavior. We have investigated whether and to what extent neuropeptide Y (NPY), agouti-related protein (AGRP), melanin-concentrating hormone (MCH), and prepro-orexin (prepro-OX) as well as pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) mRNA levels are affected in rat hypothalamus. An experimental model of long-term fasting rat characterized by three metabolic phases from changes in lipid and protein utilization was used. Except for prepro-OX and compared to fed group, starvation induced an increase in the orexigenic gene expressions that was much more marked in phase 3 (by 2.5-, 8.1-, and 13.5-fold for MCH, AGRP, and NPY, respectively) than in phase 2 (by about 1.5-2.2-fold as an average) of fasting. AGRP and NPY mRNA levels were inversely related to body fat content. Anorexigenic gene expression was only slightly affected at both fasting stages. We conclude that the regulation of NPY and AGRP gene expression is primarily involved during late fasting and could mediate the concomitant enhanced drive for refeeding.

The lateral hypothalamus (LH) is implicated in the behavioral actions of drugs of abuse, but the cellular and molecular basis of this role is unclear. Recent identification of neuropeptides localized in LH neurons has allowed for more specific studies of LH function. The LH-specific peptide orexin (hypocretin) has been shown to be important in arousal and sleep regulation. However, orexin cells of the LH project broadly throughout the brain such that orexin may influence other behaviors as well. In this study, we show that orexin neurons, and not nearby LH neurons expressing melanin-concentrating hormone (MCH), have mu-opioid receptors and respond to chronic morphine administration and opiate antagonist-precipitated morphine withdrawal. cAMP response element-mediated transcription is induced in a subset of orexin cells, but not MCH cells, after exposure to chronic morphine or induction of withdrawal. Additionally, c-Fos and the orexin gene itself are induced in orexin cells in the LH during morphine withdrawal. Finally, we show that orexin knock-out mice develop attenuated morphine dependence, as indicated by a less severe antagonist-precipitated withdrawal syndrome. Together, these studies support a role for the orexin system in molecular adaptations to morphine, and demonstrate dramatic differences in molecular responses among different populations of LH neurons.

Anatomical and functional evidence suggests that the diencephalic melanin-concentrating hormone- (MCH-) containing neurons do not form a homogeneous population. In this work, the expression of the neurokinin-3 receptor (NK3) has been researched in MCH neurons which have been retrogradely labelled following fast blue injections into either the spinal cord or the cerebral cortex. The birth-date of these cortically and spinally projecting cells has been determined using the bromodeoxyuridine method. The results obtained show that neurons projecting to the spinal cord are born early (E11) and most of them (78,7%) do not express NK3, but neurons that send axons to the cerebral cortex are born later (E12-E13) and most of them (84,8%) express NK3. Both neuronal types are largely intermingled in the lateral hypothalamic area proper. These results are discussed in terms of the functional organization of the MCH neuronal population.

Recent studies have provided compelling evidence demonstrating that orexin (also known as hypocretin) neurons play a central role in the pathophysiology of narcolepsy. However, targeted deletion of orexin does not fully mimic the functional deficits induced by selective ablation of these neurons; implying that other secreted signaling molecules expressed in these neurons mediate key aspects of their function. In this study, we demonstrate that orexin neurons display robust expression of neuronal activity-regulated pentraxin (Narp), a secreted neuronal pentraxin, implicated in regulating clustering of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Furthermore, we have found that hypothalamic melanin-concentrating hormone (MCH) neurons, which form a peptidergic pathway thought to oppose the effects of the orexin system, express another neuronal pentraxin, NP1. Thus, these findings suggest that these pathways utilize neuronal pentraxins, in addition to neuropeptides, as synaptic signaling molecules.

In this study, we have demonstrated the presence of melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptor (MCHR1) transcripts in human skin. Sequence analysis confirmed that the transcripts of both genes were identical to those previously found in human brain. In culture, endothelial cells showed pro-MCH expression whereas no signal was found in keratinocytes, melanocytes, and fibroblasts. MCHR1 expression was restricted to melanocytes and melanoma cells. Stimulation of cultured human melanocytes with MCH reduced the alpha-MSH-induced increase in cAMP production. Furthermore, the melanogenic actions of alpha-MSH were inhibited by MCH. We propose that the MCH/MCHR1 signalling system is present in human skin and may have a role with the melanocortins in regulating the melanocyte.

Agouti-related protein (AgRP) is coexpressed with neuropeptide Y (NPY) in a population of neurons in the arcuate nucleus (ARC) of the hypothalamus and stimulates food intake for up to 7 days if injected intracerebroventricularly. The prolonged food intake stimulation does not seem to depend on continued competition at the melanocortin-4 receptor (MC4R), because the relatively specific MC4R agonist MTII regains its ability to suppress food intake 24 h after AgRP injection. Intracerebroventricular AgRP also stimulates c-Fos expression 24 h after injection in several brain areas, so the neurons exhibiting delayed Fos expression might be particularly important in feeding behavior. Thus we aimed to identify the neurochemical phenotype of some of these neurons in select hypothalamic areas, using double-label immunohistochemistry. AgRP-injected rats ingested significantly more chow (10.2 +/- 0.6 g) vs. saline controls (3.4 +/- 0.7 g) in the first 9 h (light phase) after injection. In the lateral hypothalamus (particularly the perifornical area) 23 h after injection, AgRP induced significantly more Fos vs. saline in orexin-A (OXA) neurons (25.6 +/- 4.9 vs. 4.8 +/- 3.1%), but not in melanin-concentrating hormone (MCH) or cocaine- and amphetamine-regulated transcript (CART) neurons. In the ARC, AgRP induced significantly more Fos in CART (40.6 +/- 5.9 vs. 13.4 +/- 1.8%) but not NPY neurons. In the paraventricular nucleus, there was no significant difference in Fos expression induced by AgRP vs. saline in oxytocin and CART neurons. We conclude that the long-lasting hyperphagia induced by AgRP is correlated with and possibly partially mediated by hyperactive OXA neurons in the lateral hypothalamus and CART neurons in the ARC, but not by NPY and MCH neurons. The substantial increase in light-phase food intake by AgRP supports a role for the arousing effects of OXA. Activation of CART neurons in the ARC (which likely coexpress proopiomelanocortin) could indicate attempts to activate counterregulatory decreases in food intake.

Adrenal tumors, such as pheochromocytomas, are known to express various peptides and their receptors. Prolactin-releasing peptide (PrRP) is a novel neuropeptide isolated from bovine hypothalamic tissues. In the present study, expression of PrRP receptor was studied in the human brain, pituitaries, adrenal glands and tumor tissues of adrenocortical tumors, pheochromocytomas, a ganglioneuroblastoma and neuroblastomas by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. The presence of immunoreactive-PrRP in the adrenal glands and in these tumor tissues was studied by radioimmunoassay. Human brain tissues and pituitaries were obtained at autopsy. Normal portions of adrenal glands and tumor tissues were obtained at surgery. RT-PCR analysis showed expression of PrRP receptor in the human brain, pituitaries, normal portions of adrenal glands and various tumor tissues. Northern blot analysis showed high expression of PrRP receptor only in tumor tissues of pheochromocytomas, indicating that PrRP receptor expression is high in pheochromocytomas. Immunoreactive-PrRP was detected in normal portions of adrenal glands (0.162+/-0.024 pmol/g wet weight, n=4, mean+/-S.E.M.), four out of six cases of pheochromocytomas (0.050-7.9 pmol/g wet weight), one neuroblastoma and some adrenocortical tumors. The present study has shown that PrRP receptor mRNA was widely expressed in the brain tissues, pituitaries, adrenal glands and various tumors. The high expression of PrRP receptor in pheochromocytomas suggests potential pathophysiological roles of PrRP in these tumors.

Human decidua and decidualized endometrial cells produce prolactin (PRL). Several growth factors and cytokines have been shown to regulate decidual PRL release, but a specific PRL-releasing substance remains to be characterized. Prolactin-releasing peptide (PrRP) is a peptide isolated from the brain and distinguished by its potent and specific stimulation of PRL release by cultured pituitary cells. Here, we demonstrate that human decidua expresses immunoreactive PrRP as well as the mRNAs encoding PrRP and its receptor. First trimester deciduas were obtained from women undergoing elective termination of pregnancy. Tissue specimens were stained by immunohistochemistry using a rabbit anti-human PrRP-31 antibody, and PrRP was localized in both epithelial cells of the decidual glands and in stromal cells, with diffuse distribution and no special relation with the neighbourhood of blood vessels. In primary cultures of decidual stromal cells, PrRP and PrRP receptor gene expression were detected using RT-PCR, and the identity of the PCR products was further confirmed by restriction enzyme digestion. The effect of PrRP on decidual PRL release was also evaluated, and there was a significant increase in PRL production (135 +/- 4% of control levels, P < 0.05) after incubation of decidual stromal cells with synthetic PrRP. The expression of PrRP and PrRP receptor in human decidual cells and the ability of PrRP to induce PRL secretion by cultured decidual cells suggests that this peptide may be a novel local modulator of decidual PRL release.

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear. Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.

The fatty acid synthase inhibitor, C75, acts centrally to reduce food intake and body weight in mice. Here we report the effects of C75 on the expression of key orexigenic [neuropeptide Y (NPY), agouti-related protein (AgRP), and melanin-concentrating hormone] and anorexigenic [pro-opiomelanocortin (POMC) and cocaine-amphetamine-related transcript (CART)] neuropeptide messages in the hypothalami of lean and obese (ob/ob) mice. In lean mice, C75 rapidly and almost completely blocked food intake and prevented fasting-induced up-regulation of hypothalamic AgRP and NPY mRNAs, as well as down-regulation of CART and POMC mRNAs. Thus, in lean mice C75 seems to interrupt the fasting-induced signals that activate expression of NPY and AgRP and suppression of POMC and CART. In obese mice, C75 rapidly suppressed food intake, reduced body weight, and normalized obesity-associated hyperglycemia and hyperinsulinemia. Like its effect in lean mice, C75 prevented the fasting-induced increase of hypothalamic NPY and AgRP mRNAs in obese mice, but had no effect on the expression of POMC and CART mRNAs. The suppressive effect of C75 on food intake in lean mice seems to be mediated both by NPY/AgRP and POMC/CART neurons, whereas in obese mice the effect seems to be mediated primarily by NPY/AgRP neurons. In both lean and obese mice, C75 markedly increased expression of melanin-concentrating hormone and its receptor in the hypothalamus.

Several lines of investigation suggest that the hypothalamic neuropeptide melanin-concentrating hormone (MCH) regulates body weight in mammals. Obese mice lacking functional leptin overexpress the MCH message in the fed or fasted state. Acute intracerebroventricular injection of MCH increases energy intake in rats. Mice lacking the MCH gene are lean. To test the hypothesis that chronic overexpression of MCH in mice causes obesity, we produced transgenic mice that overexpress MCH (MCH-OE) in the lateral hypothalamus at approximately twofold higher levels than normal mice. On the FVB genetic background, homozygous transgenic animals fed a high-fat diet ate 10% more and were 12% heavier at 13 weeks of age than wild-type animals, and they had higher systemic leptin levels. Blood glucose levels were higher both preprandially and after an intraperitoneal glucose injection. MCH-OE animals were insulin-resistant, as demonstrated by markedly higher plasma insulin levels and a blunted response to insulin; MCH-OE animals had only a 5% decrease in blood glucose after insulin administration, compared with a 31% decrease in wild-type animals. MCH-OE animals also exhibited a twofold increase in islet size. To evaluate the contribution of genetic background to the predisposition to obesity seen in MCH-OE mice, the transgene was bred onto the C57BL/6J background. Heterozygote C57BL/6J mice expressing the transgene showed increased body weight on a standard diet, confirming that MCH overexpression can lead to obesity.

The mRNA expression of neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the UHR-1/GPR10 receptor were examined using in situ hybridization in rat peripheral tissues. In the hypophysis, modest expression of PrRP and receptor mRNA were seen in the anterior lobe. The trigeminal ganglion was devoid of expression signals. PrRP and UHR-1/GPR10 receptor mRNA:s were found in the adrenal medulla and PrRP mRNA was found in the pancreas. NPFF mRNA was detected in the spleen. In the testis and epididymis, PrRP and UHR-1/GPR10 receptor mRNA:s were detected. The results suggest a limited expression of mammalian RF-amide peptides in the peripheral organs.

We examined whether corticotropin-releasing factor (CRF) was involved in orexin-induced grooming and face-washing behaviors, and whether orexin was involved in the stress reaction. Administration of alpha-helical CRF, CRF antagonist, alone had no behavioral effect, but it blocked the orexin-induced grooming and face-washing behaviors in rats. The level of corticosterone increased in a dose-dependent manner 15 min after icv injection of orexin, and it remained high for at least 60 min. In 2-month-old rats, 1 h of immobilization stress increased orexin mRNA levels, but not the melanin-concentrating hormone (MCH) mRNA, in the lateral hypothalamic area (LHA). In 6-month-old rats, 30 min of cold stress increased the expression of orexin mRNA in the LHA. Unlike in the 2-month-old rats, immobilization stress did not change orexin mRNA expression in 6-month-old rats. These results suggest that CRF is involved in orexin-induced behaviors, and that orexin may play an important role in some stress reactions., (Copyright 2000 Academic Press.)

Using in situ hybridization, the mRNA levels encoding neuropeptide Y (NPY), agouti gene-related protein (AGRP), proopiomelanocortin (POMC), melanin-concentrating hormone (MCH) and hypocretin/orexin (HC/ORX) were investigated in the rat arcuate nucleus (Arc) and lateral hypothalamic area (LHA) 2 h after a single dose of the glucose antimetabolite 2-deoxy-D-glucose (2-DG; 600 mg/kg) or of the fatty acid oxidation inhibitor mercaptoacetate (MA; 600 mumol/kg). Two hours after 2-DG or MA injection food intake was significantly increased. NPY and AGRP mRNA levels in the Arc were increased by 2-DG but not affected by MA, and MCH mRNA levels in the LHA were increased by both antimetabolites. These results suggest that Arc neurons expressing NPY and AGRP are regulated by changes in glucose, but not fatty acid availability, whereas both factors affect MCH neurons in the LHA.

Intracranial lesions may affect hypothalamic-pituitary (HP) function and growth in several ways, depending on the location of the lesion within this area, the presence or absence of secondary hydrocephalus, and/or treatment of the lesion by surgery and/or radiotherapy. The lesion may cause a deficiency of HP hormones or, conversely, activation of the HP-gonadal axis leading to precocious puberty. Growth hormone (GH) deficiency is the most frequent endocrine abnormality that results from the lesions of the HP area. There has been progress in diagnosis, patterns of replacement therapy and the administration of biosynthetic GH in association with gonadotropin-releasing hormone analogues in precocious puberty. The major problem in these patients is the dramatic increase in their weight, which frequently occurs after surgery and increases their psychosocial and physical disabilities. It may be due to the hyperinsulinism caused by the lesion. This hyperinsulinism may be the factor that replaces GH in stimulating growth factor production and leads to normal growth in some of the patients.

Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.

The peptidergic melanin-concentrating hormone (MCH) system was investigated by immunocytochemistry in several birds. MCH perikarya were found in the periventricular hypothalamic nucleus near the paraventricular organ and in the lateral hypothalamic areas. Immunoreactive fibers were very abundant in the ventral pallidum, in the nucleus of the stria terminalis, and in the septum/diagonal band complex, where immunoreactive pericellular nets were prominent. Many fibers innervated the whole preoptic area, the lateral hypothalamic area, and the infundibular region. Some fibers also reached the dorsal thalamus and the epithalamus. The median eminence contained only sparse projections, and the posterior pituitary was not labeled. Thus, in birds, a neurohormonal role for MCH is not likely. Immunoreactive fibers were observed in other regions, such as the intercollicular nucleus, stratum griseum periventriculare (mesencephalic tectum), central gray, nigral complex (especially the ventral tegmental area), reticular areas, and raphe nuclei. Although no physiological investigation concerning the role of MCH has been performed in birds, the distribution patterns of the immunoreactive perikarya and fibers observed suggest that MCH may be involved in functions similar to those described in rats. In particular, the projections to parts of the limbic system (ventropallidal ganglia, septal complex, hypothalamus, dorsal thalamus, and epithalamus) and to structures concerned with visceral and other sensory information integration suggest that MCH acts as a neuromodulator involved in a wide variety of physiological and behavioral adaptations (arousal) with regard to feeding, drinking, and reproduction., (Copyright 1999 Wiley-Liss, Inc.)

In rats, melanin-concentrating hormone (MCH) neurons are mainly located within the lateral hypothalamic area (LHA). This area is known to be involved in the control of feeding and to contain glucose-sensitive cells. As a role for MCH in the regulation of food intake has been reported, we investigated the effects of 2-deoxyglucose (2DG) on MCH expression in cultured LHA slices, to verify if MCH neurons are sensitive to local glucoprivation through a modulation of MCH synthesis. After a 2-10 h 2DG incubation, competitive reverse transcription-polymerase chain reaction (RT-PCR) did not show any variation of MCH mRNA; no change was also observed in MCH immunocytochemical labeling. A slight decrease of MCH mRNA (5-15%) after a 17 h 2DG treatment might be due to a general degradation of neurons induced by long-term glucoprivation. In conclusion, we suggest that MCH neurons are not the glucose-sensitive cells previously described in the LHA and that the signals inducing their previously reported response to glycemia variations do not arise from the LHA itself.