Dynamic Regulation of Hypothalamic DMXL2, KISS1, and RFRP Expression During Postnatal Development in Non-Human Primates.

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.

Distribution of LPXRFa, a gonadotropin-inhibitory hormone ortholog peptide, and LPXRFa receptor in the brain and pituitary of the tilapia.

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.

Suckling-induced Fos activation and melanin-concentrating hormone immunoreactivity during late lactation.

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.

Glucocorticoids are required for meal-induced changes in the expression of hypothalamic neuropeptides.

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.

Sleep alterations in an environmental neurotoxin-induced model of parkinsonism.

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.

Isolation of tumour stem-like cells from benign tumours.

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.

Glucagon-like peptide 1 excites hypocretin/orexin neurons by direct and indirect mechanisms: implications for viscera-mediated arousal.

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 hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism.

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.

A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep.

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.

Hypothalamic levels of NPY, MCH, and prepro-orexin mRNA during pregnancy and lactation in the rat: role of prolactin.

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.

Involvement of the lateral hypothalamic peptide orexin in morphine dependence and withdrawal.

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.

Melanin-concentrating hormone and its receptor are expressed and functional in human skin.

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.

Expression of prolactin-releasing peptide and its receptor in the human adrenal glands and tumor tissues of adrenocortical tumors, pheochromocytomas and neuroblastomas.

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.

Neurochemical phenotype of hypothalamic neurons showing Fos expression 23 h after intracranial AgRP.

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.

Hypothalamic-pituitary function and growth in children with intracranial lesions.

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.

17beta-estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study.

Melanin-concentrating hormone (MCH) and neuropeptide-E-I (NEI) regulate several behaviors and neuroendocrine functions in rats. Possible influence of these peptides on sexual behavior and reproduction in mammals other than rodents prompted us to investigate: 1) The sites of synthesis of MCH and NEI in the brain of a non-human primate (M. fascicularis); 2) The effect of 17 beta-estradiol (E2) benzoate (E2B) on pro-MCH-derived peptide concentrations in the hypothalamus of the ovariectomized (OVX) cynomolgus monkeys (M. fascicularis). Expression of MCH mRNA and peptides was examined by Northern blotting, RT-PCR and RP-HPLC/RIA. Our results demonstrate that the MCH gene is predominantly expressed in hypothalamus of macaque. E2B exposure of OVX monkeys provoked parallel phasic variations in the MCH-immunoreactivity (IR) and NEI-IR. NEI-IR and to a lesser extent MCH-IR, showed a transient increase (associated with the estradiol peak) at 30 h with a final rise of both MCH-IR and NEI-IR observed at the time (72 h post E2B) of the luteinizing hormone (LH) surge. RP-HPLC analysis of peptide extracts revealed the presence, in addition to mature MCH and NEI, of different MCH-IR and NEI-IR forms in the hypothalami of control and E2B-treated monkeys. Taken together, our results indicated that hypothalamic MCH and NEI contents are regulated after E2B treatment and they suggest the possible involvement of these peptides in the regulation of the pre-ovulatory midcycle LH surge in primates.

Synthesis of new peptides with prolactin-releasing activity by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction.

A newly isolated peptide from bovine hypothalamus with prolactin-releasing activity (prolactin-releasing peptide; PrRP) was synthesized by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction, together with rat and human homologs. The peptides were expressed in the form of fusion proteins with basic fibroblast growth factor mutein, which were purified by heparin-affinity chromatography. The fusion proteins were cleaved at the cysteine residues of the junction site by cyanylation, followed by treatment with ammonia for C-terminal amidation. Purification of the resulting crude peptides was performed using chromatography on a gel-filtration column, a cation-exchange column, and a reversed-phase column. As an example, about 90 mg of bovine PrRP (bPrRP) was obtained from 201 of culture bloth. The purified b PrRP showed full biological activities in binding to its receptor expressed on CHO cells and releasing arachidonic acid metabolite from the same cells, while the C-terminal acid form of bPrRP had little of these activities. These results indicate that the C-terminal amide structure is very important for expressing biological activity. The peptides obtained here might be very useful for studies on their biological significance and roles in vivo.

Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide involved in the regulation of food-intake behaviour and stress response in mammals. Expression of the MCH gene predominates in hypothalamic neurons. Mechanisms governing the regulation of expression of MCH gene in established cell lines were not explored yet. Here, we analysed the actions of nerve growth factor (NGF), dexamethasone, forskolin and lithium on MCH mRNA levels in the PC12 pheochromocytoma cell line. We compared them with those observed on tyrosine hydroxylase (TH) mRNA, constitutively expressed in PC12 cells, and neurotensin (NT) mRNA, taken as a control. In untreated cells, MCH RNA species of high molecular weight were found. Exposure of cells at a combination of NGF and lithium resulted in decreased expression of these MCH RNAs and in the transient production of mature MCH mRNA. Strikingly, after short exposure of PC12 cells to NGF, lithium per se elicited a marked increase in MCH mRNA levels whilst it exerted a potent inhibitory action on TH mRNA expression. Detailed investigations revealed that lithium enhanced MCH mRNA expression through post-transcriptional mechanisms whereas it regulated TH gene expression mainly at the level of transcription. These results demonstrate that lithium, an agent widely used for treatment of manic depressive illness, can exert an opposite effect on MCH and TH mRNA production in PC12 cells. The MCH gene system in NGF-treated PC12 cells provides a good opportunity for studying the effect of lithium on gene expression at post-transcriptional levels in a neuron-like cellular model.

[Interactions of neuromediators and neurohormones on dendritic cells, monocytes and macrophages (out of the central nervous system)]. / Interactions des neuromédiateurs et neurohormones sur les cellules dendritiques, les monocytes et les macrophages (hors système nerveux central).

Immune cells are modulated by neurotransmitters and hormones. Apart Langerhans cells, studies about dendritic cells and these peptides are very rare. But their effects on monocytes or macrophages are known. Substance P, VIP, CGRP, prolactin, ACTH are among the most important. These effects are supported by an anatomical reality: connexions between nerve and immune cells. Immune cells are capable to product neuromediators and hormones. Neuroimmunology is probably the next great subject of research about dendritic cells.

Insulin treatment stimulates the rat melanin-concentrating hormone-producing neurons.

Melanin-concentrating hormone (MCH) is involved in the regulation of body colour in teleost fish. A peptide highly homologous to salmon MCH has been found in the rat brain, but its physiological functions have not yet been precisely defined. The location of MCH neurons in the lateral hypothalamus (LHT) of the rat suggests possible implication in feeding behaviour. In the present study, immunohistochemical and in situ hybridization methods were used to investigate MCH gene expression following insulin injections. Five hours after insulin injection, a significant increase in the abundance and staining intensity of MCH immunoreactive perikarya and fibres was observed. Concurrently the level of MCH mRNA significantly increased (50%). Insulin-treatment also induced a marked and progressive increase in the number and staining intensity of nuclei detected by a Fos antiserum in LHT and other brain areas. Double labelling technique demonstrated that very few if any MCH neurons exhibited Fos-like immunoreactivity. These results demonstrate that an insulin-treatment stimulates MCH neuron activity without the mediation of the proto-oncogene c-fos. The mechanisms triggering this activation remain to be elucidated.