Nucleus accumbens melanin-concentrating hormone signaling promotes feeding in a sex-specific manner.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide produced in the lateral hypothalamus and zona incerta that increases food intake. The neuronal pathways and behavioral mechanisms mediating the orexigenic effects of MCH are poorly understood, as is the extent to which MCH-mediated feeding outcomes are sex-dependent. Here we investigate the hypothesis that MCH-producing neurons act in the nucleus accumbens shell (ACBsh) to promote feeding behavior and motivation for palatable food in a sex-dependent manner. We utilized ACBsh MCH receptor (MCH1R)-directed pharmacology as well as a dual virus chemogenetic approach to selectively activate MCH neurons that project to the ACBsh. Results reveal that both ACBsh MCH1R activation and activating ACBsh-projecting MCH neurons increase consumption of standard chow and palatable sucrose in male rats without affecting motivated operant responding for sucrose, general activity levels, or anxiety-like behavior. In contrast, food intake was not affected in female rats by either ACBsh MCH1R activation or ACBsh-projecting MCH neuron activation. To determine a mechanism for this sexual dimorphism, we investigated whether the orexigenic effect of ACBsh MCH1R activation is reduced by endogenous estradiol signaling. In ovariectomized female rats on a cyclic regimen of either estradiol (EB) or oil vehicle, ACBsh MCH1R activation increased feeding only in oil-treated rats, suggesting that EB attenuates the ability of ACBsh MCH signaling to promote food intake. Collective results show that MCH ACBsh signaling promotes feeding in an estrogen- and sex-dependent manner, thus identifying novel neurobiological mechanisms through which MCH and female sex hormones interact to influence food intake.

Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome.

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Effect of cortisol on gonadotropin inhibitory hormone (GnIH) in the cinnamon clownfish, Amphiprion melanopus.

Hypothalamic peptides, gonadotropin-releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH), play pivotal roles in the control of reproduction and gonadal maturation in fish. In the present study we tested the possibility that stress-mediated reproductive dysfunction in teleost may involve changes in GnRH and GnIH activity. We studied expression of brain GnIH, GnIH-R, seabream GnRH (sbGnRH), as well as circulating levels of follicle stimulating hormone (FSH), and luteinizing hormone (LH) in the cinnamon clownfish, Amphiprion melanopus. Treatment with cortisol increased GnIH mRNA level, but reduced sbGnRH mRNA and circulating levels of LH and FSH in cinnamon clownfish. Using double immunofluorescence staining, we found expression of both GnIH and GnRH in the diencephalon region of cinnamon clownfish brain. These findings support the hypothesis that cortisol, an indicator of stress, affects reproduction, in part, by increasing GnIH in cinnamon clownfish which contributes to hypothalamic suppression of reproductive function in A. melanopus, a protandrous hermaphroditic fish.

Gonadotrophin-Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure, Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone.

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6)  m and 10(-8)  m) of synthetic C. dimerus (cd-) LPQRFa-1 and LPQRFa-2 on the release of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased ß-luteinising hormone (LH) and ß-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of ß-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits ß-LH and ß-FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd-LPQRF-2 could act as an ß-FSH-releasing factor in this fish species.

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.

Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry.

Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiple-charged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.

A new approach of light microscopic immunohistochemical triple-staining: combination of Fos labeling with diaminobenzidine-nickel and neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes.

OBJECTIVE: The aim of the present study was to introduce a new approach of the light microscopic immunohistochemical triple-staining enabling to study the differences in the activity of at least two different phenotypes of neurons on the same histological section. For this purpose combination of Fos (a product of the immediate early gene) labeling with nickel intensified diaminobenzidine (DAB-Ni) and two neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes on cryo-processed 35-40 µm thick free-floating brain sections was selected. METHODS: The parallel occurrence of three antibodies studied, i.e. Fos, hypocretin (HCRT), and melanin-concentrating hormone (MCH), was studied by a new methodic approach utilizing combination of Fos immunolabeled with DAB-Ni and HCRT and MCH labeled with Alexa488 and Alexa555 fluorescent dyes, respectively. Fos stimulation was induced by a single immobilization (IM0) for 120 min. Then, the rats were sacrificed, the brains removed, soaked with 30% sucrose in 0.1 M phosphate buffer (PB), cryo-sectioned throughout the hypothalamus into 35-40 µm thick coronal sections, collected, and washed in the same buffer for 10-15 min. Fos was revealed by avidin-biotin-peroxidase (ABC) complex and visualized by diaminobenzidine chromogen containing nickel chloride salt. HCRT and MCH neurons were visualized by the above mentioned fluorescent dyes. Evaluation of the Fos and fluorescent staining was performed in the computerized Axo Imager Carl Zeiss microscope using light and fluorescent illuminations. RESULTS: All the antibodies used showed clear immunoreactive staining. Fos staining occurred in the form of black color located in the cell nuclei. HCRH and MCH neuropeptides showed clear green and red fluorescence in the cell perikarya, respectively. The final merged picture showed Fos protein in the activated green HCRT or red MCH neurons in the form of white nuclei. CONCLUSIONS: The present study clearly demonstrate that the combination of Fos labeling with DAB-Ni and neuropeptides labeled with Alexa488 and Alexa555 on cryo-processed 35-40 µm thick free-floating brain sections is an excellent approach providing further advantages for quick and reproducible triple immuno-staining enabling to compare the activity of at least two phenotypes of neurons on the same section. KEYWORDS: Alexa488 and Alexa555 fluorescent dyes, Fos, hypocretin, melanin-concentrating hormone, cryostat sections, triple labeling immunohistochemistry, rat.

Hypothalamo-pituitary sarcoidosis: a multicenter study of 24 patients.

AIM: To assess clinical features, treatment and outcome of Hypothalamo-pituitary (HP) sarcoidosis and to determine whether HP is associated with a particular clinical phenotype of sarcoidosis. DESIGN: Multicentric retrospective study. METHODS: Retrospective chart review. Each patient was matched with two controls. RESULTS: Twenty-four patients were identified (10 women, 14 men). Their median age at the sarcoidosis diagnosis was 31.5 years (range: 8-69 years). HP involvement occurred in the course of a previously known sarcoidosis in 11 cases (46%), whereas it preceded the diagnosis in 13 patients (54%). All but two patients had anterior pituitary dysfunction, 12 patients presented with diabetes insipidus. The most common hormonal features were gonadotropin deficiency (n=21), TSH deficiency (n=15) and hyperprolactinemia (n=12). Magnetic Resonance Imaging (MRI) revealed infundibulum involvement (n=8), pituitary stalk thickness (n=12) and involvement of the pituitary gland (n=14). All but two patients received prednisone. After a mean follow-up of 4 years, only two patients recovered from hormonal deficiencies. MRI abnormalities improved or disappeared in 12 cases under corticosteroid. There was no correlation between the hormonal dysfunctions and the radiologic outcomes. Patients with HP sarcoidosis had significantly more frequent sinonasal localizations and neurosarcoidosis and required a systemic treatment more frequently than controls. CONCLUSION: Although HP sarcoidosis is unusual, physicians should be aware that such specific localization could be the first manifestation of sarcoidosis. HP involvement is associated with general severity of sarcoidosis. MRI abnormalities can improve or disappear under corticosteroid treatment, but most endocrine defects are irreversible.

Effects of background color on GnRH and MCH levels in the barfin flounder brain.

Effects of background color on gonadotropin-releasing hormone (GnRH) and melanin-concentrating hormone (MCH) levels in the brain of the barfin flounder Verasper moseri were monitored to investigate the interaction of GnRH and MCH in the brain. Fish were reared in white or black tanks from one month after hatching for about 7 months. MCH levels in the brain and pituitary were higher in the white tank fish. In contrast, chicken GnRH-II (cGnRH-II) levels in the brain were higher in the black tank fish. No significant differences between background colors were observed in the brain concerning salmon GnRH and seabream GnRH levels. Furthermore, six-month-old fish that had been reared in white tank were transferred to another white or black tank. Brain cGnRH-II levels were higher in black tank fish than those in white tank at 2 and 7 days after the transfer. Double-staining immunohistochemistry showed that some cGnRH-II-immunoreactive (ir) fibers were in close contact with MCH-ir cell bodies in the hypothalamus. These results indicate that background color affects not only MCH levels but also cGnRH-II levels in the brain and suggest that cGnRH-II may play a role in the regulation of MCH neural function, food intake, in the brain of the barfin flounder.

Increase of the trophoblast giant cells with prolactin-releasing peptide (PrRP) receptor expression in p53-null mice.

Trophoblast giant cells in the mouse placentas are polyploid cells that form as a result of endoreduplication. The giant cells form the outermost layer of the extraembryonic compartment and produce a number of pregnancy-specific hormones, including prolactin family members. Here we demonstrate that trophoblast giant cells are increased, and display upregulation of prolactin releasing peptide (PrRP) receptor in the p53-null (p53(-/-)) embryonic placentas. At day 13.5 of gestation, the weight of p53(-/-) placentas was less than that of both wild-type and p53(+/-) placentas. In p53(-/-) placentas, the spongiotrophoblast layer was significantly decreased in thickness, and the trophoblast giant cells were observed not only in the outer layer of placentas but in both the spongiotrophoblast layer and the labyrinthine layer. The giant cells spread over the spongiotrophoblast and labyrinthine layer in p53(-/-) placentas displayed more intensive expression of immunoreactive PrRP receptor than in wild-type placentas. Previous studies indicated that the association between PrRP and PrRP receptor physiologically involves in the expression and secretion of the peptide hormones, including prolactin and growth hormones. These results suggest that p53 may regulate the differentiation of trophoblast giant cells, and may control the physiological PrRP stimuli in mouse placentas.

Expression of molecular equivalent of hypothalamic-pituitary-adrenal axis in adult retinal pigment epithelium.

We have investigated expression of molecular elements of the hypothalamic-pituitary-adrenal (HPA) axis in the human retinal pigment epithelium (RPE) cells. The presence of corticotropin-releasing factor (CRF); urocortins I, II and III; CRF receptor type 1 (CRFR1); POMC and prohormone convertases 1 and 2 (PC1 and PC2) mRNAs were shown by RT-PCR; the protein products were detected by ELISA, western blot or immunocytochemical methods in an ARPE-19 cell line derived from an adult human donor. CRFR2 was below the level of detectability. The CRFR1 was functional as evidenced by CRF stimulation of cAMP and inositol triphosphate production as well as by ligand induction of transcriptional activity of inducible cis-elements cAMP responsive element (CRE), activator protein 1 responsive element (AP-1) and POMC promoter) in ARPE-19 using luciferase reporter assay. Immunoreactivities representative of CRF, pre-urocortin, CRFR1 receptor and ACTH were also detected in mouse retina by in situ immunocytochemistry. Finally, using RT-PCR, we detected expression of genes encoding four key enzymes participating in steroids synthesis (CYP11A1, CYP11B1, CYP17 and CYP21A2) and showed transformation of progesterone into cortisol-immunoreactivity in cultured ARPE-19 cells. Therefore, we suggest that ocular tissue expresses CRF-driven signalling system that follows organisational structure of the HPA axis.

Pre-analytic considerations for the proper assessment of hormones of the hypothalamic-pituitary axis in epidemiological research.

There is growing epidemiological interest in hormones as predictors of chronic diseases. The correct handling and analysis of hormones can be cumbersome, and great care must be taken in these processes in order to gain the most information possible. Given differences in sampling, processing, and stability of the various hormonal assays, we sought to provide a comprehensive review to aid future epidemiological research. We have coupled a thorough literature search with our own analytical experience to outline common laboratory problems one must consider in analyzing the hormones of the hypothalamic-pituitary axis. In addition, we describe the benefits and limitations of using alternative media--including urine, saliva, and blood spots on filter paper--to measure endocrine hormones in epidemiological studies.

Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals.

Successful reproduction requires maintenance of the reproductive axis within fine operating limits through negative feedback actions of sex steroids. Despite the importance of this homeostatic process, our understanding of the neural loci, pathways, and neurochemicals responsible remain incomplete. Here, we reveal a neuropeptidergic pathway that directly links gonadal steroid actions to regulation of the reproductive system. An RFamide (Arg-Phe-NH2) peptide that inhibits gonadotropin release from quail pituitary was recently identified and named gonadotropin-inhibitory hormone (GnIH). Birds are known to have specialized adaptations associated with gonadotropin-releasing hormone (GnRH) regulation to optimize reproduction (e.g., encephalic photoreceptors), and the existence of a hypothalamic peptide inhibiting gonadotropins may or may not be another such specialization. To determine whether GnIH serves as a signaling pathway for sex steroid regulation of the reproductive axis, we used immunohistochemistry and in situ hybridization to characterize the distribution and functional role of this peptide in hamsters, rats, and mice. GnIH-immunoreactive (GnIH-ir) cell bodies are clustered in the mediobasal hypothalamus with pronounced projections and terminals throughout the CNS. In vivo GnIH administration rapidly inhibits luteinizing hormone secretion. Additionally, GnIH-ir neurons form close appositions with GnRH cells, suggesting a direct means of GnRH modulation. Finally, GnIH-ir cells express estrogen receptor-alpha and exhibit robust immediate early gene expression after gonadal hormone stimulation. Taken together, the distribution of GnIH efferents to neural sites regulating reproductive behavior and neuroendocrine secretions, expression of steroid receptors in GnIH-ir nuclei, and GnIH inhibition of luteinizing hormone secretion indicate the discovery of a system regulating the mammalian reproductive axis.

Gonadotropin-inhibitory peptide in song sparrows (Melospiza melodia) in different reproductive conditions, and in house sparrows (Passer domesticus) relative to chicken-gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) regulates reproduction in all vertebrates. Until recently, an antagonistic neuropeptide for gonadotropin was unknown. The discovery of an RFamide peptide in quail that inhibits gonadotropin release in vitro raised the possibility of direct hypothalamic inhibition of gonadotropin release. This peptide has now been named gonadotropin-inhibitory hormone (GnIH). We investigated GnIH presence in the hypothalamus of two seasonally breeding songbird species, house sparrows (Passer domesticus) and song sparrows (Melospiza melodia). Using immunocytochemistry (ICC), GnIH-containing neurones were localized in both species in the paraventricular nucleus, with GnIH-containing fibres visible in multiple brain locations, including the median eminence and brainstem. Double-label ICC with light microscopy and fluorescent ICC with confocal microscopy indicate a high probability of colocalization of GnIH with GnRH neurones and fibres within the avian brain. It is plausible that GnIH could be acting at the level of the hypothalamus to regulate gonadotropin release as well as at the pituitary gland. In a photoperiod manipulation experiment, GnIH-containing neurones were larger in birds at the termination of the breeding season than at other times, consistent with a role for this neuropeptide in the regulation of seasonal breeding. We have yet to elucidate the dynamics of GnIH synthesis and release at different times of year, but the data imply temporal regulation of this peptide. In summary, GnIH has the potential to regulate gonadotropin release at more than one level, and its distribution is suggestive of multiple regulatory functions in the central nervous system.

Melanin-concentrating hormone system in the brain and skin of the cichlid fish Cichlasoma dimerus: anatomical localization, ontogeny and distribution in comparison to alpha-melanocyte-stimulating hormone-expressing cells.

Distribution and development of the melanin-concentrating hormone (MCH) system were examined by immunocytochemistry of the brain, pituitary gland and skin of the South American cichlid fish Cichlasoma dimerus. In adults, the most prominent group of MCH-ir perikarya was located in the nucleus lateralis tuberis (NLT). Outside the NLT, in the posterior hypothalamic region, a group of small neurons was found between the third ventricle and the lateral ventricular recess with delicate immunoreactive fibers that did not seem to contribute to the pituitary innervation. MCH-ir perikarya were identified at day 4 after hatching (AH) in a proliferating zone of the hypothalamic floor. Pituitary innervation could be detected at this stage. Another group of small MCH-ir neurons, only detected in pre-juvenile stages, originated close to the third ventricle in the medial hypothalamic region by day 6 AH. alphaMSH-ir neurons were localized in similar regions of the NLT and in the nucleus periventricularis posterior (NPP). Free MCH-ir neuromasts were detected in the ventral and dorsal skin of larval heads. These epidermal sensory organs were in close association with blood vessels and dermal melanocytes, suggesting that MCH synthesized in larval skin might act in an endocrine way reaching different targets and/or in a paracrine mode regulating melanin concentration in dermal melanocytes.

Prolactin-releasing peptide as a novel stress mediator in the central nervous system.

A1/A2 noradrenergic neurons in the medulla oblongata are well known to mediate stress signals in the central nervous system. Stress activates A1/A2 noradrenergic neurons, and then noradrenaline (NA) stimulates ACTH secretion through hypothalamic CRH. On the other hand, PRL-releasing peptide (PrRP) was recently isolated and was found to be produced by some A1/A2 neurons and the dorsomedial hypothalamic nucleus. We previously demonstrated that PrRP neurons make synapse-like contact with hypothalamic CRH neurons. In fact, we demonstrated that the central administration of PrRP stimulates CRH-mediated ACTH secretion. Furthermore, it has been reported that PrRP neurons in A1/A2 cell groups are colocalized with tyrosine hydroxylase (TH), which is known as the marker enzyme of catecholaminergic neurons. These data strongly suggest that PrRP is related to stress-responsive signal transduction, and PrRP and NA cooperatively modulate the hypothalamo-pituitary-adrenal axis. We therefore examined the effect of water immersion-restraint stress on c-Fos protein accumulation in PrRP- and TH-immunoreactive neurons. The synergistic effects of PrRP and NA on plasma ACTH elevation were also examined. The results clearly showed that c-Fos protein accumulation dramatically increased in the nuclei of A1/A2 and dorsomedial hypothalamic nucleus PrRP neurons. In addition, it was revealed that c-Fos protein was specifically expressed in the PrRP/TH double positive cells in the A1/A2 cell groups. We also demonstrated that the central administration of PrRP and NA in combination at subactive (noneffective) doses clearly induced plasma ACTH elevation. Here we report that PrRP is a novel and important mediator of the hypothalamo-pituitary-adrenal axis for the stress response.

Differential neuronal expression and projections of melanin-concentrating hormone (MCH) and MCH-gene-overprinted-polypeptide (MGOP) in the rat brain.

The rat melanin-concentrating hormone (MCH) gene may produce, through alternative splicing, either the precursor of MCH and neuropeptide EI, two neuropeptides coexpressed in the zona incerta (ZI) and lateral hypothalamus (LHA), or a putative protein we named previously MCH-gene-overprinted-polypeptide (MGOP). First, we investigated the distribution and relative expression of MCH and MGOP mRNA in the rat brain by Northern blotting, RT-PCR and in situ hybridization. MGOP gene transcripts were detected mainly in the hypothalamus only by RT-PCR. Second, different antisera were raised toward the C-terminus of MGOP and used to identify the translational products. In the rat brain, no MGOP-processed peptide could be detected based on RP-HPLC coupled to specific RIA. A polypeptide of 14 kDa was found in the secretory pathway of transfected monkey COS7 cells expressing recombinant MGOP. In the rat hypothalamus, a specific protein of 12 kDa was identified by Western blot analysis. Finally, distribution of MGOP-immunoreactivity (IR) was investigated in the rat brain. Colocalization studies demonstrated that 98% of the MGOP-expressing perikarya in ZI/LHA also synthesized MCH. In addition, numerous, strongly stained MGOP-containing neurons were encountered in the hypothalamic periventricular nucleus. Perikarya labelled with MGOP antiserum were also found scattered in the cortex, caudate putamen, amygdala and lateral septal nucleus. MCH was not detected in these MGOP-containing neurons. Strikingly, dense staining of terminals was observed with MGOP antiserum but not with MCH antibodies in the suprachiasmatic, ventromedial and arcuate nuclei, and also in the external layer of the median eminence. These results demonstrated that MGOP and MCH-IR overlapped in LHA/ZI but displayed a differential distribution in other areas. Based on this cerebral distribution, MGOP may act as a new secreted protein in regulating many neuroendocrine functions, such as nursing, feeding and growth control in associated behavioural components.