Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Melanin-concentrating hormone peptidergic system: Comparative morphology between muroid species.

Melanin-concentrating hormone (MCH) is a conserved neuropeptide, predominantly located in the diencephalon of vertebrates, and associated with a wide range of functions. While functional studies have focused on the use of the traditional mouse laboratory model, critical gaps exist in our understanding of the morphology of the MCH system in this species. Even less is known about the nontraditional animal model Neotomodon alstoni (Mexican volcano mouse). A comparative morphological study among these rodents may, therefore, contribute to a better understanding of the evolution of the MCH peptidergic system. To this end, we employed diverse immunohistochemical protocols to identify key aspects of the MCH system, including its spatial relationship to another neurochemical population of the tuberal hypothalamus, the orexins. Three-dimensional (3D) reconstructions were also employed to convey a better sense of spatial distribution to these neurons. Our results show that the distribution of MCH neurons in all rodents studied follows a basic plan, but individual characteristics are found for each species, such as the preeminence of a periventricular group only in the rat, the lack of posterior groups in the mouse, and the extensive presence of MCH neurons in the anterior hypothalamic area of Neotomodon. Taken together, these data suggest a strong anatomical substrate for previously described functions of the MCH system, and that particular neurochemical and morphological features may have been determinant to species-specific phenotypes in rodent evolution.

Feeding state and age dependent changes in melanin-concentrating hormone expression in the hypothalamus of broiler chickens.

We aimed to quantify the gene expression changes of the potent orexigenic melanin-concentrating hormone (MCH) in chicken (Gallus gallus) hypothalamus with quantitative real-time polymerase chain reaction (qPCR), and for the first time determine peptide concentrations with a novel radioimmunoassay (RIA) under different feeding status. Three different experimental conditions, namely ad libitum feeding; fasting for 24 h; fasting for 24 h and then refeeding for 2 h, were applied to study changes of the aforementioned target and its receptor (MCHR4) gene expression under different nutritional status. The relative changes of MCH and MCHR4 were also studied from 7 to 35 days of age. Expression of PMCH and MCHR4 along the gastrointestinal tract (GIT) was also investigated. We found that expression of both targets was significant in the hypothalamus, while only weak expression was detected along the GIT. Different nutritional states did not affect the PMCH and MCHR4 mRNA levels. However, fasting for 24 h had significantly increased the MCH-like immunoreactivity by 25.65%. Fasting for 24 h and then refeeding for 2 h had further significantly increased the MCH peptide concentration by 32.51%, as compared to the ad libitum state. A decreasing trend with age was observable for both, the PMCH and MCHR4 mRNA levels, and also for the MCH-like immunoreactivity. Correlation analysis did not result in a significant correlation between MCH peptide concentration and abdominal fat mass in ad libitum fed birds. In conclusion, MCH peptide concentration altered in response to 24 h fasting, which indicated that this peptide may take part in feed intake regulation of broiler chickens.

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.

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.

Melanin concentrating hormone in central hypersomnia.

BACKGROUND: Narcolepsy with cataplexy (NC) is a disabling disorder characterized by excessive daytime sleepiness and abnormal rapid eye movement (REM) sleep manifestations, due to a deficient hypocretin/orexin neurotransmission. Melanin concentrating hormone (MCH) neurons involved in the homeostatic regulation of REM sleep are intact. We hypothesized that an increased release of MCH in NC would be partly responsible for the abnormal REM sleep manifestations. METHODS: Twenty-two untreated patients affected with central hypersomnia were included: 14 NC, six idiopathic hypersomnia with long sleep time, and two post-traumatic hypersomnia. Fourteen neurological patients without any sleep disorders were included as controls. Using radioimmunoassays, we measured hypocretin-1 and MCH levels in cerebrospinal fluid (CSF). RESULTS: The MCH level was slightly but significantly lower in patients with hypersomnia (98 ± 32 pg/ml) compared to controls (118 ± 20 pg/ml). After exclusion of patients affected with post-traumatic hypersomnia the difference became non-significant. We also failed to find any association between MCH level and hypocretin level, the severity of daytime sleepiness, the number of SOREMPs, the frequency of cataplexy, and the presence of hypnagogic hallucinations or sleep paralysis. CONCLUSION: This study reports the first measurement of MCH in CSF using radioimmunoassay technology. It appears to be a non-informative tool to differentiate etiologies of central hypersomnia with or without REM sleep dysregulation.

Hormones in the mentally disturbed brain: steroids and peptides in the development and treatment of psychopathology.

One of the more fascinating recent discoveries in neuroscience is the widespread influence of hormones on brain regions and functions underlying pathological behaviors. A story is unfolding that points to critical roles played by hypothalamic - pituitary - gonadal (HPG) and hypothalamic - pituitary - adrenal (HPA) axes on a startling array of mental disorders, from depression to dementia. The influence of peptides and steroids does not end with hormones released from the two axes, however. It is now clear that the brain has adapted, "highjacked" is more descriptive, HPG and HPA hormones for uses unrelated to their original functions in reproduction and responses to stress. Findings of neuromodulatory effects of HPA and HPG hormones on monoamine, GABA, glutamate and opioid pathways and of hormone receptors and enzymes involved in hormone synthesis, particularly of steroids, in the hippocampus, amygdala and other subcortical brain regions provide the brain with multiple evolutionary means to adapt to new functions. The complexity of the metabolic cascade for the steroids also leaves open mechanisms by which endogenous errors and exogenous chemicals could be involved in the etiology of psychopathologies. The planned review will examine the recent literature for evidence of steroidal and peptidergic influences on basic biological functions and on mood disorders, anxiety and PTSD, schizophrenia, substance abuse and dementia. Emphasis will be placed on animal models, although findings with patient populations will be prominently included. Special attention will be paid to novel pathways by which the precursors and metabolites of sex steroids can influence psychopathologies. We also will speculate on promising treatments with hormone modulators that may be useful in mollifying the symptomology of the mental disorders.

Hypocretin (orexin) cell loss in Parkinson's disease.

It has recently been reported that Parkinson's disease (PD) is preceded and accompanied by daytime sleep attacks, nocturnal insomnia, REM sleep behaviour disorder, hallucinations and depression, symptoms which are frequently as troublesome as the motor symptoms of PD. All these symptoms are present in narcolepsy, which is linked to a selective loss of hypocretin (Hcrt) neurons. In this study, the Hcrt system was examined to determine if Hcrt cells are damaged in PD. The hypothalamus of 11 PD (mean age 79 +/- 4) and 5 normal (mean age 77 +/- 3) brains was examined. Sections were immunostained for Hcrt-1, melanin concentrating hormone (MCH) and alpha synuclein and glial fibrillary acidic protein (GFAP). The substantia nigra of 10 PD brains and 7 normal brains were used for a study of neuromelanin pigmented cell loss. The severity of PD was assessed using the Hoehn and Yahr scale and the level of neuropathology was assessed using the Braak staging criteria. Cell number, distribution and size were determined with stereologic techniques on a one in eight series. We found an increasing loss of hypocretin cells with disease progression. Similarly, there was an increased loss of MCH cells with disease severity. Hcrt and MCH cells were lost throughout the anterior to posterior extent of their hypothalamic distributions. The percentage loss of Hcrt cells was minimal in stage I (23%) and was maximal in stage V (62%). Similarly, the percentage loss of MCH cells was lowest in stage I (12%) and was highest in stage V (74%). There was a significant increase (P = 0.0006, t = 4.25, df = 15) in the size of neuromelanin containing cells in PD patients, but no difference in the size of surviving Hcrt (P = 0.18, t = 1.39, df = 14) and MCH (P = 0.28, t = 1.39, df = 14) cells relative to controls. In summary, we found that PD is characterized by a massive loss of Hcrt neurons. Thus, the loss of Hcrt cells may be a cause of the narcolepsy-like symptoms of PD and may be ameliorated by treatments aimed at reversing the Hcrt deficit. We also saw a substantial loss of hypothalamic MCH neurons. The losses of Hcrt and MCH neurons are significantly correlated with the clinical stage of PD, not disease duration, whereas the loss of neuromelanin cells is significantly correlated only with disease duration. The significant correlations that we found between the loss of Hcrt and MCH neurons and the clinical stage of PD, in contrast to the lack of a relationship of similar strength between loss of neuromelanin containing cells and the clinical symptoms of PD, suggests a previously unappreciated relationship between hypothalamic dysfunction and the time course of the overall clinical picture of PD.

Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon.

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH(2) from the goldfish brain. In this study, to clarify the physiological functions of these three LPXRFamide peptides (gfLPXRFa-1, -2, and -3), we analysed the localisation and hypophysiotrophic activity of these peptides using sockeye salmon, Oncorhynchus nerka, in which immunoassay systems for several anterior pituitary hormones have been developed. gfLPXRFa-immunoreactive cell bodies were detected in the nucleus posterioris periventricularis of the hypothalamus and immunoreactive fibres were distributed in various brain regions and the pituitary. We also detected gfLPXRFa-immunoreactivity in the pituitary by competitive enzyme-linked immunosorbent assay combined with reversed-phase HPLC. These three gfLPXRFamide peptides stimulated the release of FSH, LH and GH, but did not affect the release of prolactin (PRL) and somatolactin (SL) from cultured pituitary cells. These results suggest that novel fish hypothalamic LPXR-Famide peptides exist in the brain and pituitary of sockeye salmon and stimulate the release of gonadotrophins and GH from the pituitary.

Late-onset leanness in mice with targeted ablation of melanin concentrating hormone neurons.

The observation that loss of orexin (hypocretin) neurons causes human narcolepsy raises the possibility that other acquired disorders might also result from loss of hypothalamic neurons. To test this possibility for body weight, mice with selective loss of melanin concentrating hormone (MCH) neurons were generated. MCH was chosen to test because induced mutations of the MCH gene in mice cause hypophagia and leanness. Mice with ablation of MCH neurons were generated using toxin (ataxin-3)-mediated ablation strategy. The mice appeared normal but, after 7 weeks, developed reduced body weight, body length, fat mass, lean mass, and leptin levels. Leanness was characterized by hypophagia and increased energy expenditure. To study the role of MCH neurons on obesity secondary to leptin deficiency, we generated mice deficient in both ob gene product (leptin) and MCH neurons. Absence of MCH neurons in ob/ob mice improved obesity, diabetes, and hepatic steatosis, suggesting that MCH neurons are important mediators of the response to leptin deficiency. These data show that loss of MCH neurons can lead to an acquired leanness. This has implications for the pathogenesis of acquired changes of body weight and might be considered in clinical settings characterized by substantial weight changes later in life.

Sensitivity of galanin- and melanin-concentrating hormone-containing neurones to nutritional status: an immunohistochemical study in the ovariectomized ewe.

The sensitivities of galanin and melanin-concentrating hormone (MCH) neuronal systems to nutrition are poorly understood in sheep compared to rodents. The aim of this study was to describe the changes in the numbers of galanin and MCH neurones in ovariectomized ewes submitted to different nutritional levels. In the first experiment, ewes were fed ad libitum or food deprived for 24 h. In the second experiment, two groups of ewes were fed at maintenance level (group 100) or undernourished (group 40) for 167 days, after which one-half of each group was killed or refed ad libitum (group 100R and 40R) for 4 days. The MCH neuronal population located in the lateral hypothalamic area was not affected by these nutritional changes. Long-term undernutrition enhanced the number of galanin neurones located in the infundibular nucleus and the dorsal hypothalamic area (DHA), refeeding resulted in an increase of neurones in the DHA and preoptic area, but short-term starvation had no effect on any galanin subpopulations. Our data suggest that the sensitivity of MCH neuronal populations to nutrition in sheep differs from that of rodents. Various populations of galanin-containing neurones differ in sensitivity in ewes subjected to long undernutrition and refeeding but not to short starvation.

Immunocytochemical localization and ontogenic development of melanin-concentrating hormone in the brain of a pleuronectiform fish, the barfin flounder.

Melanin-concentrating hormone (MCH) was first discovered in the pituitary of chum salmon because of its role in the regulation of skin pallor. Later, it was found that MCH could also play a role as a central neurotransmitter or neuromodulator in the brain. However, knowledge of the function of MCH in fish has been restricted to certain fish species. Therefore, in the present study, the immunocytochemical localization and ontogenic development of MCH in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, were examined to obtain a better understanding of this hormone. In adult barfin flounder, MCH-immunoreactive (ir) neuronal somata were most prevalent in the magnocellular neurons of the nucleus tuberis lateralis (NLT), which project to the pituitary. In the pituitary, MCH-ir fibers were distributed in the neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis. MCH-ir neuronal somata were also present in dorsally projecting parvocellular neurons, located more posteriorly in the area above the lateral ventricular recess (LVR). LVR-MCH neurons did not seem to project to the pituitary. In the brain, MCH-ir fibers were detected not only in the hypothalamus but also in areas such as the optic tectum and thalamus. MCH-ir neuronal somata and fibers were not detected on the day of hatching. MCH-ir neuronal somata and fibers were first detected in the hypothalamus and the pituitary, respectively, 7 days after hatching. Subsequently, MCH-ir neuronal somata were observed in the NLT and in the area above the LVR 14 days after hatching. The distribution of MCH-ir neuronal somata and fibers showed a pattern similar to that in the adult fish 35-42 days after hatching. These results indicate that MCH neurons were located in the NLT and in the area above the LVR and that NLT-MCH neurons project to the pituitary. MCH neurons were first detected 7 days after hatching, suggesting that MCH plays some physiological role in the early development of barfin flounder.

The distribution of melanin-concentrating hormone in the lamprey brain.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems.

The suprachiasmatic nucleus (SCN) temporally organizes behavior in part by sustaining arousal during the wake period of the sleep/wake cycle to consolidate adaptive waking behavior. In this study, we demonstrate direct projections from the SCN, in both the rat and the human brains, to perikarya and proximal dendrites of two groups of posterior hypothalamic neurons with axonal projections that suggest they are important in the regulation of arousal, one producing hypocretins (HCT) and the other melanin-concentrating hormone (MCH). In addition, we demonstrate that both HCT and MCH-producing neurons are immunoreactive for glutamate (GLU). These observations support the hypothesis that direct projections from the SCN to the posterior hypothalamus mediate the arousal function of the circadian timing system.

Leptin receptor- and STAT3-immunoreactivities in hypocretin/orexin neurones of the lateral hypothalamus.

Hypocretins/orexins are recently characterized peptides that are synthesized in neurones of the lateral hypohalamus and stimulate food intake in rats. To clarify whether leptin may interact with hypocretin/orexin to reduce ingestive behaviour, the presence of leptin receptor-immunoreactivity in hypocretin/orexin-containing neurones was examined. Many leptin receptor-and hypocretin/orexin-immunoreactive neurones were demonstrated in the lateral hypothalamic area and perifornical region. Both direct double-labelling and elution-restaining methods showed that leptin receptor-immunoreactivity was present in the vast majority of hypocretin/orexin-containing neurones. Immunoreactivity for STAT3, a transcription factor activated by leptin, was also demonstrated in hypocretin/orexin-containing neurones. Isolated hypocretin/orexin cell bodies in the dorsal part of the lateral hypothalamic area and the ventral perifornical region were shown to contain immunoreactivity for galanin, another peptide known to affect feeding. Galanin neurones were also seen to contain leptin receptor-and STAT3-immunoreactivity. Melanin-concentrating hormone (MCH)-containing neurones constituted a cell population within the lateral hypothalamus distinct from the one containing hypocretin/orexin-immunoreactivity, as shown by elution-restaining methodology. The presence of leptin receptor-and STAT3-immunoreactivities in hypocretin/orexin-containing neurones of the lateral hypothalamus suggests that leptin may directly regulate these hypothalamic neurones, most likely via an inhibitory action on hypocretin/orexin expression and/or secretion resulting in reduced food intake.

Survival of rat MCH (melanin-concentrating hormone) neurons in hypothalamus slice culture: histological, pharmacological and molecular studies.

Hypothalamic slices containing the lateral hypothalamic area (LHA) were prepared from 6- to 8-day-old rats and maintained in stationary culture for up to 35 days in order to analyse how well the melanin-concentrating hormone (MCH) neurons survived. As previously reported for other brain areas, this method yielded a long-term well-preserved organotypic organization. Light- and electron-microscopic investigations showed that differentiation continued and that synaptic contacts developed in vitro. After a period of elimination of damaged cells and fibres, most of the remaining neurons and glial cells retained a normal morphology throughout the culture period. MCH neurons, in particular, survived well as attested by the strong immunocytochemical and in situ hybridization signals still observed after several weeks. In a comparison with the day of explantation, competitive reverse transcription/polymerase chain reaction demonstrated the remarkable stability of the level of MCH mRNA at least until the 20th day in culture; after 30 days, the clear decrease in this level seemed to be correlated with a loss of MCH neurons, rather than with a decrease in MCH expression. After 10 days of culture, the incubation of slices in the presence of the hormone leptin (50 ng/ml) resulted in a strong decrease of MCH gene expression, suggesting that MCH neurons retained their physiological properties. Thus, the LHA slice stationary culture, especially between one and three weeks (i.e. after tissue stabilization and before extensive cell loss), appears to be a suitable method for physiological and pharmacological studies of these neurons.

Attenuation of the polypeptide 7B2, prohormone convertase PC2, and vasopressin in the hypothalamus of some Prader-Willi patients: indications for a processing defect.

7B2 is a neuroendocrine chaperone interacting with the prohormone convertase PC2 in the regulated secretory pathway. Its gene is located near the Prader-Willi syndrome (PWS) region on chromosome 15. In a previous study we were able to show 7B2 immunoreactivity in the supraoptic nucleus (SON) or the paraventricular nucleus (PVN) in only three of five PWS patients. Here we report that in contrast with five other PWS patients, the neurons in the hypothalamic SON and PVN of the two 7B2-immunonegative PWS patients also failed to show any reaction using two antibodies directed against processed vasopressin (VP). On the other hand, even these two cases reacted normally with five antibodies that recognize different parts of the VP precursor. This finding pointed to a processing defect. Indeed, the same patients had no PC2 immunoreactivity in the SON or PVN, whereas PC1 immunoreactivity was only slightly diminished. In conclusion, in the VP neurons of two PWS patients, greatly reduced amounts of 7B2 and PC2 are present, resulting in diminished VP precursor processing.

Distribution of melanin-concentrating hormone (MCH)-like immunoreactivity in neurons of the diencephalon of sheep.

An immunohistochemical study with an antiserum raised against salmon melanin concentrating-hormone has demonstrated the presence of numerous melanin concentrating-hormone-immunoreactive neurons in the lateral hypothalamic areas of the sheep. The pattern of distribution of these perikarya is similar to that of rodents and primates. In sheep, however, melanin concentrating-hormone-immunoreactive neurons appeared to form two gatherings: the first is situated ventromedially to the internal capsule and the second in the dorsolateral hypothalamus. In these areas, numerous immunostained perikarya are observed. Compared to the rats, labelled neurons extended more caudally in the ventral tegmental area and more rostrally above the optic chiasma. Compared to primates, these neurons are less numerous in the periventricular area. In our study, dense networks of melanin concentrating-hormone-immunoreactive varicose fibers were observed in the supramamillary nucleus, the lateral hypothalamus, the nucleus medialis thalami and nucleus reuniens and in the bed nucleus of the stria terminalis.

Simultaneous identification of a specific gene protein product and transcript using combined immunocytochemistry and in situ hybridization with non-radioactive probes.

Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory since 1983. The first detection system uses biotinylated cRNA probes or oligoprobes in in situ hybridization along with antisera to biotin to detect the hybrid. The detection system is amplified by 2-3 layers of anti-biotin, second antibody (made against the anti-biotin) and streptavidin conjugated to horseradish peroxidase. After the mRNA is detected with a blue-black substrate (nickel intensified diaminobenzidine), the antigens are detected with immunoperoxidase techniques and orange-amber substrate. The in situ hybridization protocol can also be used at the electron microscopic level. Trouble shooting and control protocols are also described. This approach has been shown to be valuable for detection of pituitary hormones, growth factors mRNAs and antigens.

Identification, cellular localization and in vitro release of a novel teleost melanin-concentrating hormone gene-related peptide.

The melanin-concentrating hormone (MCH) precursor encodes MCH and a second peptide named neuropeptide EI (NEI) in mammals, neuropeptide EV (NEV) in salmonids and MCH gene-related peptide (Mgrp) in other fish. The primary structure of the putative Mgrp of the cichlid fish tilapia (Oreochromis mossambicus) appears to be very different from mammalian NEI and salmonid NEV. To investigate the processing and release of tilapia Mgrp (tMgrp), in the present study an antiserum was raised against synthetic tMgrp. By immunocytochemistry, tMgrp immunoreactivity was colocated with MCH immunoreactivity in the tilapia hypothalamus and pituitary. In addition, a tMgrp enzyme-linked immunosorbent assay in combination with reversed phase HPLC was used to demonstrate the presence of processed tMgrp in tilapia hypothalamus and pituitary. The release of tMgrp from neuro-intermediate lobes (NILs) of tilapia pituitaries was demonstrated after in vivo incubation of chopped NILs. Depolarizing concentrations of potassium significantly stimulated tMgrp release. Six weeks of adaptation of tilapia to white or black backgrounds had no effect on in vitro tMgrp release or on the tMgrp content of NIL and hypothalamus. Tilapia Mgrp, unlike MCH, had no effect on tilapia scale melanophores, nor did it modulate the melanin-concentrating effect of MCH. We conclude that tMgrp is processed from the MCH preprohormone, that it is released in vitro, and that the peptide has no direct role in the melanin concentration of fish scale melanophores. Therefore a neuroendocrine or neuromodulatory function is proposed for tMgrp.