Melanin-concentrating Hormone Immunoreactivity Research Articles

The Melanin-Concentrating Hormone (MCH) System: A Tale of Two Peptides.

The melanin-concentrating hormone (MCH) system is a robust integrator of exogenous and endogenous information, modulating arousal and energy balance in mammals. Its predominant function in teleosts, however, is to concentrate melanin in the scales, contributing to the adaptive color change observed in several teleost species. These contrasting functions resulted from a gene duplication that occurred after the teleost divergence, which resulted in the generation of two MCH-coding genes in this clade, which acquired distinctive sequences, distribution, and functions, examined in detail here. We also describe the distribution of MCH immunoreactivity and gene expression in a large number of species, in an attempt to identify its core elements. While initially originated as a periventricular peptide, with an intimate relationship with the third ventricle, multiple events of lateral migration occurred during evolution, making the ventrolateral and dorsolateral hypothalamus the predominant sites of MCH in teleosts and mammals, respectively. Substantial differences between species can be identified, likely reflecting differences in habitat and behavior. This observation aligns well with the idea that MCH is a major integrator of internal and external information, ensuring an appropriate response to ensure the organism’s homeostasis. New studies on the MCH system in species that have not yet been investigated will help us understand more precisely how these habitat changes are connected to the hypothalamic neurochemical circuits, paving the way to new intervention strategies that may be used with pharmacological purposes.

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

AimsMelanin-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 methodsFemale 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 findingsSuckling 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. SignificanceSuckling 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.

Melanin-concentrating hormone (MCH) immunoreactivity in non-neuronal cells within the raphe nuclei and subventricular region of the brainstem of the cat

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized within the postero-lateral hypothalamus and zona incerta. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes such as energy homeostasis and sleep. In the present report, we examined the distribution of MCH immunoreactivity in the brainstem of the cat. In addition to MCH+ axons, we found MCH-immunoreactive cells that have not been previously described either in the midbrain raphe nuclei or in the periaqueductal and periventricular areas. These MCH+ cells constituted: 1. ependymal cells that lined the fourth ventricle and aqueduct, 2. ependymal cells with long basal processes that projected deeply into the subventricular (subaqueductal) parenchyma, and, 3. cells in subventricular regions and the midbrain raphe nuclei. The MCH+ cells in the midbrain raphe nuclei were closely related to neuronal processes of serotonergic neurons. Utilizing Neu-N and GFAP immunohistochemistry we determined that the preceding MCH+ cells were neither neurons nor astrocytes. However, we found that vimentin, an intermediate-filament protein that is used as a marker for tanycytes, was specifically co-localized with MCH in these cells. We conclude that MCH is present in tanycytes whose processes innervate the midbrain raphe nuclei and adjacent subependymal regions. Because tanycytes are specialized cells that transport substances from the cerebrospinal fluid (CSF) to neural parenchyma, we suggest that MCH is absorbed from the CSF by tanycytes and subsequently liberate to act upon neurons of brainstem nuclei.

Melanin-concentrating hormone projections to areas involved in somatomotor responses

The lateral hypothalamic area (LHA) participates in the integration of sensory information and somatomotor responses associated with hunger and thirst. Although the LHA is neurochemically heterogeneous, a particularly high number of cells express melanin-concentrating hormone (MCH), which has been reported to play a role in energy homeostasis. Treatment with MCH increases food intake, and MCH mRNA is overexpressed in leptin-deficient ( ob/ ob) mice. Mice lacking both MCH and leptin present reduced body fat, mainly due to increased resting energy expenditure and locomotor activity. Dense MCH innervation of the cerebral motor cortex (MCx) and the pedunculopontine tegmental nucleus (PPT), both related to motor function, has been reported. Therefore, we postulated that a specific group of MCH neurons project to these areas. To investigate our hypothesis, we injected retrograde tracers into the MCx and the PPT of rats, combined with immunohistochemistry. We found that 25% of the LHA neurons projecting to the PPT were immunoreactive for MCH, and that 75% of the LHA neurons projecting to the MCx also contained MCH. Few MCH neurons were found to send collaterals to both areas. We also found that 15% of the incerto-hypothalamic neurons projecting to the PPT expressed MCH immunoreactivity. Those neurons preferentially innervated the rostral PPT. In addition, we observed that the MCH neurons express glutamic acid decarboxylase mRNA, a gamma-aminobutyric acid (GABA) synthesizing enzyme. We postulate that MCH/GABA neurons are involved in the inhibitory modulation of the innervated areas, decreasing motor activity in states of negative energy balance.

Human Th2 cells selectively express the orexigenic peptide, pro-melanin-concentrating hormone

Th1 and Th2 cells represent the two main functional subsets of CD4(+) T helper cell, and are defined by their cytokine expression. Human Th1 cells express IFNgamma, whilst Th2 cells express IL-4, IL-5, and IL-13. Th1 and Th2 cells have distinct immunological functions, and can drive different immunopathologies. Here, we show that in vitro-differentiated human Th2 cells highly selectively express the gene for pro-melanin-concentrating hormone (PMCH), using real-time RT-PCR, enzyme immunoassay, and Western blot analysis. PMCH encodes the prohormone, promelanin-concentrating hormone (PMCH), which is proteolytically processed to produce several peptides, including the orexigenic hormone melanin-concentrating hormone (MCH). PMCH expression by Th2 cells was activation responsive and increased throughout the 28-day differentiation in parallel with the expression of the Th2 cytokine genes. MCH immunoreactivity was detected in the differentiated Th2 but not Th1 cell culture supernatants after activation, and contained the entire PMCH protein, in addition to several smaller peptides. Human Th1 and Th2 cells were isolated by their expression of IFNgamma and CRTH2, respectively, and the ex vivo Th2 cells expressed PMCH upon activation, in contrast to the Th1 cells. Because Th2 cells are central to the pathogenesis of allergic diseases including asthma, expression of PMCH by activated Th2 cells in vivo may directly link allergic inflammation to energy homeostasis and may contribute to the association between asthma and obesity.

Evidence for a Stimulatory Action of Melanin‐Concentrating Hormone on Luteinising Hormone Release Involving MCH1 and Melanocortin‐5 Receptors

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

Changes of melanin-concentrating hormone related to LHRH release in the median eminence of rats

The present research was carried out to study the distribution of melanin-concentrating hormone (MCH) fibers in the median eminence of rats and to evaluate if changes in the MCH content of the median eminence could be related to the release of LHRH. Immunocytochemical studies in the median eminence of males and estrous females showed the presence of MCH fibers, mainly in its internal layer. Diestrous and proestrous animals displayed MCH immunoreactivity in both the internal and external layers of the median eminence. Longitudinal sections of the median eminence in proestrous animals showed that MCH-immunoreactive (ir) density is higher at 12 than at 9 h in both layers of the median eminence. MCH was assayed by radioimmunoassay in median eminences of males and in females in all stages of the estrous cycle at 10 h. It was observed that the content of MCH at diestrus-1 and -2 was higher than in estrus and in male rats. In the day of proestrus, MCH and LHRH were assayed at 10, 12, 13, 14, 15 and 17 h. At 12 h, the content of MCH and LHRH showed the maximal values. At 13 h, MCH content showed a decline, while LHRH was still high. At 14 h, the LHRH content started to decrease. The present results suggest that MCH is involved in the regulation of LHRH release in the female rat.

Neurons synthesizing melanin-concentrating hormone identified by selective reporter gene expression after transfection in vitro: transmitter responses.

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

Neuronal organization of the melanin‐concentrating hormone system in primitive actinopterygians: Evolutionary changes leading to teleosts

Hypothalamic melanin-concentrating hormone (MCH) neurones occur in all vertebrates and have an apparent neuromodulatory role. In teleost fish, however, MCH is used also as a neurohypophysial hormone, controlling skin color, and as a hypophysiotrophic peptide. This work describes the central location of immunoreactive MCH perikarya and their projections to the pituitary in a range of ancestral fish to determine the phylogenetic stage when the peptide adopted these roles. In all actinopterygians examined, including polypteriformes, chondrosteans, holosteans, and teleosts, immunoreactive fibers were abundant in the median eminence or, in the case of teleosts, within the pars distalis itself, suggesting MCH acquired a hypophysial regulatory role early in vertebrate evolution. MCH fibers appeared to be absent from the posterior neurohypophysis of the polypteriform Calamoichthys but were evident in this region in the chondrostean Acipensor, the holosteans Lepisosteus and Amia, and all teleosts, suggesting its use as a neurohypophysial hormone. The ability of MCH to induce skin pallor seems to have arisen at a later stage, probably in the preholosteans. This hormonal role coincides with the migration of MCH perikarya away from the ventricular surface and their enlargement into magnocellular neurones. In the higher teleosts, magnocellular hypothalamo-neurohypophysial neurones predominate in size and number, whereas smaller periventricular MCH neurones associated with the paraventricular organ, that are prominent in lampreys, early actinopterygians and tetrapods, are reduced in teleosts. The data suggest that, in teleost fish, earlier functions of the peptide may have become subordinate to its novel pigmentary role.

Expression of MCH and POMC Genes in Rainbow Trout (Oncorhynchus mykiss) during Ontogeny and in Response to Early Physiological Challenges

The expression of the neuropeptide melanin-concentrating hormone (MCH) in two groups of hypothalamic neurones (NLT- and LVR-MCH neurones), and POMC in the pituitary corticotropes and melanotropes, has been examined in rainbow trout larvae using immunocytochemistry and quantitativein situhybridization. The aim was to establish at what stage in ontogeny these cells first respond to two physiological challenges—background color and stress. Trout reared in black or white trays showed adaptive skin pigmentary changes at 10 days posthatching, when fish in a pale environment abruptly exhibited melanin aggregation from a prior dispersed state, although the pigment cells were already competent to respond to adrenalin and MCHin vitroat 3 days. Immunoreactive MCH was detectable in the neurohypophysis at hatching and MCH mRNA in the NLT-MCH neurones (which project to the pituitary) was enhanced at 7 days in the white-reared trout. Immunostainable POMC was also present in the pars intermedia at hatching but their POMC mRNA was unaffected by tank color until 28 days, when it was enhanced in the black-reared trout. It is suggested that early pigment concentration depends on neural signals from the sympathetic nervous system in conjunction with MCH from the NLT rather than on a reduction in αMSH secretion from the pars intermedia. MCH mRNA in the LVR-MCH neurones was increased on a pale environment only 28 days after hatching, suggesting that these cells play little role in the early adaptive pigment response. Previous studies on the ontogeny of cortisol secretion indicate the hypothalamopituitary–interrenal axis can respond to stress by about 14 days. However, the pituitary ACTH cells showed no stress-induced changes in POMC mRNA until 28 days. ACTH release may therefore be dissociated from POMC transcription in the early stages of development. The LVR- and NLT-MCH neurones were both stimulated by stress, LVR-MCH mRNA responding by 14 days and NLT-MCH mRNA by 21 days. Melanotrope POMC mRNA was reduced by stress but the physiological significance of this is not known.

Exploring the expression of the melanin-concentrating hormone messenger RNA in the rat lateral hypothalamus after goldthioglucose injection

Melanin-concentrating hormone (MCH) is expressed in a large neuronal population of the rat lateral hypothalamus. This area is known to be implicated in the regulation of thirst and hunger and to contain glucose-sensitive cells. In the present study, we investigated the effects of goldthioglucose (GTG), a toxic form of glucose, on the expression of the MCH gene in the rat lateral hypothalamus by immunocytochemistry, in situ hybridization and competitive RT-PCR. We observed that the MCH immunoreactivity and the level of MCH mRNA were not changed after intraperitoneal GTG injection (0.35 mg/g body weight). These results together with previous data suggest that the glucose-sensitive cells of the lateral hypothalamus are different from the MCH neurons and remain to be identified.

Developmental and stage-dependent expression of melanin-concentrating hormone in mammalian germ cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide predominantly expressed in the hypothalamus of mammals. This peptide modulates the stress response and regulates many goal-oriented behaviors in the rat brain. MCH mRNA and peptides generated from the precursor, namely MCH and neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI), were also found in rodent peripheral tissues including those in adult testis. In the present study, we first examined the cellular distribution and content of MCH gene products and peptide in the testes of adult rats. Using reverse-transcription polymerase chain reaction with MCH gene primers and in situ hybridization with specific 33P-labeled oligoprobes, we characterized the MCH RNA species. Pro-MCH products were revealed through use of immunoperoxidase detection and an RIA with specific MCH and NEI antisera. Both MCH gene transcripts and MCH peptide were found within germ cells at the periphery of some of the seminiferous tubules of adult rats. We further investigated temporal expression of MCH in 7-microns sections of testes from adult rats and mice. MCH was predominantly found in nuclei of spermatogonia at stages II-IV of spermatogenesis and in nuclei of leptotene and zygotene spermatocytes (at stages IX- XIII). MCH was markedly absent in preleptotene spermatocytes at the stage VII-VIII, and MCH immunoreactivity was no longer detectable as spermatocytes underwent the pachytene step. MCH immunoreactivity was also found in some peritubular cells, mainly at stages II-IV. Finally, we studied MCH expression during puberty in the rat, in sterile mutant mice, and in one adult man. We found predominant staining with MCH antiserum over nuclei of immature germ cells as early as 10 days postpartum in the rat and further confirmed the absence of staining except for that in spermatogonia and early spermatocytes during rat postnatal development. MCH distribution was found to be similar in normal and sterile mutant mice, suggesting that MCH expression is not dependent upon the early steps of spermiogenesis. MCH immunoreactivity was found to be confined to the nuclei of spermatogonia and early spermatocytes in the adult man. Our results indicate that MCH is present predominantly within nuclei of spermatogonia and primary spermatocytes in three mammalian species and that its expression is under strong stage-specific and developmental regulation. This peptide may play a role during stem cell renewal and/or differentiation of early spermatocytes.

Developmental Changes in Melanin-Concentrating Hormone in Rana temporaria

Melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic neurons. In bony fish, such as trout, MCH acts as a neurohypophysial hormone which, once released into the circulation, acts on pigmented skin cells with the result that the fish turns pale to camouflage itself against a light colored background. In other animals the role of MCH is not clearly established but it appears to be a neuromodulator/transmitter within the central nervous system rather than a hormone. The present study examines MCH function in the grass frog, Rana temporaria. Using immunocytochemistry the location and morphology of irMCH neurons were followed, from tadpole to adult frog. In adult R. temporaria a group of MCH neurons appeared to comprise small and large-celled populations located in the ventral and dorsal infundibular regions, respectively. A group of MCH neurons in the preoptic area is proposed, although the perikarya were rarely immunostainable. Immunoreactive fibers were seen in various areas of the brain, including the olfactory lobes, optic tecta, habenular nucleus, and spinal cord. Immunoreactive MCH cells were only visible in midmetamorphic climax stages, and cellular morphology suggested low secretory activity until the animal first emerged onto land at which time nuclear size and granulation increased significantly. No such increase was observed in equivalent stages of the South African clawed toad, Xenopus laevis, an animal which is fully aquatic throughout its life in contrast to R. temporaria which is terrestrial. These results suggest that increased neuronal activity in Rana emerging from water may reflect a role of MCH in osmotic balance.

Melanin-Concentrating Hormone in Human and Rat

Melanin-concentrating hormone (MCH) is a neuropeptide originally isolated from chum salmon pituitaries. To explore physiological roles of MCH in mammals, we studied the regional distribution of immunoreactive MCH in the rat tissues and the presence of immunoreactive MCH in human adrenal glands, adrenal tumors and plasma by radioimmunoassay, and the expression of MCH mRNA in rat tissues and human brain tissues by Northern blot analysis. Immunoreactive MCH was present in every region of rat brain and neurointermediate lobe of the pituitary gland, with the highest concentrations found in the hypothalamus (48.3 +/- 6.6 pmol/g wet weight, mean +/- SEM, n = 6). The immunoreactive MCH in rat hypothalamus, frontal lobe, and pons and medulla oblongata was eluted in the position of synthetic human/rat MCH in reverse-phase high-performance liquid chromatography. No immunoreactive MCH was detected in the rat peripheral tissues. Northern blot analysis showed that a single species of MCH mRNA (approximately 1 kb) was expressed specifically in the rat and human hypothalamus, but not detectable in other regions of brain or rat peripheral tissues. Immunoreactive MCH was not detected in human adrenal glands (< 0.5 pmol/g wet weight, n = 9) or adrenal tumors including pheochromocytomas. Immunoreactive MCH were not detected in plasma obtained from human healthy subjects (< 0.25 pmol/l) and rat (< 0.25 pmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Melanin-concentrating hormone in the human brain

The presence of human melanin-concentrating hormone (MCH) was studied in the human brain by radioimmunoassay and immunocytochemistry. Immunoreactive MCH concentrations in the human brain ranged from 0.07 to 19.7 pmol/g wet weight. High performance liquid chromatography of the hypothalamus showed a large immunoreactive peak in the position of human/rat MCH, which was eluted 9 min later than that of salmon MCH. Free-floating sections (40 μm) of the hypothalamus were immunostained. Positive MCH immunostaining was found in perifornical, tuberomammillary, and posterior nuclei. Numerous MCH-immunoreactive nerve fibers were observed throughout the hypothalamus. The presence of high concentrations of MCH in the human brain, in particular in the hypothalamus, suggests that MCH is a neurotransmitter, a neuromodulator, or a neurohormone in man.

Development of a sensitive solid-phase radioimmunoassay for melanin-concentrating hormone

A two-step solid-phase radioimmunoassay for melanin-concentrating hormone (MCH) was developed for direct determination of the hormone in plasma samples. To this end, synthetic MCH was coupled to bovine thyreoglobulin and the complex was injected into rabbits. Specific antisera of high titer were obtained which did not crossreact with other hormones. The IgGs were chemically linked to immunobeads, an acrylamide/acrylic acid polymer matrix. In the first step, plasma MCH was immunoextracted by incubation of diluted plasma samples with anti-MCH immunobeads. In the second step, the washed polymer was incubated with radioiodinated MCH tracer for titration of non-occupied sites. This procedure made it possible to determine as little as 4 pg MCH per ml of plasma. Application of the radioimmunoassay to plasma levels of black or white background-adapted trout showed a marked difference in circulating MCH: while trout on a black background contained a mean value of 29 ± 5.6 pg/ml, animals on a white background had 106 ± 19 pg/ml. These findings strengthen the hypothesis that MCH is directly involved in the control of color change of teleost fishes. By contrast, there was no detectable salmonid MCH immunoreactivity in rat or human plasma.

Melanin-concentrating hormone (MCH) is colocalized with α-melanocyte-stimulating hormone (α-MSH) in the rat but not in the human hypothalamus

Melanin-concentrating hormone (MCH)-containing neurons have recently been localized in the dorsolateral region of the rat hypothalamus, an area where the second α-MSH system is found which contains only α-MSH and none of the pro-opiomelanocortin (POMC)-related peptides. In order to study the morphological relationships between the MCH and α-MSH neuronal systems, we have studied the immunocytochemical localization of both MCH and α-MSH in the rat hypothalamus. The same study was also performed in the human hypothalamus where there is only one α-MSH system which contains α-MSH as well as the other POMC-related peptides (first α-MSH system). In the rat dorsolateral hypothalamus, we could demonstrate that most neuronal cell bodies stained for MCH also contained immunoreactive α-MSH. In the human hypothalamus, neuronal cell bodies stained for MCH were observed only in the periventricular area whereas cell bodies containing α-MSH were exclusively located in the infundibular (arcuate) nucleus. In the rat, immunoelectron microscopy showed labelling for MCH in the dense core vesicles of positive neurons and double-staining techniques clearly demonstrated that both immunoreactive MCH and α-MSH could be consistently detected in the same dense core vesicles. These ultrastructural studies then suggest that these two peptides should be released simultaneously from neurons located in the rat dorsolateral hypothalamus.