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 effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone.

The adipose hormone, leptin, not only restrains appetite, but also influences energy expenditure. One such influence is to promote sexual maturation and fertility. The neuromodulatory circuits that mediate this effect are not well known but the present study suggests that one mediator could be melanin-concentrating hormone (MCH). We show that the long-form receptor (Ob-Rb) is expressed in the zona incerta of the rat and that administration of leptin (both 0.5 microg and 1.0 microg/side) into this area of ovariectomized, oestrogen-primed rats stimulated the release of luteinizing hormone (LH) within 1 h, the effect enduring for a further 1 h. Injections of leptin into the arcuate nucleus induced a smaller, transient rise in LH while injections into the paraventricular and ventromedial nuclei were without effect. MCH neurones are present in the zona incerta and administration of this hormone into the medial preoptic area (mPOA) stimulates LH release, therefore we investigated the possibility that MCH might mediate this effect of leptin. An injection of MCH antiserum into mPOA prevented the rise in LH normally induced by leptin injected into the zona incerta. In addition, melanocortin receptor antagonists ([D-Arg8]ACTH(4-10) and [Ala6]ACTH(4-10)), previously shown to inhibit the stimulatory effect of MCH on LH release, also inhibited the effect of leptin. We propose that one route by which leptin may promote reproductive activity is by enhancing MCH release from fibres within the mPOA. Speculative mechanisms for the action of MCH include the following possibilities: MCH may be acting on the specific MCH receptor which in turn interacts with a melanocortin or melanocortin-like receptor; MCH may bind directly to one of the melanocortin receptors; or melanocortin antagonists may interact with the MCH receptor.

Rainbow trout (Oncorhynchus mykiss) urotensin-I: structural differences between urotensins-I and urocortins.

In bony fishes, both corticotropin-releasing factor (CRF) and urotensin-I play a role in the regulation of interrenal glucocorticoid release. The rainbow trout, Oncorhynchus mykiss, is a useful model for understanding the mechanisms of stress and the hypothalamo-pituitary-interrenal axis because of its phylogenetic position at the base of the euteleostei and its popularity as a food fish. Urotensin-I may act as a glucocorticoid releaser in a mechanism phylogenetically older than that of CRF. The structural and functional relationships of trout urotensin-I have been investigated. The transcript was cloned from a trout brain hypothalamic cDNA library. A single positive clone was isolated and sequenced. It possesses 3218 bases and has the longest 3' untranslated region of all urotensins-I and CRF transcripts found to date. In comparison to the other fish orthologues, it has the closest sequence identity to the mammalian urocortins. The transcript appears to be differentially processed in brain and urophysis as determined by Northern blot analysis and the presence of polyadenylation signals in the 3' untranslated region. Synthetic trout urotensin-I activated both human CRF-R1 and -R2 receptor-transfected CHO cells with a potency similar to that of white sucker (Catostomus commersoni) urotensin-I. Both fish neuropeptides possessed an order of magnitude less potency than human urocortin in CRF-R2 transfected cells.

Diurnal changes in the expression of genes encoding for arginine vasotocin and pituitary pro-opiomelanocortin in the rainbow trout (Oncorhynchus mykiss): correlation with changes in plasma hormones.

Using quantitative in-situ hybridization, this study monitored diurnal changes in the abundance of the gene transcripts of two corticotropin-releasing peptides, arginine vasotocin (AVT) and isotocin in hypothalamic neurones, and of pro-opiomelanocortin (POMC) mRNA in the pituitary of the rainbow trout (Oncorhynchus mykiss). A significant diurnal pattern of gene expression was only displayed in the hypothalamus by the parvocellular AVT neurones of the preoptic nucleus. Abundance of AVT mRNA in these neurones was low at lights on (06.00 h), increased during the morning to reach a plateau of peak values between 14.00 h and 22.00 h, and then declined during the dark phase. This pattern was the inverse of that shown by plasma cortisol values. Changes in AVT transcript abundance are also considered in terms of the reported diurnal change in circulating AVT concentration. Pituitary and hypothalamic AVT peptide content did not change. Transcripts of both POMC genes (POMC-A and POMC-B) were monitored in pituitary corticotropes and melanotropes. Only POMC-A mRNA was detected in corticotropes where it showed no diurnal change in abundance. Transcripts of both POMC genes were found in the melanotropes, although, judging from autoradiographic intensity, POMC-A mRNA predominated. Both genes showed diurnal differences in their transcription with POMC-A mRNA showing peak values at 10.00 h and a nadir at 02.00 h, while POMC-B mRNA showed an inverse pattern. The results indicate that the two POMC genes can be independently regulated.

The influence of repeated stress on the release of melanin-concentrating hormone in the rainbow trout.

Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white-adapted fish and not in fish kept in black-coloured tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress.

The effect of rearing rainbow trout on black or white backgrounds on their secretion of melanin-concentrating hormone and their sensitivity to stress.

Rainbow trout were reared in black or off-white coloured tanks for up to 18 months of age to achieve maximum differences in the synthesis of the neuropeptide, melanin-concentrating hormone (MCH). White-reared fish had greatly increased MCH concentrations in their pituitary glands, in their MCH perikarya and in the presumptive neuromodulatory fibres of the dorsal hypothalamus/thalamus when compared with black-reared and commercially reared trout. Following transfer to brighter white tanks, white-reared fish showed a significant increase in plasma MCH concentration and a reduction of MCH in the pituitary and MCH perikarya. The additional challenge of repeated stress further increased plasma MCH concentration in these fish and also reduced MCH in the dorsal hypothalamus/thalamus. In black-reared fish transferred to white tanks, plasma MCH concentrations were significantly raised after transfer, although they were lower after 11 days than in white-reared counterparts. Transfer from black to white background caused a fall in the MCH concentration in all regions--pituitary gland, perikarya and dorsal hypothalamus/thalamus; if transfer was accompanied by repeated stress, the hormone in the pituitary gland and MCH perikarya became so depleted that plasma MCH concentrations declined. Within each experimental situation (control, background transfer and transfer with stress) there was in inverse correlation between plasma MCH concentrations of black- and white-reared fish and the cortisol concentration. MCH had no direct effect on the secretion of cortisol by interrenal tissue but incubated hypothalami, in which endogenous MCH had been immunoabsorbed, provided evidence that MCH can depress the release of corticotrophin-releasing bioactivity.

Localisation and identification of melanocyte-stimulating hormones in the fish brain.

The existence of melanocyte-stimulating hormone (MSH) in fish brains was investigated by a range of techniques: radioimmunoassay, HPLC, bioassay, and immunocytochemistry. Immunoreactive alpha MSH (ir alpha MSH) was detected by radioimmunoassay in all regions of carp and trout brains, with the highest concentration in the basal hypothalamus. In trout, ir alpha MSH cell bodies were located by immunocytochemistry only periventricularly, in the medial basal hypothalamus near the third ventricle, whereas in the carp ir alpha MSH staining was seen both in periventricular cells and also in some of the magnocellular neurones in the lateral hypothalamus. When white-adapted fish were transferred to a black tank for 6 days, the melanin-concentrating hormone (MCH) content of the basal hypothalamus of both carp and trout increased 2- and 4.6-fold, respectively, but the alpha MSH content did not change in either species. Analysis by HPLC of pituitary gland, hypothalamic, and optic tectal extracts revealed that the pituitary contains desacetyl, monoacetyl, and diacetyl alpha MSH, although the ratio of these forms differed in the two species. The hypothalamus and optic tectum, however, contained predominantly the desacetyl form of alpha MSH. Bioassays for MSH in the HPLC fractions revealed the existence of presumptive beta MSH in both the pituitary and hypothalamus. An argument is advanced that the periventricular ir alpha MSH neurones are homologous with the proopiomelanocortin cells of the arcuate nucleus in mammals, and that the immunocytochemical alpha MSH-like activity in the MCH neurones may not be authentic alpha MSH.

Hypothalamo-pituitary-interrenal responses to opioid substances in the trout. I. Effects of morphine on the release in vitro of corticotrophin-releasing activity from the hypothalamus and corticotrophin from the pituitary gland.

The effects of morphine and naloxone on the secretion in vitro of corticotrophin (ACTH) and and corticotrophin-releasing factor (CRF) by the pars distalis and hypothalamus, respectively, have been studied in the trout. The spontaneous in vitro secretion of corticotrophin by the pars distalis is depressed significantly by the addition of high concentrations of morphine (10(-6)-10(-7) mol/litre) to the incubation medium. The effect is naloxone reversible. Morphine does not influence the response of the pituitary tissue to exogenous CRF41, suggesting that the inhibitory influence of the opiate is exerted primarily on the CRF nerve terminals within the pars distalis and not on the corticotrophs. At considerably lower concentrations (10(-10)-10(-8) mol/litre) morphine stimulates the release of CRF from the isolated trout hypothalamus in vitro. Its effects are dose-dependent and antagonized by naloxone. The results suggest that two anatomically and pharmacologically distinct populations of opioid receptors mediate opposing actions of morphine on the hypothalamo-pituitary-corticotrophic system in the trout.

Hypothalamo-pituitary-interrenal responses to opioid substances in the trout. II. Effects of morphine and D-Ala2, Met5-enkephalinamide on plasma cortisol titres in vivo.

The influence of morphine, D-Ala2, Met5-enkephalinamide (DALA), and naloxone on plasma cortisol titres has been studied in vivo in fingerling and adult trout. The responses were complex and variable. A single ip injection of morphine or DALA into fingerlings usually resulted in a rise in plasma cortisol after 0.5 hr followed by a fall below control values within 2 hr. In similar experiments with adult trout, only an inhibitory effect was observed. Naloxone reduced the rise in plasma cortisol following saline injection, but only when the hypothalamo-pituitary-interrenal response was intense. The antagonist also blocked the morphine-induced rise in cortisol secretion. Prolonged morphine treatment diminished both the postinjection and stress-induced secretion of cortisol in adult fish. Morphine had no effect on the spontaneous or ACTH-induced secretion of cortisol by interrenal tissue incubated in vitro. The results support the concept of inhibitory and stimulatory sites of action by opiates and opioid substances on the hypothalamo-pituitary-interrenal axis. These findings are discussed with reference to the action of morphine on hypothalamic and pituitary tissue of the trout in vitro and with the opioid control of hypothalamo-pituitary-adrenal function in mammals.

Melanin concentrating hormone inhibits the release of alpha MSH from teleost pituitary glands.

A radioimmunoassay was developed for salmonid melanin concentrating hormone (MCH) and used to measure immunoreactive (ir)MCH in the hypothalamus and pituitary of trout (Salmo gairdneri) and eels, (Anguilla anguilla) maintained under different regimes of background color. In trout, 95% of the total irMCH was located in the pituitary gland. The amount of MCH in both pituitary and hypothalamus was increased when white-adapted trout were transferred to a black background. In eels, a similar change of background led to an accumulation of MCH in the pituitary but not in the hypothalamus. The results suggest that MCH is released from the neurohypophysis in association with physiological color change. Neurointermediate lobes of trout and eels released both ir alpha MSH and irMCH when they were cultured in vitro. The release of alpha MSH was significantly enhanced when endogenous MCH was immunoabsorbed by MCH antiserum added to the culture medium. The results indicate that MCH can induce pallor in fish not only by its peripheral effect on the melanophores but also by an inhibitory action on the release of alpha MSH from the pituitary.

Evidence for the participation of a melanin-concentrating hormone in physiological colour change in the eel.

The hormonal and nervous control of colour change in the eel has been investigated. The only bioactive forms of MSH found in eel pituitary extracts or secreted by eel pituitary cultures were forms of alpha-MSH; no beta-MSH was detected. After transfer of eels from a black to a white background, the melanin concentration in skin melanophores was accompanied by a rapid decline in plasma alpha-MSH titres. Hypophysectomy resulted in melanin concentration, and pituitary extracts injected into hypophysectomized eels caused melanin dispersion. This effect was eliminated if the pituitary extracts were first incubated with a specific alpha-MSH antiserum or if the antiserum was injected into the hypophysectomized eel. However, injection of alpha-MSH antiserum into intact, black-adapted eels failed to result in melanin concentration although the same antiserum was effective in causing pallor in black-adapted toads. Partially purified preparations of teleost melanin-concentrating hormone (MCH), free from catecholamines, induced melanin concentration when injected into black-adapted eels and this effect was significantly potentiated by injections of alpha-MSH antiserum. The denervation of melanophores on the pectoral fin had only a slight effect on the responses of the melanophores to humoral agents. It is concluded that the control of physiological colour change in the eel is largely hormonal, and involves the antagonistic effects of alpha-MSH and a melanin-concentrating agent which is probably MCH.

Further observations on the distribution and properties of teleost melanin concentrating hormone.

The distribution of melanocyte concentrating hormone (MCH) bioactivity was mapped in the trout brain from cryostat sections cut in several planes. Most of the bioactivity occurred in the ventral third of the hypothalamus, with about 30% of the activity in the dorsal hypothalamus. The bioactivity was rapidly lost if the hypothalami were extracted in dilute acid, with a final extraction pH of 5.2. This loss, which can be avoided if the extract is heated, is presumed to be the result of hypothalamic enzyme activity. Preliminary chemical characterisation indicates that the molecule is a small basic peptide, of less than 2000 daltons (Da) and with an isoelectric point greater than 9.5. MCH bioactivity was also found in the hypothalamus but not the pituitary of Lampetra, Rana, Xenopus, and the rat. The activity from Xenopus and Lampetra had a similar Rf value to MCH from trout during polyacrylamide gel electrophoresis. Partially purified MCH of trout origin, free from MSH bioactivity, induced melanin concentration in eel melanophores but Xenopus melanophores failed to respond.