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.     

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.     

Immunohistochemical localization of thyrotropin-releasing hormone in the brain of carp, Cyprinus carpio

The localization of immunoreactive thyrotropin-releasing hormone (IR-TRH) in the forebrain and pituitary of carp was studied immunohistochemically using the peroxidase-antiperoxidase technique. In the hypothalamus. IR-TRH was present in the neuronal processes extending from the preoptic nucleus (NPO) to the nucleus recessus lateralis (NRL). Cell bodies appeared to be present in the inside of the medial NRL. Most of these neurons were fusiform and bipolar. Immunoreactive-beaded fibers streamed from the anterior part of the NRL toward the nucleus posterioris periventriculas and nucleus lateral tuberis pars posterioris. Vertical strands of the beaded fibers ran in the nucleus lateral tuberis pars anterioris. In the pituitary, the reaction product was found in the neural lobe, where intense immunoreactivity was evident along neural fibers entering the intermediate lobe. Staining could be detected only rarely in the anterior lobe. IR-TRH-beaded fibers were present in the olfactory stalk as well as in the caudal and inner parts of the olfactory bulb. In contrast to the high concentration of IR-TRH in the olfactory bulb, immunohistochemical data from this work indicated weak immunoreactivity in this region.     

Melanin-concentrating hormone (MCH) immunoreactive hypophysial neurosecretory system in the teleost Poecilia latipinna: light and electron microscopic study

Neurons containing immunoreactivity for melanin-concentrating hormone (MCH) were located in the brain of the teleost Poecilia latipinna by light microscopic (peroxidase antiperoxidase) and electron microscopic (immunogold) methods. Neuronal cell bodies were found in the tuberal hypothalamus, mostly within the nucleus lateralis tuberis, pars lateralis, containing MCH-immunoreactive granules up to 150 nm in diameter. From here bundles of immunoreactive fibers could be traced through the preoptic area as far forward as the olfactory bulb, and through the posterior hypothalamus up into the pretectal thalamus and midbrain. The main projection was, however, to the neurohypophysis, where MCH fibers were observed to form contacts with pituicytes, basement membranes around blood vessels, and the endocrine cells of the pars intermedia. Occasionally MCH-immunoreactive terminals were also seen near the corticotrophs of the rostral pars distalis. These results support the hypothesis that MCH may act as a systemic hormone, a central neurotransmitter, and a modulator of pituitary function.     

Beta-endorphin inhibits hypoglycemia-induced gene expression of corticotropin-releasing factor in the rat hypothalamus.

Endogenous opioid peptides have a role in the regulation of the hypothalamic-pituitary-adrenal axis. Recently, beta-endorphin (EP) has been thought to inhibit CRF release in vivo and in vitro. In the present study we examined the effects of central administration of EP on ACTH secretion and gene expression of both CRF in the hypothalamus and POMC in the anterior pituitary gland (AP) during basal and insulin-induced hypoglycemia in pentobarbital-anesthetized rats. Administration of EP in the lateral ventricle decreased basal CRF levels in the median eminence and inhibited basal and hypoglycemia-induced ACTH secretion in a dose-dependent manner. Hypoglycemia-induced POMC mRNA levels in the AP and CRF mRNA levels in the hypothalamus were also dose-dependently inhibited by the administration of EP. The inhibitory effect of EP was reversed by naloxone. These results suggest that 1) central administration of EP acts through the opioid receptor to inhibit hypoglycemia-induced CRF gene expression in the hypothalamus and CRF release, which results in a decrease in ACTH secretion and POMC mRNA levels in the AP; and 2) the active site of EP is the CRF neuron in the paraventricular nucleus.     

Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.     

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 teleost melanin-concentrating hormone -- a pituitary hormone of hypothalamic origin.

A simple bioassay method for the teleost melanin-concentrating hormone (MCH) is described. Using this assay and also the Anolis bioassay for melanocyte-stimulating hormone (MSH) the work compares the relative concentrations of MSH and MCH in the pituitary of various teleost species and their pattern of distribution after polyacrylamide gel electrophoresis. Neurointermediate lobes from trout were cultured and various factors [cold, cycloheximide, ethyleneglycol bis(β-aminoethyl ether)N,N′-tetraacetic acid) (EGTA), and high K ion concentration] were examined for their effect on MSH and MCH secretion. EGTA and high K⁺ both inhibited MSH release; cold and cycloheximide appeared to reduce MSH synthesis. Whereas these effects on MSH were predictable and consistent, the response of MCH was erratic and unpredictable. In contrast to MSH, there was no evidence for MCH synthesis in vitro. The hypothalamus of the trout contains as much MCH as the pituitary, and the concentration of MCH in the hypothalamus varies with the background colour on which the fish is kept. The evidence as a whole suggests that MCH is an hypothalamic secretion which is stored and released by the neurohypophysis.     

Beta-endorphin-like immunoreactivity in the forebrain and pituitary of the teleost Clarias batrachus (Linn.)

The organization of beta-endorphin-like immunoreactivity in the olfactory system, forebrain, and pituitary of the teleost Clarias batrachus was investigated. Immunoreactivity was prominently seen in the sensory neurons and basal cells in the olfactory epithelium and in some cells in the periphery and center (granule cells) of the olfactory bulb. Immunoreactive fibers in the olfactory nerve enter the olfactory nerve layer of the olfactory bulb and branch profusely to form tufts organized as spherical neuropils in the glomerular layer. While fascicles of immunoreactive fibers were seen in the medial olfactory tracts, the lateral olfactory tracts showed individual immunoreactive fibers. Immunoreactive fibers in the medial olfactory tract extend into the telencephalon and form terminal fields in discrete telencephalic and preoptic areas; some immunoreactive fibers decussate in the anterior commissure, while others pass into the thalamus. While neurons of the nucleus lateralis tuberis revealed weak immunoreactivity, densely staining somata were seen at discrete sites along the wall of the third ventricle. Although a large population of immunoreactive cells was seen in the pars intermedia of the pituitary gland, few were seen in the rostral pars distalis and proximal pars distalis; immunoreactive fibers were seen throughout the pituitary gland.     

Immunocytochemical localization of LH-RH in the brain and pituitary of the catfish, Clarias batrachus (Linn.)

An elaborate organization of luteinizing hormone-releasing hormone (LH-RH) immunoreactive (ir) cells and fibers was encountered in the olfactory system of Clarias batrachus. In addition to the ir structures in the olfactory nerve, peripheral area of the olfactory bulb, and the medial olfactory tract (MOT), ir cells and fibers were prominently seen in the lamellae of the olfactory organ. Perikarya showing varying degrees of intensity of immunoreaction were observed along the base of the forebrain in the nucleus preopticus basalis lateralis, nucleus preopticus periventricularis, nucleus preopticus, nucleus lateralis tuberis pars posterior, and the pituitary. Some cells were also noticed in the midbrain tegmentum. A well-defined system of ir fibers from the MOT penetrated the telencephalon and curved dorsocaudally into the pars supracommissuralis above the anterior commissure (AC); while some fibers decussate in the AC, others extended posteriorly into the diencephalon. A fairly dense network of beaded ir fibers was seen in the basal forebrain, conspicuous around the organum vasculosum laminae terminalis and caudally traceable as far as the neurohypophysis; some immunostained fibers appear to be directly contacting with the cells of the proximal pars distalis. Fibers were also witnessed in the optic chiasma and in the inner plexiform layer of the retina. Solitary fibers were noticed in certain circumscribed telencephalic areas, caudal hypothalamus, posterior commissure, midbrain tegmentum, cerebellum, and ventral medulla oblongata. The highly organized LH-RH containing system in C. batrachus is indicative of its elaborate role in synchronization of the reproductive processes and the environmental cues.     

Effect of salt loading on proopiomelanocortin (POMC) messenger ribonucleic acid levels, POMC biosynthesis, and secretion of POMC products in the mouse pituitary gland

The effect of salt loading on POMC-derived peptide secretion, POMC mRNA levels, and POMC biosynthesis in the mouse pituitary gland was investigated. Plasma alpha MSH levels decreased to 56.9% of the control value after 2 days of salt loading. Concomitantly, POMC mRNA levels and POMC biosynthesis in the intermediate lobe decreased to 47.7% and 66.4% of the control value, respectively. After 4 days of salt loading, plasma alpha MSH levels, POMC mRNA, and POMC biosynthesis returned to control levels. ACTH secretion increased to 142.2% of the control value after 2 days of salt loading, but decreased to 73.4% of the control levels after 4 days. After 9 days, ACTH secretion was still low; however, by 12 days, plasma ACTH levels in salt-loaded mice were not significantly different from control levels. POMC mRNA levels in the anterior lobe increased to 205.0% of the control value after 2 days of salt loading, continued to be high after 4 days, but returned to control levels by 9 days. However, POMC biosynthesis was not altered under these conditions during the entire period of salt loading. Thus, POMC mRNA levels and biosynthesis were modulated differently in the intermediate and anterior lobes of the mouse pituitary gland during salt loading.     

Glucagon-like immunoreactivity in the forebrain and pituitary of the teleost, Clarias batrachus (Linn.)

The organization of glucagon-like immunoreactivity (GLI) in the olfactory system, forebrain, and pituitary was investigated in the teleost Clarias batrachus. Weak to moderate GLI was seen in some olfactory receptor neurons and basal cells of the olfactory epithelium. Intense GLI was seen in the olfactory nerve fascicles that ran caudally to the bulb, spread over in the olfactory nerve layer, and profusely branched in the glomerular layer to form tufts organized as spherical neuropils; some of the immunoreactive fibers seem to closely enfold the mitral cells. In the inner cell layer of the bulb, some granule cells were intensely immunoreactive. Although there were thick fascicles of immunoreactive fibers in the medial olfactory tracts (MOT), the lateral olfactory tracts were generally devoid of immunoreactivity. Immunoreactive fibers in the medial olfactory tract penetrated into the telencephalon from its rostral pole and entered into the area ventralis telencephali/pars ventralis where the compact fiber bundles loosen somewhat and course dorsocaudally into the area ventralis telencephali/pars supracommissuralis just above the anterior commissure. While some immunoreactive fibers decussated in the anterior commissure, fine fibers were seen in the commissure of Goldstein. Isolated immunoreactive fibers of the medial olfactory tract were traced laterally into the area dorsalis telencephali/pars lateralis ventralis and mediodorsally into the area dorsalis telencephali/pars medialis. However, a major component of the MOT continued dorsocaudally into the thalamus and terminated in the habenula. Two immunoreactive neuronal groups and some isolated cells were seen in the periventricular region of the thalamus. Although nucleus preopticus showed no immunoreactivity, some neurons of the nucleus lateralis tuberis displayed moderate GLI. Several immunoreactive cells were seen in the pars intermedia of the pituitary gland; few were encountered in the rostral pars distalis and proximal pars distalis. Immunoreactive fibers were seen throughout the pituitary gland.     

Interleukin-1 stimulates corticotropin-releasing factor gene expression in rat hypothalamus

To examine the effect of interleukin-1 (IL-1) on CRF and POMC gene expression, recombinant human IL-1 alpha and -beta were ip injected in rats. The plasma ACTH level showed a dose-related increase at 2 h after the injection of 0.5 and 2 micrograms IL-1 alpha and -beta, and also showed a sustained increase from 1 h until 5 h after the injection of 2 micrograms of IL-1 beta. CRF contents in the medial basal hypothalamus and ACTH contents in the anterior pituitary (AP) decreased at 2 h after the injection of 2 micrograms of IL-1 alpha and -beta, and such decreased levels were maintained until 5 h after the injection of 2 micrograms of IL-1 beta. The levels of CRF mRNA in the hypothalamus and POMC mRNA in AP significantly increased 3 h after the injection of 2 micrograms IL-1 alpha and -beta, and these levels were still higher at 5 h after the injection of 2 micrograms of IL-1 beta compared with those of the control. There was no significant change in the ACTH content and POMC mRNA levels in the intermediate-posterior pituitary or the hypothalamus or in the CRF contents and CRF mRNA levels in the cerebral cortex. These results indicate that acute administration of IL-1 alpha and -beta stimulates gene expression of hypothalamic CRF and CRF release, which causes the stimulation of ACTH release and POMC gene expression in AP.     

Effects of hypothalamic and thalamic lesions on prolactin secretion in goldfish (Carassius auratus)

The effects of hypothalamic and thalamic lesions on serum and pituitary prolactin levels in goldfish were studied. A radioimmunoassay was used to measure prolactin levels. Lesions in the nucleus lateral tuberis (NLT) pars anterioris, NLT pars posterioris, NLT pars inferioris, nucleus anterior tuberis, or in the nucleus preopticus did not affect serum or pituitary polactin levels compared to sham or normal control groups. Lesions in the NLT pars lateralis caused a significant increase in serum prolactin, but had no effect on pituitary prolactin levels. This suggests that the NLT pars lateralis is the origin of a factor that normally inhibits prolactin release from the pituitary. Large lesions that destroyed the medial thalamic and anterior hypothalamic region dorsal to the NLT caused a significant decrease in serum prolactin, but had no significant effect on pituitary prolactin levels. It is suggested that the lesions in this region may have interrupted neural afferents to the NLT pars lateralis that normally inhibit secretion of a prolactin inhibitory factor.     

Localization, expression and control of adrenocorticotropic hormone in the nucleus preopticus and pituitary gland of common carp (Cyprinus carpio L.)

Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.     

Rat melanin concentrating hormone does not modify the release of CRH-41 from rat hypothalamus or ACTH from the anterior pituitary in vitro

It has been suggested that melanin concentrating hormone (MCH) possesses potent corticotrophin (ACTH) inhibitory activity, on the basis of the inhibitory effects displayed by salmon MCH on ACTH release from either trout or rat isolated pituitary fragments. Recently, rat MCH has been characterised, and this prompted us to investigate the putative inhibitory activity of synthetic rat MCH on basal and stimulated ACTH secretion from freshly-dispersed rat pituitary cells or incubated rat pituitary fragments, as well on KCl (28 mmol/l) or noradrenaline-evoked release of corticotrophin releasing hormone-41 (CRH-41) from rat hypothalamic explants in vitro. There were no effects of rat MCH on either CRH-41 or ACTH release in vitro.     

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.     

Localization of growth hormone releasing factor-like immunoreactivity in the hypothalamo-hypophyseal system of the frog (Rana temporaria) and the sea bass (Dicentrarchus labrax)

Using two different antisera, one raised against total human pancreatic growth hormone releasing factor (hpGRF) coupled through a two-step glutaraldehyde method and the other one raised against rat hypothalamic growth hormone releasing factor 1-10 (rGRF1-10), GRF-like immunoreactivity was localized in the hypothalamo-hypophyseal system of the frog (Rana temporaria) and the sea bass (Dicentrarchus labrax). In the frog immunoreactive neurons were found in the nucleus preopticus, pars magnocellularis. The immunopositive fibers were localized in the lateral wall of the preoptic recess, the pars ventralis of the tuber cinereum, the internal and external zone of the median eminence, and the neural lobe. Positive-stained neurons in the sea bass were located in the preoptic nucleus, in the pars magnocellularis as well as in the pars parvocellularis, and in the nucleus lateralis tuberis, pars rostralis. GRF-ir nerve fibers, originating in the hypothalamus, projected to the rostral and proximal pars distalis, the posterior neurohypophysis, and the pars intermedia (PI). Double stainings with anti-GRF and anti-ACTH or anti-trout GH showed some close relationship between GRF immunoreactive nerve fibers and adenohypophyseal cell types. In the PI both the MSH and the PI "PAS" positive cells seemed to be directly innervated by the GRF-ir axons. These results show that a GRF-like system is present in the hypothalamo-hypophyseal system of amphibians and teleosts and that in these lower vertebrates GRF-like material may be secreted directly in the systemic circulation. The function of this GRF, however, is not yet clear.     

Hypothalamic control of ACTH secretion in goldfish. III. Hypothalamic cortisol implant studies.

The implantation of pellets containing 0.3 μg of cortisol into the third ventricle adjacent to the nucleus lateral tuberis (NLT) or into the preoptic recess of the third ventricle adjacent to the nucleus preopticus (NPO) significantly suppressed the increase in circulating levels of corticosteroids that occurs in goldfish as a result of a sham-injection stress. Pellet implants containing 0.5 μg of cortisol in the third ventricle adjacent to the NPO or the lateral telencephalon posterior to the anterior commissure also suppressed the stress response of goldfish. Pellets containing 0.5 μg of cortisol implanted into the pituitary gland, optic tectum, or lateral telencephalon rostral to the anterior commissure, or into the third ventricle in the posterior or dorsomedial hypothalamus, had no effect on the stress response. These results indicate that the NLT and preoptic-telencephalon regions are negative-feedback sites of corticosteroids to suppress ACTH secretion in the goldfish.