Immunocytochemical demonstration of melanin-concentrating hormone and proopiomelanocortin-like products in the brain of the trout and carp

Immunocytochemistry on frozen sections revealed that in both the trout and the carp, parvocellular neurones located in the medial basal hypothalamus (medial nucleus lateralis tuberis) were immunostained by antisera against three molecules known to be derived from the proopiomelanocortin (POMC) molecule, viz: alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and salmonid NPP--the whole N-terminal sequence preceding ACTH in the POMC precursor. Axons from these neurones extended into various regions of the brain but did not appear to project into the pituitary gland. Antiserum against salmonid melanin-concentrating hormone (MCH) immunostained magnocellular neurones in the lateral basal hypothalamus (lateral nucleus lateralis tuberis). Axons from some of these neurones projected into the brain while other axons extended into the pituitary gland. In the carp, but not in the trout, some MCH neurones were also immunostained by antisera against alpha MSH but not by antisera against the other POMC molecules.     

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.     

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.     

Increased melanin concentrating hormone receptor type I in the human hypothalamic infundibular nucleus in cachexia

Melanin-concentrating hormone (MCH) exerts a positive regulation on appetite and binds to the G protein-coupled receptors, MCH1R and MCH2R. In rodents, MCH is produced by neurons in the lateral hypothalamus with projections to various hypothalamic and other brain sites. In the present study, MCH1R was shown, by immunocytochemistry, to be present in the human infundibular nucleus/median eminence, paraventricular nucleus, lateral hypothalamic area, and perifornical area, although in the latter two regions, only a few MCH1R-containing cells were found. In addition, MCH1R staining was found in nerve fibers in the periventricular nucleus, dorsomedial and ventromedial nucleus, suprachiasmatic nucleus, and tuberomammillary nucleus. A significant 1.6 times increase in the number of MCH1R cell body staining was found in the infundibular nucleus in postmortem brain material of cachectic patients, compared with matched controls, supporting a role for this receptor in energy homeostasis in the human.     

Immunohistochemical localization of GRF-containing neurons in rat brain

Brains from 4 normal and 4 colchicine-treated rats were studied for the presence of growth hormone-releasing factor (GRF) using an antibody directed against rat hypothalamic GRF (rGRF). Noncolchicine-treated animals showed intense staining in the external layer of the median eminence. Rats pretreated with intraventricular colchicine for 48 h showed localization of GRF-containing cell bodies in the arcuate nucleus, the perifornical area, the lateral basal hypothalamic region and lateral to the ventromedial nucleus and the premammillary nucleus. This pattern of distribution is consistent with the postulated role of GRF in hypothalamic regulation of growth hormone secretion. Our results confirm the predominant localization of GRF perikarya in the arcuate nucleus of the rat which has been noted in studies using antibodies directed against both human pancreas GRF (hpGRF) and rGRF, and demonstrate a unique pattern of rGRF-positive cell bodies elsewhere in the hypothalamus. No rGRF perikarya or processes were seen outside the hypothalamus.     

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.     

Ontogenesis of growth hormone-releasing hormone neurons in the rat hypothalamus

The ontogenesis of growth hormone releasing hormone (GH-RH) containing neurons in the rat hypothalamus has been studied by immunohistochemistry, using a specific anti-rat GH-RH serum. Immunoreactive fibers were first detected in the prospective median eminence on day 18 of gestation. During the subsequent 3 days, they rapidly increased in distribution and intensity of staining within this structure. On day 21, positive fibers were also visible in a plexus within the arcuate nucleus. In 1-day-old rats treated with colchicine, positive perikarya were distributed in several hypothalamic nuclei, including the arcuate nucleus, dorsomedial nucleus, basal lateral hypothalamus, and perifornical region. The distribution was similar to that previously described in adult rats. The intensity of staining in the various hypothalamic regions increased during early postnatal life to levels nearly comparable to those in adult rats by 30 days. These findings showing the early appearance of GH-RH-positive terminals in the median eminence and the wide distribution of the perikarya at an early stage of postnatal life support the view that hypothalamic GH-RH serves an important role in the regulation of growth hormone secretion during late prenatal and early neonatal periods.     

Melanin-concentration hormone updated functional considerations.

The melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic perikarya whose fibers project to most regions of the brain and into the spinal cord. Its role as a neurohypophyseal color-change hormone is peculiar to teleost fish, but recent studies in mammals suggests that MCH itself, and other peptides derived from the same precursor, may participate in multiple functions in the central nervous system, modulating behavior and the perception of sensory information. Recent hybridization studies in mammals have greatly increased our understanding of the response of the MCH system to environmental factors, such as osomotic challenge, lactation, stress, and changes in corticosteroid levels. Further studies in lower vertebrates are needed to highlight the physiologically important functions that have led to the structural conservation of the MCH peptide during vertebrate evolution.     

Somatotropin Release-Inhibiting Factor and Galanin Innervation in the Hypothalamus and Pituitary of Seabream (Sparus aurata)

The distribution of galanin (GAL) and somatotropin-release-inhibiting-factor (SRIF) immunoreactivity in the hypothalamus and pituitary of the sea bream (Sparus aurata) was studied by immunocytochemistry. An extensive system of neurons immunoreactive with antisera to the two peptides was identified throughout the brain with staining particularly in the hypothalamus. In the hypothalamus, GAL immunoreactive perikarya were detected principally in the nucleus preopticus and nucleus tuberis. Major nerve tracts were observed to sweep down from the hypothalamic nuclei and reached the pituitary via the preoptico-hypophysial tract. Many of the fibers had varicose swellings indicating they were secretory. SRIF immunoreactivity was distributed similarly to GAL but the network of nerve fibers was less dense; no colocalization of these two peptides was seen. SRIF immunoreactive perikarya were present in the preoptic nucleus, the tuberal nucleus, and the basolateral hypothalamus. These perikarya were large and densely staining and were predominately bipolar, although some multipolar perikarya were observed. In the pituitary GAL and SRIF immunoreactivities were confined principally to the pars distalis where fibers infiltrated between growth hormone, prolactin, and adrenocorticotrophic cells. More of the fibers were immunoreactive for SRIF than for GAL. There was no immunoreaction for GAL or SRIF in any of the pituitary cells. There is thus morphological evidence for a neuroendocrine control of the pars distalis by GAL and SRIF and for a possible functional interaction between these two systems.     

Extrahypothalamic neurophysin-containing perikarya, fiber pathways and fiber clusters in the rat brain.

Immunoreactive neurophysin (Np) is demonstrated by the immunoperoxidase technique not only within neurones of the classical hypothalamo-neurohypophyseal system, but also in extrahypothalamic perikarya and fibers. Np-positive perikarya are found in the triangular nucleus of the septum. Np fibers join the stria terminalis (ST) at the level of the anterior hypothalamus, and course medially in the ST to the central nucleus of the amygdala. Np fibers are found in the brain stem and spinal cord. Fine caliber Np fibers from the suprachiasmatic nucleus ascend to the medial dorsal thalamus and lateral septum. The presence of Np within neurones implies the presence of either vasopressin or oxytocin. Np-positive fibers in extrahypothalamic sites may interact with non-neurosecretory neurones involved in neuroendocrine regulation, or may serve as yet unknown functions.     

Melanin-concentrating hormone: unique peptide neuronal system in the rat brain and pituitary gland.

A unique neuronal system was detected in the rat central nervous system by immunohistochemistry and radioimmunoassay with antibodies to salmon melanin-concentrating hormone (MCH). MCH-like immunoreactive (MCH-LI) cell bodies were confined to the hypothalamus. MCH-LI fibers were found throughout the brain but were most prevalent in hypothalamus, mesencephalon, and pons-medulla regions. High concentrations of MCH-LI were measured in the hypothalamic medial forebrain bundle (MFB), posterior hypothalamic nucleus, and nucleus of the diagonal band. Reversed-phase high-performance liquid chromatography of MFB extracts from rat brain indicate that MCH-like peptide from the rat has a different retention time than that of the salmon MCH. An osmotic stimulus (2% NaCl as drinking water for 120 hr) caused a marked increase in MCH-LI concentrations in the lateral hypothalamus and neurointermediate lobe. The present studies establish the presence of MCH-like peptide in the rat brain. The MCH-LI neuronal system is well situated to coordinate complex functions such as regulation of water intake.     

microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate

Hagfish and lampreys are the only living representatives of the jawless vertebrates (agnathans), and compared with jawed vertebrates (gnathostomes), they provide insight into the embryology, genomics, and body plan of the ancestral vertebrate. However, this insight has been obscured by controversy over their interrelationships. Morphological cladistic analyses have identified lampreys and gnathostomes as closest relatives, whereas molecular phylogenetic studies recover a monophyletic Cyclostomata (hagfish and lampreys as closest relatives). Here, we show through deep sequencing of small RNA libraries, coupled with genomic surveys, that Cyclostomata is monophyletic: hagfish and lampreys share 4 unique microRNA families, 15 unique paralogues of more primitive microRNA families, and 22 unique substitutions to the mature gene products. Reanalysis of morphological data reveals that support for cyclostome paraphyly was based largely on incorrect character coding, and a revised dataset is not decisive on the mono- vs. paraphyly of cyclostomes. Furthermore, we show fundamental conservation of microRNA expression patterns among lamprey, hagfish, and gnathostome organs, implying that the role of microRNAs within specific organs is coincident with their appearance within the genome and is conserved through time. Together, these data support the monophyly of cyclostomes and suggest that the last common ancestor of all living vertebrates was a more complex organism than conventionally accepted by comparative morphologists and developmental biologists.     

The distribution of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of the rhesus monkey. Light microscopic studies using immunoperoxidase technique.

Neural structures containing LHRH were characterized in the hypothalamus of the rhesus monkey by four different antisera to the hormone and an immunoperoxidase technique. Immunoreactive perikarya were present in a continuum from the septal-preoptic region anteriorly to the premammillary nucleus posteriorly. These cells were more concentrated in the pericommissural and tubero-infundibular regions. Reactive axons in the median eminence appeared to originate from the positive perikarya in the medial basal hypothalamus; this projection forms a tubero-infundibular tract containing LHRH. In addition, substantial numbers of fibers which entered the median eminence continued down the infundibular stalk and into the posterior pituitary. Other axons appeared to originate in the pericommisural region and projected to the organum vasculosum of the lamina terminalis. Scattered positive fibers were also present in other hypothalamic areas, especially in the periventricular zone and medical mammillary nucleus.     

Neuroglobin in the rat brain: localization

Neuroglobin (Ngb) is a neuronal hemeprotein similar to myoglobin and hemoglobin and shares their capability for oxygen binding. It has thus been proposed that Ngb acts as an oxygen reservoir or combats reactive oxygen species. In the present study, we investigated the Ngb expression pattern in the rat brain using immunohistochemistry, in situ hybridization, and quantitative real-time PCR (qRT-PCR). This revealed the interesting finding that Ngb expression is restricted to a few neurone populations, many of which are involved in the sleep-wake cycle, circadian regulation or food regulation. In the forebrain we found intense Ngb expression in neurones in the piriform cortex, the central and medial amygdala, the medial preoptic area, the suprachiasmatic nucleus (SCN), the hypothalamic paraventricular nucleus, the perifornical nucleus, the lateral hypothalamus. Within the mid- and hindbrain Ngb expressing neurones were found in the laterodorsal tegmental nucleus, the pedunculo pontine tegmental nucleus, the locus coeruleus, and the lateral parabrachial nucleus. In the medulla oblongata Ngb expressing neurones were found in the nucleus of the solitary tract. The qRT-PCR data showed a diurnal variation of Ngb mRNA in the SCN, having a peak in the day time (light-period) and nadir during night (dark-period).     

Immunocytochemical studies of luteinizing hormone-releasing hormone in brains of agnathan fishes. II. Patterns of immunoreactivity in larval and maturing Western brook lamprey (Lampetra richardsoni).

An immunocytochemical unlabeled antibody enzyme technique has been applied to localize mammalian-like immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in central nervous tissues of Western brook lamprey (Lampetra richardsoni). In larval animals faint specific immunocytochemical staining was found over perikarya in the posterior preoptic nucleus (PON), in fiber tracts passing ventro-posteriorly into the infundibulum, and in nerve endings in the neurohypophysis (NH). In brains of nonreproductive adults, faint staining of cell bodies was observed throughout the PON; staining of NH was intense. In reproductive adults, heavy staining was noted in PON perikarya, in beaded axons containing Herring body-like dilatations, and in NH. Perikarya with ir-LH-RH were distinct from those, presumably vasotocinergic ones, which stained with aldehyde fuchsin; specific immunocytochemical staining was absent in other brain regions and in control preparations. A change in number of cells stained, and in quality of staining was coincident with metamorphosis and sexual maturation; mechanisms by which ir-LH-RH might be transported from the preoptico-neurohypophysial system to the pars distalis to exert any physiological actions have not been established.     

Calcitonin-immunoreactive cells are present in the brains of some cyclostomes.

In the brains of four species of cyclostomes (two species each of lampreys and hagfishes), immunoreactive calcitonin-producing cells (iCT cells) were located immunohistochemically by the peroxidase-antiperoxidase method using anti-salmon calcitonin antiserum. In the case of both the adults and the ammocoetes of the brook lamprey (Lampetra reissneri) which lives in freshwater throughout its life, iCT cells were found in two distinct areas: in the pars ventralis hypothalami of the diencephalon and in the torus semicircularis of the mesencephalon. The iCT cells in the diencephalon are classified as bipolar nerve cells, and those in the mesencephalon are classified as multipolar nerve cells. In both the anadromous and catadromous arctic lamprey (Lampetra japonica), iCT cells were present only in the diencephalon, and those were bipolar nerve cells. There seemed to be no differences in the numbers and the immunostainability of the iCT cells, despite the different environments inhabited by the lampreys. In the hagfishes (Eptatretus burgeir and Paramyxine atami) that inhabit seawater throughout their lives, iCT cells were also found only in the diencephalon, although they were very few in number and exhibited poor immunostainability.