The vertebrate diencephalic MCH system: a versatile neuronal population in an evolving brain.

Neurons synthesizing melanin-concentrating hormone (MCH) are described in the posterior hypothalamus of all vertebrates investigated so far. However, their anatomy is very different according to species: they are small and periventricular in lampreys, cartilaginous fishes or anurans, large and neuroendocrine in bony fishes, or distributed over large regions of the lateral hypothalamus in many mammals. An analysis of their comparative anatomy alongside recent data about the development of the forebrain, suggests that although very different, MCH neurons of the caudal hypothalamus are homologous. We further hypothesize that their divergent anatomy is linked to divergence in the forebrain - in particular telencephalic evolution.

Ontogenetic expression of CART-peptides in the central nervous system and the periphery: a possible neurotrophic role?

Little attention has been devoted to the expression of CART during development. However, a few studies in the central nervous system and periphery provide a clear indication that these peptides may play significant roles during histogenesis, and may have trophic actions.

Early detection of secretogranin-II (SgII) in the human fetal pituitary: immunocytochemical study using an antiserum raised against a human recombinant SgII.

Secretogranin-II (SgII) is a protein contained within secretory granules of mainly gonadotrophs. The purpose of this study was to determine whether SgII immunoreactivity (SgII-IR) in the human fetal pituitary was temporally related to gonadotropin immunoreactivity. A specific antihuman SgII antiserum was thus required. A complementary DNA clone with an open reading frame for human (h) SgII was synthesized by reverse transcription-polymerase chain reaction from pituitary total RNA. This clone was used to obtain the SgII polypeptide (-9 to 152) as a fusion protein, in a heterologous expression prokaryotic system. Antisera against the fusion protein were raised in rabbits and checked for specificity and sensitivity through Western blotting. Human fetal pituitaries from week 6 of gestation onward were used for immunocytochemical studies. Consecutive semithin sections were treated with the specific antisera against hSgII, beta-endorphin, and hPRL and with monoclonal antibodies to hCG alpha, hLH, and hFSH. SgII immunoreactivity appeared at week 8 and was restricted to pituitary cells expressing beta-endorphin (100% colocalization). At week 9, FSH-positive cells did not contain SgII. From week 10, gonadotrophs progressively exhibited SgII-IR, up to 50% of that in FSH-containing cells at week 26. The granin was never found in PRL cells whatever the stage of development. The present data demonstrate that SgII-IR is detected very early in fetal life; however, the positive cells are not gonadotrophs, but corticotrophs. Within gonadotrophs, SgII appears subsequent to hormones. At birth, more than 90% of SgII-IR cells are represented by corticotrophs and gonadotrophs.

Melanin-concentrating hormone-producing neurons in birds.

The peptidergic melanin-concentrating hormone (MCH) system was investigated by immunocytochemistry in several birds. MCH perikarya were found in the periventricular hypothalamic nucleus near the paraventricular organ and in the lateral hypothalamic areas. Immunoreactive fibers were very abundant in the ventral pallidum, in the nucleus of the stria terminalis, and in the septum/diagonal band complex, where immunoreactive pericellular nets were prominent. Many fibers innervated the whole preoptic area, the lateral hypothalamic area, and the infundibular region. Some fibers also reached the dorsal thalamus and the epithalamus. The median eminence contained only sparse projections, and the posterior pituitary was not labeled. Thus, in birds, a neurohormonal role for MCH is not likely. Immunoreactive fibers were observed in other regions, such as the intercollicular nucleus, stratum griseum periventriculare (mesencephalic tectum), central gray, nigral complex (especially the ventral tegmental area), reticular areas, and raphe nuclei. Although no physiological investigation concerning the role of MCH has been performed in birds, the distribution patterns of the immunoreactive perikarya and fibers observed suggest that MCH may be involved in functions similar to those described in rats. In particular, the projections to parts of the limbic system (ventropallidal ganglia, septal complex, hypothalamus, dorsal thalamus, and epithalamus) and to structures concerned with visceral and other sensory information integration suggest that MCH acts as a neuromodulator involved in a wide variety of physiological and behavioral adaptations (arousal) with regard to feeding, drinking, and reproduction.

Diencephalic neurons producing melanin-concentrating hormone are influenced by local and multiple extra-hypothalamic tachykininergic projections through the neurokinin 3 receptor.

As melanin-concentrating hormone (MCH) neurons express the neurokinin 3 receptor (NK3) in the rat diencephalon, their innervation by tachykininergic fibers, the origin of this innervation and the effect of a NK3 agonist on MCH mRNA expression were researched. The obtained results show that the tachykininergic system develops complex relationships with MCH neurons. Overall, MCH cell bodies appeared targeted by both NKB- and SP-inputs. These afferents have multiple hypothalamic and extra-hypothalamic origins, but a local (intra-lateral hypothalamic area) origin from small interneurons was suspected as well. MCH cell bodies do not express NK1, but around 2.7% of the MCH neurons contained SP after colchicine injection. Senktide, a NK3 agonist, produced an increase of the MCH mRNA expression in cultured hypothalamic slices. This effect was reversed by two NK3 antagonists. Tachykinins enhance MCH mRNA expression, and, thus, may modulate the effect of MCH in functions such as feeding and reproductive behaviors in which this peptide has been experimentally involved.

Rat diencephalic neurons producing melanin-concentrating hormone are influenced by ascending cholinergic projections.

Innervation of diencephalic neurons producing melanin-concentrating hormone by choline acetyltransferase-containing axons was examined using double immunohistochemistry. In the rostromedial zona incerta and perifornical regions of the lateral hypothalamic area, many choline acetyltransferase-positive fibers were detected in the immediate vicinity of melanin-concentrating hormone perikarya and their proximal dendrites. Putative contact sites were less abundant in the far lateral hypothalamus, and only scattered close to the third ventricle. After injections of the retrograde tracer FluoroGold, most of these projections appeared to originate in the pedunculopontine and laterodorsal tegmental nuclei. Finally, to determine the putative effect of acetylcholine on the melanin-concentrating hormone neuron population, the cholinergic agonist carbachol was added to the medium of hypothalamic slices in culture. Using competitive reverse transcriptase-polymerase chain reaction, carbachol was found to induce a rapid increase in the melanin-concentrating hormone messenger RNA expression. This response was abolished by both atropine, a muscarinic antagonist, and hexamethonium, a nicotinic antagonist. Thus, the bulk of these results indicates that the diencephalic melanin-concentrating hormone neurons are targeted by activating ascending cholinergic projections.

Evidence of melanin-concentrating hormone-containing neurons supplying both cortical and neuroendocrine projections.

In the rat, melanin-concentrating hormone-containing projections are detected in the median eminence and in the neural lobe of the pituitary. After vascular injections of the retrograde tracers fluorogold or fastblue, melanin-concentrating hormone neurons are retrogradely labeled in the rostromedial zona incerta and adjacent perifornical region. These neurons may be the source of the melanin-concentrating hormone projections toward the median eminence and posterior pituitary, and may release their secretory products into the bloodstream. After fastblue injections in the cerebral cortex and vascular fluorogold injections, some melaninconcentrating hormone neurons contain both tracers, indicating that they send collaterals in the cerebral cortex and in the median eminence/posterior pituitary. No such collaterals have been described for the classical neuroendocrine systems. The melanin-concentrating hormone system is thought to play a role in arousal in correlation with specific goal oriented behaviors such as feeding or reproduction. Some MCH neurons may be involved in such functions by modulating directly cortical activity as well as being neuroendocrine.

QSOX sulfhydryl oxidase in rat adenohypophysis: localization and regulation by estrogens.

The expression of the rat quiescin sulfhydryl oxidase (rQSOX) and its putative regulation by estrogens were investigated in the adenohypophysis. Immunohistochemical observations revealed that rQSOX protein is abundantly expressed throughout the anterior lobe of the pituitary, and can be found in almost all the different cell populations. However, as shown by double immunohisto-chemistry, the cells displaying the strongest rQSOX labeling belong to a subset of gonadotrophs. Immunoelectron microscopy showed that, in adenohypophyseal cells, the protein is linked to the membranes of the rough endoplasmic reticulum, the Golgi apparatus and to dense-core secretory granules. These results are consistent with the secretion of the protein and its presumed role in the extracellular matrix. According to its sulfhydryl oxidase function, rQSOX could also participate in the intracellular folding of secreted proteins or hormones like LH and FSH and act as an endogenous redox modulator of hormonal secretion. A semiquantitative RT-PCR analysis of rQSOX level across the estrous cycle and the fact that chronic administration of 17 beta-estradiol to ovariectomized rats led to a sustained up-regulation of rQSOX in the pituitary suggest that rQSOX expression is controlled by sex hormone levels. Further investigations are needed in order to elucidate its precise roles in that gland and the mechanisms of its regulation.

Down regulation of melanin concentrating hormone in virally induced obesity.

Obesity is a complex disease involving genetic components and environmental factors and probably associated with the dysregulation of central homeostasis normally maintained by the hypothalamic neuroendocrine/neurotransmitter network. We previously reported that canine distemper virus (CDV), which is closely related to human measles virus, can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection. Here, using differential display PCR, we demonstrate specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected-obese mice. Although ppMCH was down-regulated in all infected mice during the acute stage of infection, this was only seen during the late stage of infection in infected-obese mice. In addition, ppMCH mRNA and protein expression in the lateral hypothalamus was decreased in the absence of neuronal death. These results show the importance of ppMCH in the establishment and maintenance of obesity and the involvement of a virus as an environmental factor.

Immunocytochemical detection of the neurokinin B receptor (NK3) on melanin-concentrating hormone (MCH) neurons in rat brain.

The presence of the neurokinin B receptor (NK3 receptor) in the rat lateral hypothalamus and the zona incerta was previously reported. The aim of the present study was to define its cellular localization in these areas. Investigations, coupling immunocytochemical and in situ hybridization techniques, focussed on two neuron populations: the melanin-concentrating hormone (MCH) neurons and a population of neurons recognized by an ovine prolactin antiserum (PRL-ir neurons). While PRL-ir neurons did not exhibit NK3 immunoreactivity, 57% +/- 6% of MCH neurons were strongly stained by the NK3 antiserum. These results suggest that neurokinin B is involved in the regulation of MCH neuron activity via the NK3 receptor; they provide new bases for further investigations on MCH role in the control of food and water intake.

Occurrence of secretogranin II in the prolactin-immunoreactive neurons of the rat lateral hypothalamus: an in situ hybridization and immunocytochemical study.

The occurrence of secretogranin II in a neuron population of the rat lateral hypothalamus specifically detected by an anti-serum to ovine prolactin was examined. As this population was previously reported to synthesize dynorphin, the distribution of neurons recognized by ovine prolactin-, dynorphin B- and secretogranin II anti-sera was investigated on adjacent sections of hypothalami. The prolactin immunoreactive neurons were the only cells in the lateral hypothalamus to be stained by secretogranin II anti-serum. Moreover, coupling immunocytochemical detection and in situ hybridization with an oligonucleotide probe complementary to secretogranin II mRNA showed that these neurons expressed the secretogranin II gene. These new findings should help to study the physiological role of the prolactin immunoreactive neurons of the lateral hypothalamus.

Alteration of the expression of the hypocretin (orexin) gene by 2-deoxyglucose in the rat lateral hypothalamic area.

Following an i.p. injection of 2-deoxyglucose (2DG), a nonmetabolizable analogue of glucose known to induce intracellular glucopenia, a progressive decrease in the level of hypocretin (Hcrt)/orexin mRNA was observed in the rat lateral hypothalamus while the melanin-concentrating hormone (MCH) expression in neighbouring neurons remained unaffected. This result together with the previously reported stimulation of Hcrt expression by insulin confirms that Hcrt neurons, but not MCH neurons, are sensitive to glucose availability and suggests that they respond through different mechanisms and/or different pathways to intracellular glucopenia and hypoglycemic conditions.

Isolation of FMRFa-like peptides from the DB-containing connective tissue of Helix aspersa.

The endocrine dorsal bodies (DB) of Helix aspersa are innervated by axons from the central nervous system, which establish synapse-like structures (SLS) with the DB cells. Previous immunocytochemical studies suggested the presence of FMRFa-like substances in nerves of the DB area and in SLS. This paper reports on biochemical attempts undertaken in order to investigate the nature of these substances: the use of HPLC and RIA confirms the presence of three FMRFa-like peptides in the DB-containing connective tissue among which one is probably the FMRFa itself.

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.

Insulin treatment stimulates the rat melanin-concentrating hormone-producing neurons.

Melanin-concentrating hormone (MCH) is involved in the regulation of body colour in teleost fish. A peptide highly homologous to salmon MCH has been found in the rat brain, but its physiological functions have not yet been precisely defined. The location of MCH neurons in the lateral hypothalamus (LHT) of the rat suggests possible implication in feeding behaviour. In the present study, immunohistochemical and in situ hybridization methods were used to investigate MCH gene expression following insulin injections. Five hours after insulin injection, a significant increase in the abundance and staining intensity of MCH immunoreactive perikarya and fibres was observed. Concurrently the level of MCH mRNA significantly increased (50%). Insulin-treatment also induced a marked and progressive increase in the number and staining intensity of nuclei detected by a Fos antiserum in LHT and other brain areas. Double labelling technique demonstrated that very few if any MCH neurons exhibited Fos-like immunoreactivity. These results demonstrate that an insulin-treatment stimulates MCH neuron activity without the mediation of the proto-oncogene c-fos. The mechanisms triggering this activation remain to be elucidated.

The synthesis of melanin-concentrating hormone is stimulated by ventromedial hypothalamic lesions in the rat lateral hypothalamus: a time-course study.

The activity of melanin-concentrating (MCH) neurons, was investigated by immunocytochemical and hybridocytochemical techniques in male rats bearing limited lesions of the ventromedial hypothalamic nuclei (VMN). 2 days after operation, the abundance of immunoreactive cell bodies and fibres and the intensity of labelling seemed slightly decreased in lesioned rats as compared to controls while no significant difference could be detected in MCH gene expression. After 8 days, synthesis, storage and transport of MCH appeared strongly stimulated and this stimulation lasted until the end of the experiment (day 35), suggesting that VMN plays a physiological role in controlling MCH neuron activity.

Ontogenic development of prolactin immunoreactive neurons in the rat lateral hypothalamus.

The present study investigated the ontogenic expression of a prolactin-like substance (oPRL-ir) in rat hypothalamus from embryonic day (E) 17 to postnatal day (P) 29. By immunocytochemistry, the oPRL-ir peptide was only detected from P3. As in adults, labeled neurons were found exclusively in the lateral hypothalamic area. By in situ hybridization, with a cocktail of oligonucleotides complementary to the PRL mRNA, no labeling was observed in the hypothalamus, although dense labeling was obtained over the pituitary. With reverse-transcriptase polymerase chain reaction, a 408 bp band, presumably corresponding to an oPRL mRNA, was detected from PO in the LHA, but also in other brain regions. These results suggest that the oPRL-ir neurons do not contain oPRL. The nature of the oPRL-ir peptide is still unknown, but its late onset of expression may be related to its putative involvement in feeding behavior.

Activation of the rat melanin-concentrating hormone neurons by ventromedial hypothalamic lesions.

The occurrence of a melanin-concentrating hormone-like peptide (MCH) was previously reported in the lateral hypothalamus of the rat. The sequence of this peptide was determined but its role as well as its regulation remain unclear. In the present study, we examined the effects of minor electrolytic lesions of the ventromedial nuclei (VMN) on MCH neurons by using immunocytochemical and in situ hybridization procedures. We report that VMN lesions resulted in (1) a clear elevation in the number and staining intensity of MCH immunoreactive perikarya and fibres, (2) a significant increase in the level of hybridocytochemical signal obtained with an oligonucleotide probe complementary to rMCH mRNA. These data provide evidence for a role of VMN in modulating the MCH gene a peptide expression.

Insulin-like peptide(s) in the central nervous system of the snail Helisoma duryi.

The central nervous system of the snail Helisoma duryi contains porcine insulin-immunoreactive cells. Most of these cells are known as neurosecretory mediodorsal cells (MDC), and are involved in regulation of growth. There are about 25-40 large and small MDC in each cerebral ganglion near the commissure. Besides the MDC, 6-8 insulin-immunoreactive cells are also seen in the left parietal ganglion and the visceral ganglion. Insulin-immunoreactive material can be traced from the MDC perikarya into their axons in the median lip nerve. The elementary granules of the MDC are 150-250 nm in diameter, and are insulin-immunoreactive. Insulin-like material is released by the CNS in vitro, and can be measured by radioimmunoassay using an antiserum against human insulin. High potassium and 4-aminopyridine increase such release in vitro. It is likely that insulin-like peptide is a growth hormone in H. duryi.

Prolactin immunoreactive neurons of the rat lateral hypothalamus: immunocytochemical and ultrastructural studies.

A population of neurons immunoreactive to an antiserum (AS) raised against ovine prolactin (LHPLI neurons) was previously described in the rat perifornical areas and lateral hypothalamus. In the present paper, we demonstrate by complementary immunocytochemical studies using AS to various biologically active peptides or neurotransmitters that these neurons are also detected by AS to bradykinin and to dynorphin B. Electron microscope examination shows that the LHPLI neurons are peptidergic neurons synthesizing apparently only one type of secretory granules. Numerous synapses on their perikarya and processes reflect the complexity of their relationships with other neuron populations, which have yet to be mapped and elucidated.