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.

Double immunostaining for oxytocin (OT) and Fos was used to study the oxytocinergic system of the rat hypothalamic paraventricular nucleus (PVN) following intraperitoneal insulin injections. The expression of c-fos in the PVN appeared about 3 h after insulin treatment and was very high after 5 h while no labelling was observed in isotonic saline-injected animals. Twelve to 18% of OT neurons expressed Fos-like immunoreactivity and these activated neurons were found in both the magno- and the parvocellular compartments of the PVN suggesting that the OT neuron responses to insulin induced disturbances are complex and involve hormonal as well as autonomic pathways.

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 and peptide expression.

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.

Dynorphin B (DYN B) immunoreactivity was recently reported in a population of prolactin (PRL)-immunoreactive neurons of the rat lateral hypothalamus (LH). By coupling immunocytochemistry and in situ hybridization using two synthetic oligonucleotide probes complementary to DYN mRNA, a hybridization signal was observed over the neurons of the LH exhibiting both DYN- and PRL-like immunoreactivities. Our results clearly demonstrate that these neurons contain the mRNA encoding preproDYN and are able to synthesize authentic DYN B in colocalization with a peptide related to PRL.

The role of the lateral hypothalamus (LHT) in the regulation of feeding behavior has long been established. The major contribution of LHT glucose-sensitive neurons, activated by low glucose concentration, is well accepted. Two peptidergic neuron populations, whose perikarya are exclusively located within this area, have been recently described. They respectively produce peptides derived from the melanin-concentrating hormone (MCH) precursor and prolactin-like immunoreactive peptide (PLI). In the present study, hypoglycemia-induced neuron stimulation was assessed in the rat LHT, by using Fos immunocytochemistry 1 to 5 h after a single insulin injection. In control animals, very scarce Fos-like immunoreactivity (FLI)-containing nuclei were observed in LHT. Insulin treatment induced an important and progressive increase in the number and staining intensity of neuron nuclei detected by Fos antiserum. Five hours after injection, few MCH neurons exhibited FLI, but about 80% of PLI-containing neurons expressed FLI and more than 50% of the FLI-containing nuclei belonged to PLI neurons. It was thus concluded that PLI-expressing neurons, which dramatically responded to insulin treatment, might correspond to a subpopulation of the glucose-sensitive LHT neurons.

Using an antiserum (AS) raised against dynorphin B (DYN B), we revealed immunoreactive neurons in different nuclei of the rat hypothalamus as well as a population of neurons scattered in the lateral areas of the posterior hypothalamus. This population corresponds to that previously shown to be specifically recognized by an ovine prolactin (PRL) AS. Our results suggest that in these neurons, DYN B and PRL AS reacted with different epitopes and that these epitopes could be carried by distinct unidentified peptides.

Using a co-culture model, we showed that diffusible factors from arcuate nucleus (AN) specifically increased the number and the size of hypothalamic neurons producing melanin-concentrating hormone (MCH). In this model neurite outgrowth and contacts between MCH neurons and dopaminergic neurons were also prominently increased, as compared to control lateral areas of the posterior hypothalamus (LH) primary cultures. These effects were mediated in part by AN glia but also by neurons of both fetal and adult AN. AN glia produced diffusible factor(s) mainly responsible for an important MCH neurite outgrowth and expressed inhibiting factors, preventing the adhesion of LH cells on AN glial cells. Furthermore, we report here a nerve growth factor-like effect from Broca's diagonal band on MCH hypothalamic neurons.

Annals of the New York Academy of SciencesVolume 680, Issue 1 p. 511-516 Morphofunctional Studies on the Neurons Producing Melanin-Concentrating Hormone D. FELLMANN, D. FELLMANN Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorP. Y. RISOLD, P. Y. RISOLD Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorM. BAHJAOUI, M. BAHJAOUI Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorN. COMPAGNONE, N. COMPAGNONE Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorJ. L. BRESSON, J. L. BRESSON Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorM. C. CLAVEQUIN, M. C. CLAVEQUIN Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorJ. CARDOT, J. CARDOT Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorA. GOUGET, A. GOUGET Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorD. LENYS, D. LENYS Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorC. BUGNON, C. BUGNON Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this author D. FELLMANN, D. FELLMANN Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorP. Y. RISOLD, P. Y. RISOLD Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorM. BAHJAOUI, M. BAHJAOUI Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorN. COMPAGNONE, N. COMPAGNONE Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorJ. L. BRESSON, J. L. BRESSON Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorM. C. CLAVEQUIN, M. C. CLAVEQUIN Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorJ. CARDOT, J. CARDOT Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorA. GOUGET, A. GOUGET Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorD. LENYS, D. LENYS Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this authorC. BUGNON, C. BUGNON Laboratoire d'Histologie CNRS URA 561 Faculté de Médecine 25030 Besancon Cedex, FranceSearch for more papers by this author First published: May 1993 https://doi.org/10.1111/j.1749-6632.1993.tb19724.xCitations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume680, Issue1The Melanotropic PeptidesMay 1993Pages 511-516 RelatedInformation

Antisera (AS) raised against rat melanin-concentrating hormone (rMCH) and against two additional peptides sequences derived from the rat MCH precursor (neuropeptide glutamic acid-isoleucineamide (NEI), and neuropeptide glycine-glutamic acid (NGE)) exclusively stained the hypothalamic neurons previously described using AS to salmon MCH, human somatocrinin 1-37 (GRF37) and alpha-MSH. Liquid phase and dot-blot controls for specificity indicated that rMCH-, NEI- and NGE-AS bound epitopes recognized by sMCH-, alpha-MSH- and GRF-37-AS, respectively. The distinct intracellular patterns of immunoreactivity obtained in control animals with rMCH-, NGE- and NEI-AS, as well as the changes observed after intracerebroventricular injection of colchicine matched previous findings using sMCH-, GRF37- and alpha-MSH-AS.

A serum-free medium culture was developed in order to study the secretory behavior of neurons producing the melanin-concentrating hormone (MCH) precursor. The present results show that our culture conditions (supplemented RPMI 1640, poly-D-lysine substrate) are efficient in promoting attachment and growth of MCH neurons dissociated from rat fetal hypothalamus. These neurons acquire a differentiation stage in which neuropeptides of interest to us are expressed in a pattern similar to that observed on tissue sections: (1) coexpression of salmon MCH, growth-hormone-releasing factor (GRF37), alpha-melanocyte-stimulating hormone and acetylcholinesterase immunoreactivities, and (2) different intracellular distribution of salmon MCH and 1-37 sequence of GRF37 staining. Neurite growth was rapid and interneuronal connections were observed early. These observations suggest that our model of defined medium culture is suitable for functional investigations on MCH neurons.

48 hrs. after an intra-cerebroventricular injection of colchicine (100 micrograms), antisera to three putative peptides included in the rat melanin-concentrating hormone (MCH) precursor, strongly stained the secretory granules accumulated in perikarya. In control rats, these antisera stained endoplasmic reticulum, Golgi apparatus, or neurosecretory granules respectively. Colchicine also induced a dramatic decrease in hybridization signal obtained with a probe complementary to the prepro-MCH-mRNA. Similarly, colchicine induced a strong increase in vasopressin immunoreactivity in neurons of the paraventricular and supraoptic nuclei, and a strong decrease of the vasopressin precursor mRNA. These results demonstrated that, in two peptidergic neuron populations of the rat hypothalamus, colchicine lowers mRNAs and impairs neuropeptide protein synthesis, consecutively to the accumulation of neurosecretory granules in perikarya.

The authors compared the growth of limbal and central explants removed from human cornea. Epithelial growth began on the third day, was greater during the first week and was subsequently slightly invaded by fibroblasts. This study demonstrated that peripheral corneal epithelial cells growth better than central cells. In this study, the cytologic and histologic features of the cells were examined. However, epithelial immunofluorescence staining by anti-cytokeratin antibodies was rather poor. This in vitro system may provide a suitable model for epithelial human corneal cell culture, allowing further biocompatibility studies.

In order to identify the neuropeptide related to melanin-concentrating hormone (MCH) synthetized by neurons of the posterior hypothalamus in the mammals, we have screened rat hypothalamus and rat brain cDNA expression libraries using MCH antiserum. We isolated 5 distinct immunopositive recombinants with cross-hybridizing cDNA inserts. One of them hybridized to RNAs exclusively located in neurons stained by the same antiserum, as seen by successively performing in situ hybridization and then an immunocytochemical technique on the same section. Sequencing of this MCH-like cDNA is in progress.

An oligonucleotide probe corresponding to the 9 C-terminal residues encoded by a complementary DNA of a rat peptide related to salmon melanin concentrating hormone (MCH) was synthetized. It specifically hybridized to the neurons stained by antisera to MCH in the rat posterior hypothalamus, as seen by coupling in situ hybridization and immunocytochemical methods. This result validates our sequence determination. This oligonucleotide will be useful to establish the complete sequence of the rat MCH precursor molecule. It will also constitute a valuable tool to study physiological or experimentally-induced changes in the expression of the rat MCH gene.

We have cloned and sequenced DNAs complementary to the mRNA encoding the precursor of the rat melanin-concentrating hormone. This allowed us to elucidate the primary structure of the 96 C-terminal residues of this precursor. It contains three possible sites for enzymatic cleavage enabling the generation of MCH and of two additional neuropeptides. These three peptides can bind one of our antisera raised against human somatocrinin 1-37, alpha-melanotropin and salmon melanin-concentrating hormone, which immunocytochemically stain the same neuron population in the hypothalamus.

Using an antiserum (AS) raised against rat cerebral acetylcholinesterase (AChE), we revealed a neuron population in lateral and dorsal areas of the posterior rat hypothalamus. These neurons were previously described using antibodies to human growth hormone-releasing factor(1-37) (GRF-37), alpha-melanotropin (alpha-MSH) and melanin-concentrating hormone (MCH). Different intracytoplasmic distributions of the immunodeposits were observed depending on the used serum. Ultrastructural investigations demonstrated that AChE-AS labeled rough endoplasmic reticulum and nuclear envelope in control rats. MCH-AS stained Golgi apparatus in control animals and secretory granules in colchicine-injected rats. GRF-37-AS always revealed secretory granules, and alpha-MSH-AS gave the same staining only after colchicine injection.

An antiserum raised against synthetic salmon melanin-concentrating hormone (MCH) reveals an extensive neuronal system in the posterior lateral areas of the human hypothalamus. These neurons correspond to those previously described in the rat, which are characterized by expression of MCH-like, α-melanotropin-like and human growth hormone-releasing factor (1–37)-like immunoreactivities.