Bernadette Griffond

Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area

The recent identification of two peptides named hypocretins (Hcrt), and expressed in neurons of the rat tuberal lateral hypothalamus (LHA) previously detected by an ovine prolactin antiserum, led us to revisit some experimental procedures intented to understand the physiological roles of these neurons. In the present study, rats received intraperitoneal injections of insulin and/or glucose. Immunocytochemical observations and quantitation of in situ hybridization signals pointed out a clear stimulation of Hcrt neurons following the sole injection of insulin in hypoglycemic but not in hyperglycemic conditions. This result, together with the robust appetite boosting effect of Hcrt reported elsewhere, suggests the involvement of Hcrt neurons in the control of food intake.

Preprohypocretin (orexin) and prolactin-like immunoreactivity are coexpressed by neurons of the rat lateral hypothalamic area.

Recently, two new neuropeptides named hypocretins I and II (Hcrt) have been described in the rat lateral hypothalamus. The distribution of the neurons expressing these new peptides is similar to that of neurons described in the literature as prolactin (PRL) immunoreactive neurons and specifically labeled by an antiserum (AS) raised against ovine prolactin (oPRL). In this study, we report that every neuron labeled by immunohistochemistry or in situ hybridization for Hcrt is also labeled by the oPRL-AS, and conversely. Furthermore, the labeling of the oPRL-AS is inhibited when this AS is pre-incubated with the 104-109 fragment of the preproHcrt, thus indicating that the oPRL-AS is recognizing an epitope carried by this particular fragment of the preproHcrt. Our previous experimental work on these neurons showed that they may be involved in energy metabolism and water balance, which is in agreement with the current literature about Hcrt functions.

Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear. Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.

Characterization of subpopulations of neurons producing melanin-concentrating hormone in the rat ventral diencephalon.

Neurons producing melanin‐concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the ‘cocaine‐ and amphetamine‐regulated transcript’ (CART) peptide, while another expresses neither one of these two molecules. MCH+/CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/CART– axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART– projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions.

Colocalization of prolactin- and dynorphin-like substances in a neuronal population of the rat lateral hypothalamus

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.

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.

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.

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.

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. J. Comp. Neurol. 411:239–256, 1999.

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.