Pierre-Yves Risold

Ontogenetic development of the diencephalic MCH neurons: a hypothalamic 'MCH area' hypothesis.

The ontogeny of rat diencephalic melanin‐concentrating hormone (MCH) neurons has been analysed, using the bromodeoxyuridine method to determine the period of birth of these neurons, and using in situ hybridization and immunohistochemistry to study their chemical differentiation. The spatiotemporal pattern of MCH neuron generation is complex, although it is broadly lateromedial with a peak between embryonic days (E) 12 and E13. The first expression of the MCH gene has been detected on E13 in neurons in the presumptive lateral hypothalamic area. But the adult‐like pattern was observed from E18. Medial‐most MCH neurons express the peptide CART (cocaine‐amphetamine‐regulated transcript) from E18, and the receptor neurokinin 3 (NK3) from between postnatal day (P) 0 and P5. These results are discussed and compared with data from the literature to better understand the organization of the ‘MCH‐containing area’.

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.

Distribution and genesis of the RFRP-producing neurons in the rat brain: comparison with melanin-concentrating hormone- and hypocretin-containing neurons.

Prepro-RFRP-containing neurons have recently been described in the mammalian brain. These neurons are only found in the tuberal hypothalamus. In this work, we have provided a detailed analysis of the distribution of cells expressing the RFRP mRNA, and found them in seven anatomical structures of the tuberal hypothalamus. No co-expression with melanin-concentrating hormone (MCH) or hypocretin (Hcrt), that are also described in neurons of the tuberal hypothalamus, was observed. Using the BrdU method, we found that all RFRP cell bodies are generated between E13 and E14. Thus, RFRP neurons form a specific cell population with a complex distribution pattern in the tuberal hypothalamus. However, they are generated in one peak. These observations are discussed with data concerning the distribution and genesis of the MCH and Hcrt cell populations that are also distributed in the tuberal hypothalamus.

Time of genesis determines projection and neurokinin-3 expression patterns of diencephalic neurons containing melanin-concentrating hormone.

Anatomical and functional evidence suggests that the diencephalic melanin‐concentrating hormone‐ (MCH‐) containing neurons do not form a homogeneous population. In this work, the expression of the neurokinin‐3 receptor (NK3) has been researched in MCH neurons which have been retrogradely labelled following fast blue injections into either the spinal cord or the cerebral cortex. The birth‐date of these cortically and spinally projecting cells has been determined using the bromodeoxyuridine method. The results obtained show that neurons projecting to the spinal cord are born early (E11) and most of them (78,7%) do not express NK3, but neurons that send axons to the cerebral cortex are born later (E12–E13) and most of them (84,8%) express NK3. Both neuronal types are largely intermingled in the lateral hypothalamic area proper. These results are discussed in terms of the functional organization of the MCH neuronal population.

FAD-linked sulfhydryl oxidase QSOX: topographic, cellular, and subcellular immunolocalization in adult rat central nervous system.

The distribution of the sulfhydryl oxidase QSOX in the rat brain was mapped using immunohistochemistry. QSOX is specifically expressed by neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons express QSOX, different intensities of labeling were observed depending on the area: the strongest labeling was observed in the olfactory bulbs, isocortex, hippocampus, basal telencephalon, several thalamic and hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. This study also describes the ultrastructural localization of QSOX in neuronal cells and demonstrates that the enzyme is associated with the Golgi apparatus. Finally, selected double immunohistochemistry showed that in the hypothalamus the highest levels of QSOX labeling were colocalized in neuron populations that express disulfide‐bounded neuropeptides. These observations are consistent with a role of the enzyme in secreted peptide/protein folding. Data presented herein will serve as a basis for further investigations of the physiological function of QSOX in the central nervous system. J. Comp. Neurol. 473:334–363, 2004.

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.

A Comparative Analysis Shows Morphofunctional Differences between the Rat and Mouse Melanin-Concentrating Hormone Systems

Sub-populations of neurons producing melanin-concentrating hormone (MCH) are characterized by distinct projection patterns, birthdates and CART/NK3 expression in rat. Evidence for such sub-populations has not been reported in other species. However, given that genetically engineered mouse lines are now commonly used as experimental models, a better characterization of the anatomy and morphofunctionnal organization of MCH system in this species is then necessary. Combining multiple immunohistochemistry experiments with in situ hybridization, tract tracing or BrdU injections, evidence supporting the hypothesis that rat and mouse MCH systems are not identical was obtained: sub-populations of MCH neurons also exist in mouse, but their relative abundance is different. Furthermore, divergences in the distribution of MCH axons were observed, in particular in the ventromedial hypothalamus. These differences suggest that rat and mouse MCH neurons are differentially involved in anatomical networks that control feeding and the sleep/wake cycle.

MCH and feeding behavior-interaction with peptidic network.

Numerous works associate the MCH peptide, and the hypothalamic neurons that produce it, to the feeding behavior and energy homeostasis. It is commonly admitted that MCH is an orexigenic peptide, and MCH neurons could be under the control of arcuate NPY and POMC neurons. However, the literature data is not always concordant. In particular questions about the intrahypothalamic circuit involving other neuropeptides and about the mechanisms through which MCH could act are not yet clearly answered. For example, which receptors mediate a MCH response to NPY or alpha-MSH, does MCH act alone, is there any local anatomical organization within the tuberal LHA? A review of the current literature is then needed to help focus attention on these unresolved and often neglected issues.

Cell-specific localization of the sulphydryl oxidase QSOX in rat peripheral tissues

Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.

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.