The rostromedial zona incerta is involved in attentional processes while adjacent LHA responds to arousal: c-Fos and anatomical evidence.

Neurons producing melanin-concentrating hormone (MCH) are located in the tuberal lateral hypothalamus (LHA) and in the rostromedial part of the zona incerta (ZI). This distribution suggests that rostromedial ZI shares some common features with the LHA. However, its functions with regard to arousal or feeding, which are often associated with the LHA, have not been thoroughly investigated. This study analyses the responses in the tuberal LHA and adjacent rostromedial ZI after experiments related to arousal, exploration, food teasing and ingestive behavior. Specific aspects of the connections of the rostromedial ZI were also studied using retrograde and anterograde tract-tracing approaches. The rostromedial ZI is activated during exploratory and teasing experiments. It receives specific projections from the frontal eye field and the anterior pole of the superior colliculus that are involved in gaze fixation and saccadic eye movements. It also receives projections from the laterodorsal tegmental nucleus involved in attention/arousal. By contrast, the tuberal LHA is activated during wakefulness and exploratory behavior and reportedly receives projections from the medial prefrontal and insular cortex, and from several brainstem structures such as the periaqueductal gray. We conclude that the rostromedial ZI is involved in attentional processes while the adjacent tuberal LHA is involved in arousal.

Different distributions of preproMCH and hypocretin/orexin in the forebrain of the pig (Sus scrofa domesticus).

Neurons producing melanin-concentrating hormone (MCH) or hypocretin/orexin (Hcrt) have been implicated in the sleep/wake cycle and feeding behavior. Sleep and feeding habits vary greatly among mammalian species, depending in part of the prey/predatory status of animals. However, the distribution of both peptides has been described in only a limited number of species. In this work, we describe the distribution of MCH neurons in the brain of the domestic pig. Using in situ hybridization and immunohistochemistry, their cell bodies are shown to be located in the posterior lateral hypothalamic area (LHA), as expected. They form a dense cluster ventro-lateral to the fornix while only scattered cells are present dorsal to this tract. By comparison, Hcrt cell bodies are located mainly dorsal to the fornix. Therefore, the two populations of neurons display complementary distributions in the posterior LHA. MCH projections are, as indicated by MCH-positive axons, very abundant in all cortical fields ventral to the rhinal sulcus, as well as in the lateral, basolateral and basomedial amygdala. In contrast, most of the isocortex is sparsely innervated. To conclude, the distribution of MCH cell bodies and projections shows some very specific features in the pig brain, that are clearly different of that described in the rat, mouse or human. In contrast, the Hcrt pattern seems more similar to that in these species, i.e. more conserved. These results suggest that the LHA anatomic organization shows some very significant interspecies differences, which may be related to the different behavioral repertoires of animals with regard to feeding and sleep/wake cycles.

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.

The development of the MCH system.

Although a great deal is published on the MCH neurons, very few works were devoted to the study of their development. However, existing literature points out two important traits: first, these neurons differentiate a MCH phenotype very early in all species studied so far, which might suggest a role for the MCH peptide during development; second, in the rat, birth date greatly influence the phenotype of MCH neurons. At least two sub-populations were described on the basis of their chemical phenotype, projection pattern and birth date. The understanding of processes involved in the differentiation of these sub-populations may help understand the medio-lateral differentiation of the tuberal hypothalamus.

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.

Hypocretin/orexin-containing neurons are produced in one sharp peak in the developing ventral diencephalon.

The birth date of hypocretin-containing neurons was analysed using the bromodeoxyuridine method in the rat. The results indicate that these neurons are generated between embryonic days 11 (E11) and E14, with a sharp peak on E12. This spatiotemporal pattern of genesis contrasts with that of the co-distributed neurons producing the melanin-concentrating hormone in the lateral hypothalamic area, which have been described as generated in one large peak from E10 to E16. These observations may be linked to the relative distribution area of both populations.

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.

Expression of the secreted FAD-dependent sulfydryl oxidase (QSOX) in the guinea pig central nervous system.

cpQSOx1 is a member of the QSOx family of proteins, expressed in the guinea pig (Cavia porcellus) and ortholog of the rat rQSOx1. In this study, in vitro experiments were conducted and showed that, as other member of this family, cpQSOx1 has a sulfydryl oxidase activity, and is a secreted protein. Then, the expression of this enzyme was researched in the guinea pig brain, as very little information exists yet on the expression of QSOx family members in the central nervous system. By immunohistochemistry, RT-PCR and in situ hybridization, cpQSOx1 is synthesized by neurons throughout the whole guinea pig central nervous system. Reticular structures as the basal forebrain, reticular thalamic nucleus and reticular nuclei of the brainstem contained the densest labeling. These results are discussed in terms of putative roles of this protein in synaptic strengthening and in redox activities.

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.

Sensitivity of galanin- and melanin-concentrating hormone-containing neurones to nutritional status: an immunohistochemical study in the ovariectomized ewe.

The sensitivities of galanin and melanin-concentrating hormone (MCH) neuronal systems to nutrition are poorly understood in sheep compared to rodents. The aim of this study was to describe the changes in the numbers of galanin and MCH neurones in ovariectomized ewes submitted to different nutritional levels. In the first experiment, ewes were fed ad libitum or food deprived for 24 h. In the second experiment, two groups of ewes were fed at maintenance level (group 100) or undernourished (group 40) for 167 days, after which one-half of each group was killed or refed ad libitum (group 100R and 40R) for 4 days. The MCH neuronal population located in the lateral hypothalamic area was not affected by these nutritional changes. Long-term undernutrition enhanced the number of galanin neurones located in the infundibular nucleus and the dorsal hypothalamic area (DHA), refeeding resulted in an increase of neurones in the DHA and preoptic area, but short-term starvation had no effect on any galanin subpopulations. Our data suggest that the sensitivity of MCH neuronal populations to nutrition in sheep differs from that of rodents. Various populations of galanin-containing neurones differ in sensitivity in ewes subjected to long undernutrition and refeeding but not to short starvation.

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.

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.

Early and transient ontogenetic expression of the cocaine- and amphetamine-regulated transcript peptide in the rat mesencephalon: correlation with tyrosine hydroxylase expression.

The ontogeny of cocaine- and amphetamine-regulated transcript (CART) expression has been analyzed by immunohistochemistry in the mesencephalon of the rat central nervous system, and compared to the pattern of tyrosine hydroxylase- (TH-) expression. CART-producing neurons were first detected on the embryonic day 11 (E11) in the ventral mesencephalic vesicle. These neurons are among the first cells of the mantle layer to differentiate. From E13, a complementary pattern of distribution was observed, dividing the mantle layer into an external TH zone and an internal CART zone. Many TH-positive neurons were found to migrate from the neuroepithelium through the area containing the CART-immunoreactive neurons to settle more laterally. These TH cells exhibited prominent leading and trailing dendrites in the immediate vicinity of CART perikarya. On E16, the number of CART neurons appeared to diminish, and they were confined near the ventricle and around the fasciculus retroflexus. On E18 and E20, only the Edinger-Westphal nucleus exhibited a strong CART staining as described in the adult brain. Thus, the very early detection of CART during prenatal ontogeny led us to speculate that this peptide might have a role in the development of specific regions of the rat brain. In particular, our observations suggest that CART-expressing neurons might help the migration of the dopaminergic neurons of the substantia nigra.

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.

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'.

[Solid cell nests and thyroid pathologies. Retrospective study of 1,390 thyroids]. / <> et pathologies thyroïdiennes. Etude retrospective de 1,390 thyroïdes.

Solid Cell Nests (SCN) of the thyroid gland were considered as embryonic remnants or as squamous metaplasia of follicular epithelium. In a retrospective study of 1 390 thyroids, SCN were found in 9.7 % of thyroids (16 % men and 8 % women, chi(2) : p <0, 01). The number of SCN rises with increasing number of sections examined. No statistical differences were found with age or thyroid disorders and presence of SCN, excluding the metaplastic theory. SCN contained in 38 % of cases, neuroendocrine cells (calcitonin and/or calcitonin gene related peptide, chromogranin A) and thyroglobulin cells was observed in 78.3 % of SCN. The different cell types of SCN fitted with the ultimobranchial remnant theory. Thus, some rare carcinomas (like mixed medullary and vesicular carcinoma or mucoepidermoid carcinoma) might originate from SCN.

An in vitro model for the study of human bone marrow angiogenesis: role of hematopoietic cytokines.

This study describes a human bone marrow endothelial cell culture in which endothelial cells are organized into capillary tubes. These endothelial cells were positive for von Willebrand Factor, expressed CD34, CD31, and L-fucose residues, took up acetylated low-density lipoproteins, contained Weibel-Palade bodies, and were ensheathed in a basal lamina (which included laminin beta1, EDa+ and EDb+ fibronectin, and collagen type iv). Pericytes expressing alpha-smooth muscle (alpha-SM) actin were spatially associated with the capillary tubes and there was a highly significant correlation between the number of capillary tubes and pericytes. In this model, basal angiogenesis was found to be vascular endothelial growth factor (VEGF)-dependent, because neutralization of endogenous VEGF induced a dramatic regression in the number of tubes. However, the presence of alpha-SM actin-expressing pericytes in the linings of endothelial tubes partially prevented the VEGF-neutralized tube regression. We also observed that nitric oxide production contributed to basal angiogenesis and that upregulation of nitric oxide increased the number of tubes. Tube numbers also decreased when antibodies neutralizing the integrin alphavbeta5 were applied to the cultures. Moreover, addition of any of the hematopoietic cytokines, erythropoietin, stem cell factor, granulocytic colony stimulating factor, or granulomonocytic colony stimulating factor induced a highly significant increase in tube formation. When erythropoietin and granulocytic colony stimulating factor were added, this increase was larger than the maximum increase observed with VEGF. Thus, we have described an in vitro model for human bone marrow angiogenesis in which pericytes and basal lamina matrix were associated with endothelial cells and formed fully organized capillary tubes. In this model, cytokines known to regulate hematopoiesis also seemed to be mediators of angiogenesis. This culture system may therefore prove to be a valuable tool for the study of hematopoietic cytokines on angiogenesis.

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.

Ultrastructural features of "solid cell nest" of the human thyroid gland: a study of 8 cases.

The ultrastructural features of solid cell nests (SCN), made of squamous cells, and associated calcitonin cells (C cells), of the thyroid gland were studied in only a few cases in humans. A study was performed on 8 paraffin-embedded SCN, postembedded in Epon, to look for their ultrastructural features. Immunohistochemical analysis using calcitonin antibody was performed on semithin sections of SCN to explore the presence of C cells. Three cases (37.5%) of SCN were positive for calcitonin, and electron-dense secretory granules were observed in the cytoplasm. In two of these cases, an increased number of C cells in the adjacent thyroid parenchyma was observed. The presence of ciliated and lymphoid cells, in addition to intracytoplasmic microvacuolar and microfollicular (microglandular) structures, was noticed. Ciliated cells have already been reported in embryonic rests of human and animals, but ultrastructurally for the first time in human SCN. The presence of microfollicular structures, intracytoplasmic microvacuolar, secretory granules features, and ciliated cells, in addition to lymphoid cell, suggests the existence of a common ultimobranchial stem cell for C cells or for one or more cell types of the thyroid gland.