Long-term undernutrition followed by short-term refeeding effects on the corticotropin-releasing hormone containing neurones in the paraventricular nucleus: an immunohistochemical study in sheep

The effect of nutritional level on the immunoreactivity of corticotropin-releasing hormone (CRH) in neurones of the hypothalamic paraventricular nucleus was described in sheep, a ruminant, whose feeding strategy differs from that of monogastric species. Two groups of ewes were underfed (40%), or fed at maintenance (100%) for 167 days, after which one-half of each group was killed or ad libitum refed (at least 150% of maintenance) for 4 days before killing. The presence of CRH in the paraventricular nucleus was examined by immunohistochemistry. The number of CRH immunoreactive neurones was increased in underfed ewes, but without modification of the plasma concentration of cortisol, indicating that the rise of CRH was not released in the portal blood nor linked to the pituitary-adrenal axis. Refeeding did not modify significantly the number of CRH immunoreactive neurones in the nucleus although these neurones were increased, only in refed ewes that were previously underfed. These data differ from those for rats and mice where CRH expression is decreased or not modified by underfeeding which could reflect different effects of undernutrition on CRH immunoreactive neurones in monogastric compared to ruminants species.     

Several subpopulations of neuropeptide Y-containing neurons exist in the infundibular nucleus of sheep: an immunohistochemical study of animals on different diets

Conversely to rodents, the involvement of hypothalamic neuropeptide Y (NPY) neurons in the control of nutrition is poorly understood in ruminants such as sheep. Therefore, the aim of this work was to describe the NPY neurons of the diencephalon in ewes submitted to different diets. In colchicine-treated animals, large populations of NPY-immunoreactive (-ir) neurons were observed in a ventral and a lateral subpopulation of the infundibular nucleus (IN), in the median eminence, the pituitary stalk, and the dorsomedian and dorsocaudal nuclei. No labeled perikaryon was observed in the magnocellular neurons of the hypothalamus, although numerous labeled fibers were noted in the neural part of the pituitary. The pattern of distribution of NPY-ir neurons in the sheep hypothalamus is similar in many ways to those of rodents, but it presents also many specific characteristics that have not been previously described. In ewes that were fasted for 24 hours, or fed ad libitum, the number of NPY-ir neurons was the same whatever the hypothalamic structures. In underfed ewes (40% of maintenance for 24 weeks), the lateral subpopulation of the IN presented a higher number of NPY-ir neurons than observed in the 100% fed ewes. Conversely, in the ventral subpopulation, the animals refed ad libitum (at least 150% of maintenance for 4 days) presented a lower number of NPY-ir neurons than the other groups. The other NPY neuronal populations of the hypothalamus were not significantly modified by the dietary treatments. For the first time, we demonstrated the presence of two functionally distinct subpopulations of NPY neurons in the sheep IN. The variations of labeled neurons were correlated with plasma nonesterified fatty acid levels but not with leptinemia.     

Oestradiol effect on galanin-immunoreactive neurones in the diencephalon of the ewe

Galanin is a neuropeptide involved in the regulation of numerous functions such as reproduction. In female rats, this peptide stimulates gonadotropin-releasing hormone (GnRH)/luteinizing hormone release and its synthesis is stimulated by oestradiol. It could therefore be an intermediary between the oestrogenic signal from the ovaries and the GnRH neurones (e.g. during the time course leading to the preovulatory GnRH surge). However, although the involvement of galanin is well-known in rodents, it is poorly understood in ewes. Using immunohistochemistry with a specific antigalanin antiserum, we detected the peptide in neurones of two groups of ovariectomized ewes treated for 6 h with subcutaneous implants, either with oestradiol (experimental group) or empty (control group). The galanin-immunoreactive neurones were counted in three areas, the preoptic area, the bed nucleus of the stria terminalis and the infundibular nucleus, using a computerized image analysis system. There was no change in the mean number of galanin-immunoreactive (GAL-ir) neurones in the infundibular nucleus (37 +/- 12 neurones/section in treated animals and 31 +/- 11 in controls) or in the bed nucleus of the stria terminalis (22 +/- 5 neurones/section in treated animals and 16 +/- 4 in controls), but the number of GAL-ir neurones was higher in the preoptic area in treated than in control ewes (35 +/- 4 versus 14 +/- 10, P < 0.001). To determine whether the neurones of the preoptic area were directly sensitive to oestradiol, we performed double immunohistochemical labelling for oestradiol receptor alpha and galanin. More than 50% of the GAL-ir neurones contained the oestradiol receptor alpha and therefore could be directly regulated by oestradiol. These results indicate that oestradiol might act directly on a GAL-ir neuronal population situated in the preoptic area, without any effect on the GAL-ir neurones of the infundibular nucleus or the bed nucleus of the stria terminalis. Because a 6-h oestradiol treatment can induce a preovulatory GnRH surge in ewes, the GAL-ir neuronal population of the preoptic area might be one of the neuronal systems by which oestradiol activates the GnRH neurones. However, although the morphological relationships between galanin and GnRH neurones have been described in rodents, they remain to be demonstrated in the ewe.     

Immunoreactive galanin expression in ovine gonadotropin-releasing hormone neurones: no effects of gender or reproductive status

The neuropeptide, galanin, has been implicated to play a significant role in numerous physiological functions, including reproduction. Studies on several species have shown that galanin enhances gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone secretion. In rodents, a subset of GnRH neurones expresses galanin in a sexually dimorphic manner and it has been suggested that this may underpin the differences in GnRH secretion observed between the sexes. However, there are few data available for other species. Previous studies in sheep have shown that the distribution of GnRH neurones overlaps with galanin cells. The primary objectives of our study were to determine whether GnRH and galanin coexist in the sheep brain and, importantly, if a sex difference is apparent in the colocalization of these two peptides. Using immunocytochemistry coupled to high temperature antigen retrieval, we found that all GnRH neurones in the ovine brain colocalize with galanin. There is also a distinct population of galanin neurones that do not secrete GnRH. In addition, the distribution of galanin-immunoreactive cells was similar to that previously reported for colchicine treated ewes and, in agreement with earlier studies, the number of GnRH neurones did not differ between rams and ewes or between ewes killed at different stages of the oestrous cycle. These results suggest that, in sheep, GnRH and galanin may be cosecreted but the functional significance of this coexpression and possible cosecretion remains to be elucidated.     

Effect of feeding on Fos protein expression in sheep hypothalamus with special reference to the supraoptic and paraventricular nuclei: an immunohistochemical study

The hypothalamus plays an important role in the control of food intake in different species, but there is little relevant information for ruminants like sheep. In order to study the putative role of several hypothalamic nuclei in food intake in sheep, Fos expression, a marker of cellular activity, was compared by immunohistochemistry between fed and unfed ewes. The expression of Fos protein was stimulated in the supraoptic nucleus of fed ewes, whereas it was increased in the paraventricular nucleus of unfed animals. In the latter nucleus, Fos immunoreactivity was mainly localized close to the third ventricle, an area corresponding to the parvocellular system of the nucleus, but never in the magnocellular system. In the paraventricular nucleus, the number of corticotrophin releasing factor-immunoreactive neurons and the number of Fos/corticotrophin releasing factor double-labelled neurons were not affected by feeding or lack of feeding. The number of Fos-immunoreactive neurons was higher in the lateral septum, the infundibular, the ventromedial and in the dorsomedial nuclei of unfed ewes than in those of fed ewes. Our results show for the first time that the dorsomedial and ventromedial nuclei are involved in the control of feeding in sheep as in rodents. The supraoptic nucleus of sheep is activated by the same conditions as in rodents but, conversely, the paraventricular nucleus is activated in unfed sheep, whereas in rodents and primates, this nucleus is activated by satiety as well as by fasting. In sheep, unlike in rodents, corticotrophin releasing factor did not appear to be involved in short-term regulation of food intake.     

Effect of nutritional stress on the hypothalamo-pituitary-gonadal axis in the growing male rat

BACKGROUND/OBJECTIVE: Nutritional dwarfing (ND) consists of a decrease in weight and height gain and delayed onset of puberty. The aim of the present investigation was to study the modifications induced in male rats by the nutritional stress of a mere 20% reduction in food intake which, however, started immediately after weaning. MATERIALS AND METHODS: At weaning, male Wistar rats were divided into two groups: Control (C) and ND. C rats were fed ad libitum with a balanced rodent diet. ND received 80% of the diet consumed by C for 4 weeks (T4); then they were fed ad libitum for another 4 (T8) and 8 weeks (T12). The rats were studied at T0, T4, T8 and T12 for the effects of nutritional stress and refeeding on nutritional status, body composition, hypothalamic-pituitary-gonadal axis, and sperm morphology and concentration. RESULTS: ND body weight and length diminished vs. C (p < 0.001). ND body fat percentage decreased 40% (p < 0.001) without change in the percentage of body protein content. The hypothalamic content of LHRH did not change. However, FSH, LH and testosterone serum levels had significantly decreased (p < 0.001) at T4 in ND rats. A 48.4 % decrease in serum leptin in the ND group was observed at T4 (p < 0.05). The absolute testicular and seminal vesicle weight was significantly decreased by ND at T4 (p < 0.001). At T4 the percentage of anomalies of caudal spermatozoa increased in about 64% (p < 0.001) of ND vs. C rats, despite the unchanged sperm concentrations. All parameters normalized during refeeding. CONCLUSION: In this model, a decrease in leptin due to nutritional stress could be responsible, at least in part, for the inhibition of reproductive function. Refeeding normalized all parameters studied.     

Interleukin-6 receptor α is co-localised with melanin-concentrating hormone in human and mouse hypothalamus

Interleukin (IL)-6 deficient mice develop mature-onset obesity. Furthermore, i.c.v. administration of IL-6 increases energy expenditure, suggesting that IL-6 centrally regulates energy homeostasis. To investigate whether it would be possible for IL-6 to directly influence the energy homeostasis via hypothalamic regulation in humans and rodents, we mapped the distribution of the ligand binding IL-6 receptor α (IL-6Rα) in this brain region. In the human hypothalamus, IL-6Rα-immunoreactivity was detected in perikarya and first-order dendrites of neurones. The IL-6Rα-immunoreactive (-IR) neurones were observed posterior to the level of the interventricular foramen. There, IL-6Rα-IR neurones were located in the lateral hypothalamic, perifornical, dorsal and posterior hypothalamic areas, the hypothalamic dorsomedial nucleus and in the zona incerta. In the caudal part of the hypothalamus, the density of the IL-6Rα-IR neurones gradually increased. Double-labelling immunofluorescent studies demonstrated that IL-6Rα immunoreactivity was localised in the same neurones as the orexigenic neuropeptide, melanin-concentrating hormone (MCH). By contrast, IL-6Rα-immunoreactivity was not observed in the orexin B-IR neurones. To determine whether the observed expression of IL-6Rα is evolutionary conserved, we studied the co-localisation of IL-6Rα with MCH and orexin in the mouse hypothalamus, where IL-6Rα-immunoreactivity was present in numerous MCH-IR and orexin-IR neurones. Our data demonstrate that the MCH neurones of the human hypothalamus, as well as the MCH and orexin neurones of the mouse hypothalamus, contain IL-6Rα. This opens up the possibility that IL-6 influences the energy balance through the MCH neurones in humans, and both MCH and orexin neurones in mice.     

Two olfactory placode derived galanin subpopulations: luteinizing hormone-releasing hormone neurones and vomeronasal cells

In adult rodents, the peptide galanin is expressed in a subpopulation of hypothalamic luteinizing hormone-releasing hormone (LHRH) neurones in an activity-dependent manner. In this investigation, we examined whether galanin mRNA expression in mice was activated coincident with LHRH mRNA expression, as LHRH neurones differentiate from the olfactory placode. Using in situ hybridization, we show (i) that galanin mRNA is coexpressed in LHRH neurones prenatally, (ii) that there is a decrease in galanin mRNA expression relative to LHRH mRNA expression once LHRH mRNA positive/galanin mRNA positive neurones migrate out of the olfactory pit and into the nasal septum, and (iii) the presence of a novel population of galanin mRNA positive/LHRH mRNA negative expressing neurones in the olfactory pit/vomeronasal organ which do not migrate into the central nervous systenm (CNS). This study demonstrates that there are at least two populations of galanin mRNA expressing neurones arising from the olfactory placode; one that remains in nasal regions, is LHRH mRNA negative and whose function is unknown, and one which is coexpressed with LHRH. In addition, the temporal expression of galanin mRNA in LHRH cells indicates that initial activation and subsequent inactivation of galanin mRNA expression is independent of synaptic CNS connections.