Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain.

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.

Evidence that projections from the bed nucleus of the stria terminalis and from the lateral and medial regions of the preoptic area provide input to gonadotropin releasing hormone (GNRH) neurons in the female sheep brain.

The arcuate nucleus/ventromedial hypothalamic nucleus (ARC/VMH) region is thought to relay estrogen feedback signals to gonadotropin-releasing hormone (GnRH) cells in the sheep brain. This region sends major projections to the lateral preoptic area (lPOA), ventral bed nucleus of the stria terminals (vBnST) and the ventro-caudal division of the median preoptic nucleus (vcMePON) with little direct input to GnRH cell bodies, suggesting interneuronal relay to GnRH neurons. The brain stem also provides input to the POA. The present study aimed to identify possible relay circuits in the POA and BnST to GnRH neurons. Biotinylated dextran amine (BDA) was injected into lPOA (n=6), vBnST (n=2), vcMePON (n=3) and periventricular nucleus (PeriV; n=1) of ewes for anterograde tracing. GnRH immunoreactive (IR) perikarya appearing to receive input from BDA-containing varicosities were identified by fluorescence microscopy, with further analysis by confocal microscopy. When BDA was injected into rostral and caudal regions of lPOA (n=3), no tracer-filled varicose fibers were found in contact with GnRH-IR perikarya. Injections into the center of the lPOA (n=3) indicated direct projections to GnRH-IR cells. Injections into the vBnST, vcMePON and PeriV indicated that cells of these regions also provide input to GnRH cells. BDA-containing varicosities found in the MPOA were immunoreactive for NPY or were GABAergic or glutamatergic when the tracer was injected into vBnST and lPOA, but not when injections were placed in the vcMePON. With injection into the PeriV, tracer-filled varicosities in the MPOA were not immunoreactive for somatostatin or enkephalin. Injection of FluoroGold into ventral POA retrogradely labeled cells in the above mentioned areas, but few were also immunoreactive for estrogen receptor-alpha. Thus, cells of the vBnST, lPOA, vcMePON and PeriV project to GnRH neurons. These cells may provide an interneuronal route to GnRH neurons from the ARC/VMH, the brain stem and other regions of the brain.

Long-term alteration in bodyweight and food restriction does not affect the gene expression of either preproorexin or prodynorphin in the sheep.

Various hypothalamic neuropeptides are involved in central regulation of food intake and expression of genes encoding these peptides changes with alterations in the bodyweight/metabolic status/nutritional status. Orexin(s) and dynorphin have been implicated in the regulation of appetite and neuroendocrine systems, but the function of these peptides is not well understood. We have employed in situ hybridization to examine the effects of long-term alterations in the bodyweight on expression of mRNA for preproorexin and prodynorphin in the putative feeding centers of the ovine hypothalamus. Expression of preproorexin was localized to the dorsomedial hypothalamic nucleus, perifornical area and lateral hypothalamic area. Cells expressing prodynorphin were localized to the periventricular, supraoptic, paraventricular, ventromedial hypothalamic nuclei and the thalamus. Small numbers of single scattered cells were seen in other brain areas. A few scattered prodynorphin-expressing cells were found in the lateral hypothalamic area but, in contrast to observations in the rat, there was no colocalization with preproorexin. Long-term alterations in the bodyweight did not influence the level of expression of preproorexin or prodynorphin. These findings suggest that orexin and dynorphin may not play a direct role in appetite regulation in sheep, although regulation at the level of the receptors for these peptides remains a possibility.

Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus.

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.

Immunohistochemical characterization of localization of long-form leptin receptor (OB-Rb) in neurochemically defined cells in the ovine hypothalamus.

Leptin, a hormone secreted from the adipose tissue, is involved in the regulation of food intake and neuroendocrine function, by modulation of the expression and/or function of various neuropeptides in the hypothalamus. The long isoform (OB-Rb) is the major signaling form of the leptin receptor in the hypothalamus. We have used double-labeling immunohistochemistry to examine the extent of OB-Rb expression in neurochemically defined cell types in the ovine hypothalamus. OB-Rb-like immunoreactivity was widespread within cells localized to the periventricular, paraventricular, supraoptic, dorsomedial hypothalamic, ventromedial hypothalamic and arcuate nuclei, as well as the median eminence, perifornical, anterior hypothalamic and lateral hypothalamic areas and the zona incerta. Double-labeling showed expression of OB-Rb in 59.6+/-6.0% neuropeptide Y-containing cells, 60.8+/-4.7% galanin-containing cells, 89.8+/-2.65% pro-opiomelanocortin-containing cells, 73.4+/-3.5% tyrosine hydroxylase-containing cells and 31.8+/-2.8% corticotropin-releasing factor-containing cells. Interestingly 100% of melanin-concentrating hormone and orexin positive cells were also OB-Rb immunoreactive. These data provide semi-quantitative information on the extent to which various cell types express OB-Rb in the hypothalamus. Expression of OB-Rb within specific neuropeptidergic neurons provides evidence for the direct action of leptin upon the various neurochemical systems that regulate food intake, neuroendocrine and autonomic function in the brain.

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus.

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals. These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.

Localization of long-form leptin receptor in the somatostatin-containing neurons in the sheep hypothalamus.

Reduction in the adiposity or dietary restriction increases plasma growth hormone (GH) concentrations, and in sheep this appears to be due, at least in part, to a reduction in the concentrations of somatostatin (SRIF) in hypophyseal portal blood. Leptin is a hormone secreted by the adipocytes and it is possible that the effects of altered adiposity or fasting on GH secretion could be due to regulation of SRIF neurons by leptin. To ascertain the extent to which leptin may act on these neurons, we have used immunohistochemistry to examine co-localization of long-form of the leptin receptor (OB-Rb) and SRIF in the sheep hypothalamus. In the hypothalamic periventricular area (PeriV), 44.5+/-10% of SRIF cells were found to co-stain for OB-Rb. In the dorsomedial hypothalamic, ventromedial hypothalamic and arcuate nuclei, 100% of SRIF immunoreactive neurons expressed OB-Rb. These findings provide a basis for the direct action of leptin on SRIF neurons. Thus, it is possible that leptin stimulates the secretion of SRIF in relatively obese individuals. The significance of the lower number of SRIF cells in the PeriV co-localizing OB-Rb expression is not clear at present.

Shapes and projections of neurons with immunoreactivity for gamma-aminobutyric acid in the guinea-pig small intestine.

The distribution of nerve cell bodies and fibres with immunoreactivity for gamma-aminobutyric acid (GABA) has been studied in the guinea-pig small intestine. Cell bodies were common in myenteric ganglia but were extremely rare in the submucosa. Reactive fibres were numerous in the tertiary component of the myenteric plexus and in the circular muscle but they were rare in both myenteric and submucous ganglia. Reactive nerve fibres were absent from the mucosa. This distribution conforms to previous descriptions. Exposure to exogenous GABA, in vitro, was used to supplement endogenous stores of GABA. The morphology of cell bodies was better defined after this treatment. Nearly all cell bodies had type-I morphology, i.e., the cells had numerous short lamellar dendrites and one axon. Most axons ran anally. Some could be traced to the tertiary component of the myenteric plexus, others to the circular muscle. Removal of the myenteric plexus from a short length of intestine caused a loss of nerve fibres from the circular muscle beneath the site of operation and a decrease in fibre density in the circular muscle that extended anally from the lesion for about 1 mm. The nerve lesions caused no significant changes in the tertiary plexus. It is concluded that GABA is contained in motor neurons supplying the longitudinal and circular muscle, and that the neurons supplying the circular muscle may be inhibitory.