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 neuro peptides implicated in feeding and drinking behaviour as well as neuroendoc rine 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 hypothal amus. ORX immunoreactive cells were distributed in the dorsomedial hypothal amic nucleus, lateral hypothalamic area, zona incerta and perifornical area ; a few cells were also observed in the anterior hypothalamic area. in cont rast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical are a; scattered cells are found in lateral hypothalamic area. ORX immunoreacti ve 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. T hirty 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 retrograd ely labelled a subpopulation of ORX cells in the lateral hypothalamic/perif ornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an in tegral role for ORX in the regulation of GnRH cells in the sheep and thus p rovide evidence of a novel mechanism whereby leptin can influence reproduct ive neuroendocrine function.