Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

Kisspeptin signaling via the kisspeptin receptor G-protein-coupled receptor-54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin-releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin-immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin-immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual-immunolabeled specimens, fine kisspeptin-immunoreactive axon varicosities formed axo-somatic, axo-dendritic and axo-axonal contacts with GnRH neurons. Dual-immunofluorescent studies established that 77% of kisspeptin-immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site-specific co-localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin-immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin-immunoreactive fibers and GnRH cells, and demonstrates co-synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

Human genetics indicate that kisspeptin and neurokinin B (NKB) signaling are necessary for generating pulsatile LH release and therefore for initiation of puberty and maintaining gonadal function. In the present study, male monkeys were employed to examine 1) whether activation of the NKB receptor (NK3R) is associated with GnRH release, and 2) hypothalamic localization of these peptides using immunofluorescence histochemistry. Agonadal juveniles, in which pituitary responsiveness to GnRH was heightened by GnRH priming, were employed to indirectly examine GnRH-releasing actions of NK3R and kisspeptin receptor agonists by tracking LH after their i.v. injection. Castrated adults were used for immunohistochemistry. Single i.v. injections of NKB or senktide (an NK3R agonist) elicited robust LH discharges that were abolished by GnRH receptor antagonism (acyline) confirming the ligands' hypothalamic action. Intermittent infusion of senktide (1-min pulse every hour for 4 h), in contrast to that of kisspeptin, failed to sustain pulsatile GnRH release. Repetitive senktide injections did not compromise the GnRH-releasing action of kisspeptin. NKB and kisspeptin were colocalized in perikarya of the arcuate nucleus and in axonal projections to the median eminence, confirming earlier findings in sheep. These results are consistent with the human genetics, and indicate that although brief activation of NK3R stimulates GnRH release, repetitive stimulation of this pathway, in contrast to that of kisspeptin receptor, fails to sustain pulsatile GnRH release. In addition, the data provide a platform for future elucidation of the interactions between NKB and kisspeptin that are required for generating pulsatile GnRH release in primates.

The fertility and survival of an individual rely on the ability of the periphery to promptly, effectively, and reproducibly communicate with brain neural networks that control reproduction, food intake, and energy homeostasis. Tanycytes, a specialized glial cell type lining the wall of the third ventricle in the median eminence of the hypothalamus, appear to act as the linchpin of these processes by dynamically controlling the secretion of neuropeptides into the portal vasculature by hypothalamic neurons and regulating blood-brain and blood-cerebrospinal fluid exchanges, both processes that depend on the ability of these cells to adapt their morphology to the physiological state of the individual. In addition to their barrier properties, tanycytes possess the ability to sense blood glucose levels, and play a fundamental and active role in shuttling circulating metabolic signals to hypothalamic neurons that control food intake. Moreover, accumulating data suggest that, in keeping with their putative descent from radial glial cells, tanycytes are endowed with neural stem cell properties and may respond to dietary or reproductive cues by modulating hypothalamic neurogenesis. Tanycytes could thus constitute the missing link in the loop connecting behavior, hormonal changes, signal transduction, central neuronal activation and, finally, behavior again. In this article, we will examine these recent advances in the understanding of tanycytic plasticity and function in the hypothalamus and the underlying molecular mechanisms. We will also discuss the putative involvement and therapeutic potential of hypothalamic tanycytes in metabolic and fertility disorders.

The secretory output of gonadotropin-releasing hormone (GnRH) neurons is critically influenced by peptidergic neurons synthesizing kisspeptins (KP) and neurokinin B (NKB) in the hypothalamic infundibular nucleus (Inf). These cells mediate negative feedback effects of sex steroids on the reproductive axis. While negative feedback is lost in postmenopausal women, it is partly preserved by the sustained testosterone secretion in aged men. We hypothesized that the different reproductive physiology of aged men and women is reflected in morphological differences of KP and NKB neurons. This sexual dimorphism was studied with immunohistochemistry in hypothalamic sections of aged human male (≥50 years) and female (>55 years) subjects. KP and NKB cell bodies of the Inf were larger in females. The number of KP cell bodies, the density of KP fibers, and the incidence of their contacts on GnRH neurons were much higher in aged women compared with men. The number of NKB cell bodies was only slightly higher in women and there was no sexual dimorphism in the regional density of NKB fibers and the incidence of their appositions onto GnRH cells. The incidences of NKB cell bodies, fibers, and appositions onto GnRH neurons exceeded several-fold those of KP-IR elements in men. More NKB than KP inputs to GnRH cells were also present in women. Immunofluorescent studies identified only partial overlap between KP and NKB axons. KP and NKB were colocalized in higher percentages of afferents to GnRH neurons in women compared with men. Most of these sex differences might be explained with the lack of estrogen negative feedback in aged women, whereas testosterone can continue to suppress KP, and to a lesser extent, NKB synthesis in men. Overall, sex differences in reproductive physiology of aged humans were reflected in the dramatic sexual dimorphism of the KP system, with significantly higher incidences of KP-IR neurons, fibers and inputs to GnRH neurons in aged females vs. males.

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.

Lactation results in negative energy balance in the rat leading to decreased gonadotrophin-releasing hormone (GnRH) release and anoestrus. Inhibited GnRH release may be a result of decreased stimulatory tone from neuropeptides critical for GnRH neuronal activity, such as kisspeptin (Kiss1) and neurokinin B (NKB). The present study aimed to identify neuronal projections from the colocalised population of Kiss1/NKB cells in the arcuate nucleus (ARH) using double-label immunohistochemistry to determine where this population may directly regulate GnRH neuronal activity. Additionally, the present study further examined lactation-induced changes in the Kiss1 system that could play a role in decreased GnRH release. The colocalised ARH Kiss1/NKB fibres projected primarily to the internal zone of the median eminence (ME) where they were in close proximity to GnRH fibres; however, few Kiss1/NKB fibres from the ARH were seen at the level of GnRH neurones in the preoptic area (POA). Arcuate Kiss1/NKB peptide levels were decreased during lactation consistent with previous mRNA data. Surprisingly, anteroventral periventricular (AVPV) Kiss1 peptide levels were increased, whereas Kiss1 mRNA levels were decreased during lactation, suggesting active inhibition of peptide release. These findings indicate ARH Kiss1/NKB and AVPV Kiss1 appear to be inhibited during lactation, which may contribute to decreased GnRH release and subsequent reproductive dysfunction. Furthermore, the absence of a strong ARH Kiss1/NKB projection to the POA suggests regulation of GnRH by this population occurs primarily at the ME level via local projections.

Previous immunohistochemical and in situ hybridization studies of sheep, goats, and rodents indicated that kisspeptin (KP), neurokinin B (NKB), and dynorphin A (DYN) are extensively colocalized in the hypothalamic arcuate nucleus, thus providing a basis for the KP/NKB/DYN (KNDy) neuron concept; in both sexes, KNDy neuropeptides have been implicated in the generation of GnRH neurosecretory pulses and in the negative feedback effects of sexual steroids to the reproductive axis. To test the validity and limitations of the KNDy neuron concept in the human, we carried out the comparative immunohistochemical analysis of the three neuropeptides in the infundibular nucleus (Inf; also known as arcuate nucleus) and stalk of young male human individuals (<37 yr). Results of quantitative immunohistochemical experiments established that the regional densities of NKB immunoreactive (IR) perikarya and fibers, and the incidence of afferent contacts they formed onto GnRH neurons, were about 5 times as high as those of the KP-IR elements. Dual-immunofluorescent studies confirmed that considerable subsets of the NKB-IR and KP-IR cell bodies and fibers are separate, and only about 33% of NKB-IR perikarya and 75% of KP-IR perikarya were dual labeled. Furthermore, very few DYN-IR cell bodies could be visualized in the Inf. DYN-IR fibers were also rare and, with few exceptions, distinct from the KP-IR fibers. The abundance and colocalization patterns of the three immunoreactivities showed similar trends in the infundibular stalk around portal blood vessels. Together these results indicate that most NKB neurons in the Inf do not synthesize detectable amounts of KP and DYN in young male human individuals. These data call for a critical use of the KNDy neuron terminology when referring to the putative pulse generator system of the mediobasal hypothalamus. We conclude that the functional importance of these three neuropeptides in reproductive regulation considerably varies among species, between sexes, and at different ages.