Lateral hypothalamic orexin and melanin-concentrating hormone neurons provide direct input to gonadotropin-releasing hormone neurons in the human.

Hypophysiotropic projections of gonadotropin-releasing hormone (GnRH)-synthesizing neurons form the final common output way of the hypothalamus in the neuroendocrine control of reproduction. Several peptidergic neuronal systems of the medial hypothalamus innervate human GnRH cells and mediate crucially important hormonal and metabolic signals to the reproductive axis, whereas much less is known about the contribution of the lateral hypothalamic area to the afferent control of human GnRH neurons. Orexin (ORX)- and melanin-concentrating hormone (MCH)-synthesizing neurons of this region have been implicated in diverse behavioral and autonomic processes, including sleep and wakefulness, feeding and other functions. In the present immunohistochemical study, we addressed the anatomical connectivity of these neurons to human GnRH cells in post-mortem hypothalamic samples obtained from autopsies. We found that 38.9 ± 10.3% and 17.7 ± 3.3% of GnRH-immunoreactive (IR) perikarya in the infundibular nucleus of human male subjects received ORX-IR and MCH-IR contacts, respectively. On average, each 1 mm segment of GnRH dendrites received 7.3 ± 1.1 ORX-IR and 3.7 ± 0.5 MCH-IR axo-dendritic appositions. Overall, the axo-dendritic contacts dominated over the axo-somatic contacts and represented 80.5 ± 6.4% of ORX-IR and 76.7 ± 4.6% of MCH-IR inputs to GnRH cells. Based on functional evidence from studies of laboratory animals, the direct axo-somatic and axo-dendritic input from ORX and MCH neurons to the human GnRH neuronal system may convey critical metabolic and other homeostatic signals to the reproducive axis. In this study, we also report the generation and characterization of new antibodies for immunohistochemical detection of GnRH neurons in histological sections.

Neuropeptide co-expression in hypothalamic kisspeptin neurons of laboratory animals and the human.

Hypothalamic peptidergic neurons using kisspeptin (KP) and its co-transmitters for communication are critically involved in the regulation of mammalian reproduction and puberty. This article provides an overview of neuropeptides present in KP neurons, with a focus on the human species. Immunohistochemical studies reveal that large subsets of human KP neurons synthesize neurokinin B, as also shown in laboratory animals. In contrast, dynorphin described in KP neurons of rodents and sheep is found rarely in KP cells of human males and postmenopausal females. Similarly, galanin is detectable in mouse, but not human, KP cells, whereas substance P, cocaine- and amphetamine-regulated transcript and proenkephalin-derived opioids are expressed in varying subsets of KP neurons in humans, but not reported in ARC of other species. Human KP neurons do not contain neurotensin, cholecystokinin, proopiomelanocortin-derivatives, agouti-related protein, neuropeptide Y, somatostatin or tyrosine hydroxylase (dopamine). These data identify the possible co-transmitters of human KP cells. Neurochemical properties distinct from those of laboratory species indicate that humans use considerably different neurotransmitter mechanisms to regulate fertility.

Hypophysiotropic gonadotropin-releasing hormone projections are exposed to dense plexuses of kisspeptin, neurokinin B and substance p immunoreactive fibers in the human: a study on tissues from postmenopausal women.

Neuronal populations that synthesize kisspeptin (KP), neurokinin B (NKB) and substance P (SP) in the hypothalamic infundibular nucleus of humans are partly overlapping. These cells are important upstream regulators of gonadotropin-releasing hormone (GnRH) neurosecretion. Homologous neurons in laboratory animals are thought to modulate episodic GnRH secretion primarily via influencing KP receptors on the hypophysiotropic fiber projections of GnRH neurons. To explore the structural basis of this putative axo-axonal communication in humans, we analyzed the anatomical relationship of KP-immunoreactive (IR), NKB-IR and SP-IR axon plexuses with hypophysiotropic GnRH fiber projections. Immunohistochemical studies were carried out on histological samples from postmenopausal women. The neuropeptide-IR axons innervated densely the portal capillary network in the postinfundibular eminence. Subsets of the fibers formed descending tracts in the infundibular stalk, some reaching the neurohypophysis. KP-IR, NKB-IR and SP-IR plexuses intermingled, and established occasional contacts, with hypophysiotropic GnRH fibers in the postinfundibular eminence and through their lengthy course while descending within the infundibular stalk. Triple-immunofluorescent studies also revealed considerable overlap between the KP, NKB and SP signals in individual fibers, providing evidence that these peptidergic projections arise from neurons of the mediobasal hypothalamus. These neuroanatomical observations indicate that the hypophysiotropic projections of human GnRH neurons in the postinfundibular eminence and the descending GnRH tract coursing through the infundibular stalk to the neurohypophysis are exposed to neurotransmitters/neuropeptides released by dense KP-IR, NKB-IR and SP-IR fiber plexuses. Localization and characterization of axonal neuropeptide receptors will be required to clarify the putative autocrine and paracrine interactions in these anatomical regions.

Colocalization of cocaine- and amphetamine-regulated transcript with kisspeptin and neurokinin B in the human infundibular region.

Kisspeptin (KP)- and neurokinin B (NKB)- synthesizing neurons of the hypothalamic arcuate nucleus play a pivotal role in the regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion. Unlike in rodents and sheep, the homologous KP and NKB neurons in the human infundibular region rarely express dynorphin- but often exhibit Substance P (SP) immunoreactivity, indicating remarkable species differences in the neurochemical phenotype of these neurons. In search for additional neuropeptides in human KP and NKB neurons, we carried out immunofluorescent studies on hypothalamic sections obtained from five postmenopausal women. Colocalization experiments provided evidence for the presence of cocaine- and amphetamine-regulated transcript (CART) in 47.9 ± 6.6% of KP-immunoreactive (IR) and 30.0 ± 4.9% of NKB-IR perikarya and in 17.0 ± 2.3% of KP-IR and 6.2 ± 2.0% of NKB-IR axon varicosities. All three neuropeptides were present in 33.3 ± 4.9% of KP-IR and 28.2 ± 4.6% of NKB-IR somata, respectively, whereas triple-labeling showed lower incidences in KP-IR (14.3 ± 1.8%) and NKB-IR (5.9 ± 2.0%) axon varicosities. CART-IR KP and NKB neurons established contacts with other peptidergic cells, including GnRH-IR neurons and also sent projections to the infundibular stalk. KP and NKB fibers with CART often contained SP as well, while being distinct from CART fibers co-containing the orexigenic peptide agouti-related protein. Presence of CART in human, but not rodent, KP and NKB neurons represents a new example of species differences in the neuropeptide repertoire of mediobasal hypothalamic KP and NKB neurons. Target cells, receptor sites and physiological significance of CART in the efferent communication of KP and NKB neurons in primates require clarification.

Orexinergic input to dopaminergic neurons of the human ventral tegmental area.

The mesolimbic reward pathway arising from dopaminergic (DA) neurons of the ventral tegmental area (VTA) has been strongly implicated in reward processing and drug abuse. In rodents, behaviors associated with this projection are profoundly influenced by an orexinergic input from the lateral hypothalamus to the VTA. Because the existence and significance of an analogous orexigenic regulatory mechanism acting in the human VTA have been elusive, here we addressed the possibility that orexinergic neurons provide direct input to DA neurons of the human VTA. Dual-label immunohistochemistry was used and orexinergic projections to the VTA and to DA neurons of the neighboring substantia nigra (SN) were analyzed comparatively in adult male humans and rats. Orexin B-immunoreactive (IR) axons apposed to tyrosine hydroxylase (TH)-IR DA and to non-DA neurons were scarce in the VTA and SN of both species. In the VTA, 15.0±2.8% of TH-IR perikarya in humans and 3.2±0.3% in rats received orexin B-IR afferent contacts. On average, 0.24±0.05 and 0.05±0.005 orexinergic appositions per TH-IR perikaryon were detected in humans and rats, respectively. The majority (86-88%) of randomly encountered orexinergic contacts targeted the dendritic compartment of DA neurons. Finally, DA neurons of the SN also received orexinergic innervation in both species. Based on the observation of five times heavier orexinergic input to TH-IR neurons of the human, compared with the rat, VTA, we propose that orexinergic mechanism acting in the VTA may play just as important roles in reward processing and drug abuse in humans, as already established well in rodents.

Substance P immunoreactivity exhibits frequent colocalization with kisspeptin and neurokinin B in the human infundibular region.

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

Morphological evidence for enhanced kisspeptin and neurokinin B signaling in the infundibular nucleus of the aging man.

Peptidergic neurons synthesizing kisspeptin (KP) and neurokinin B (NKB) in the hypothalamic infundibular nucleus have been implicated in negative sex steroid feedback to GnRH neurons. In laboratory rodents, testosterone decreases KP and NKB expression in this region. In the present study, we addressed the hypothesis that the weakening of this inhibitory testosterone feedback in elderly men coincides with enhanced KP and NKB signaling in the infundibular nucleus. This central hypothesis was tested in a series of immunohistochemical studies on hypothalamic sections of male human individuals that were divided into arbitrary "young" (21-49 yr, n = 11) and "aged" (50-67 yr, n = 9) groups. 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, exceeded several times those of the KP-IR elements. Robust aging-dependent enhancements were identified in the regional densities of KP-IR perikarya and fibers and the incidence of afferent contacts they established onto GnRH neurons. The abundance of NKB-IR perikarya, fibers, and axonal appositions to GnRH neurons also increased with age, albeit to lower extents. In dual-immunofluorescent studies, the incidence of KP-IR NKB perikarya increased from 36% in young to 68% in aged men. Collectively, these immunohistochemical data suggest an aging-related robust enhancement in central KP signaling and a moderate enhancement in central NKB signaling. These changes are compatible with a reduced testosterone negative feedback to KP and NKB neurons. The heavier KP and NKB inputs to GnRH neurons in aged, compared with young, men may play a role in the enhanced central stimulation of the reproductive axis. It requires clarification to what extent the enhanced KP and NKB signaling upstream from GnRH neurons is an adaptive response to hypogonadism or, alternatively, a consequence of a decline in the androgen sensitivity of KP and NKB neurons.

Low degree of overlap between kisspeptin, neurokinin B, and dynorphin immunoreactivities in the infundibular nucleus of young male human subjects challenges the KNDy neuron concept.

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.

A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons.

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

Glutamatergic and GABAergic innervation of human gonadotropin-releasing hormone-I neurons.

Amino acid (aa) neurotransmitters in synaptic afferents to hypothalamic GnRH-I neurons are critically involved in the neuroendocrine control of reproduction. Although in rodents the major aa neurotransmitter in these afferents is γ-aminobutyric acid (GABA), glutamatergic axons also innervate GnRH neurons directly. Our aim with the present study was to address the relative contribution of GABAergic and glutamatergic axons to the afferent control of human GnRH neurons. Formalin-fixed hypothalamic samples were obtained from adult male individuals (n = 8) at autopsies, and their coronal sections processed for dual-label immunohistochemical studies. GABAergic axons were labeled with vesicular inhibitory aa transporter antibodies, whereas glutamatergic axons were detected with antisera against the major vesicular glutamate transporter (VGLUT) isoforms, VGLUT1 and VGLUT2. The relative incidences of GABAergic and glutamatergic axonal appositions to GnRH-immunoreactive neurons were compared quantitatively in two regions, the infundibular and paraventricular nuclei. Results showed that GABAergic axons established the most frequently encountered type of axo-somatic apposition. Glutamatergic contacts occurred in significantly lower numbers, with similar contributions by their VGLUT1 and VGLUT2 subclasses. The innervation pattern was different on GnRH dendrites where the combined incidence of glutamatergic (VGLUT1 + VGLUT2) contacts slightly exceeded that of the GABAergic appositions. We conclude that GABA represents the major aa neurotransmitter in axo-somatic afferents to human GnRH neurons, whereas glutamatergic inputs occur somewhat more frequently than GABAergic inputs on GnRH dendrites. Unlike in rats, the GnRH system of the human receives innervation from the VGLUT1, in addition to the VGLUT2, subclass of glutamatergic neurons.

Sexual dimorphism of kisspeptin and neurokinin B immunoreactive neurons in the infundibular nucleus of aged men and women.

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.