Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction.

Hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH), the "master molecule" regulating reproduction and fertility, migrate from their birthplace in the nose to their destination using a system of guidance cues, which include the semaphorins and their receptors, the neuropilins and plexins, among others. Here, we show that selectively deleting neuropilin-1 in new GnRH neurons enhances their survival and migration, resulting in excess neurons in the hypothalamus and in their unusual accumulation in the accessory olfactory bulb, as well as an acceleration of mature patterns of activity. In female mice, these alterations result in early prepubertal weight gain, premature attraction to male odors, and precocious puberty. Our findings suggest that rather than being influenced by peripheral energy state, GnRH neurons themselves, through neuropilin-semaphorin signaling, might engineer the timing of puberty by regulating peripheral adiposity and behavioral switches, thus acting as a bridge between the reproductive and metabolic axes.

Complement C3 mediates early hippocampal neurodegeneration and memory impairment in experimental multiple sclerosis.

Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglia-neuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Characterization of Kisspeptin Neurons in the Human Rostral Hypothalamus.

BACKGROUND: Kisspeptin (KP) neurons in the rostral periventricular region of the 3rd ventricle (RP3V) of female rodents mediate positive estrogen feedback to gonadotropin-releasing hormone neurons and, thus, play a fundamental role in the mid-cycle luteinizing hormone (LH) surge. The RP3V is sexually dimorphic, and male rodents with lower KP cell numbers are unable to mount estrogen-induced LH surges. OBJECTIVE: To find and characterize the homologous KP neurons in the human brain, we studied formalin-fixed post-mortem hypothalami. METHODS: Immunohistochemical techniques were used. RESULTS: The distribution of KP neurons in the rostral hypothalamus overlapped with distinct subdivisions of the paraventricular nucleus. The cell numbers decreased after menopause, indicating that estrogens positively regulate KP gene expression in the rostral hypothalamus in humans, similarly to several other species. Young adult women and men had similar cell numbers, as opposed to rodents reported to have more KP neurons in the RP3V of females. Human KP neurons differed from the homologous rodent cells as well, in that they were devoid of enkephalins, galanin and tyrosine hydroxylase. Further, they did not contain known KP neuron markers of the human infundibular nucleus, neurokinin B, substance P and cocaine- and amphetamine-regulated transcript, while they received afferent input from these KP neurons. CONCLUSIONS: The identification and positive estrogenic regulation of KP neurons in the human rostral hypothalamus challenge the long-held view that positive estrogen feedback may be restricted to the mediobasal part of the hypothalamus in primates and point to the need of further anatomical, molecular and functional studies of rostral hypothalamic KP neurons.

Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.

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.

Alteration in neonatal nutrition causes perturbations in hypothalamic neural circuits controlling reproductive function.

It is increasingly accepted that alterations of the early life environment may have lasting impacts on physiological functions. In particular, epidemiological and animal studies have indicated that changes in growth and nutrition during childhood and adolescence can impair reproductive function. However, the precise biological mechanisms that underlie these programming effects of neonatal nutrition on reproduction are still poorly understood. Here, we used a mouse model of divergent litter size to investigate the effects of early postnatal overnutrition and undernutrition on the maturation of hypothalamic circuits involved in reproductive function. Neonatally undernourished females display attenuated postnatal growth associated with delayed puberty and defective development of axonal projections from the arcuate nucleus to the preoptic region. These alterations persist into adulthood and specifically affect the organization of neural projections containing kisspeptin, a key neuropeptide involved in pubertal activation and fertility. Neonatal overfeeding also perturbs the development of neural projections from the arcuate nucleus to the preoptic region, but it does not result in alterations in kisspeptin projections. These studies indicate that alterations in the early nutritional environment cause lasting and deleterious effects on the organization of neural circuits involved in the control of reproduction, and that these changes are associated with lifelong functional perturbations.

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis.

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism.

Glutamate decarboxylase 67 is expressed in hippocampal mossy fibers of temporal lobe epilepsy patients.

Recently, expression of glutamate decarboxylase-67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammon's horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self-protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals.

Expression of Functional Cannabinoid Type-1 (CB1) Receptor in Mitochondria of White Adipocytes.

Via activation of the cannabinoid type-1 (CB1) receptor, endogenous and exogenous cannabinoids modulate important biochemical and cellular processes in adipocytes. Several pieces of evidence suggest that alterations of mitochondrial physiology might be a possible mechanism underlying cannabinoids' effects on adipocyte biology. Many reports suggest the presence of CB1 receptor mRNA in both white and brown adipose tissue, but the detailed subcellular localization of CB1 protein in adipose cells has so far been scarcely addressed. In this study, we show the presence of the functional CB1 receptor at different subcellular locations of adipocytes from epididymal white adipose tissue (eWAT) depots. We observed that CB1 is located at different subcellular levels, including the plasma membrane and in close association with mitochondria (mtCB1). Functional analysis in tissue homogenates and isolated mitochondria allowed us to reveal that cannabinoids negatively regulate complex-I-dependent oxygen consumption in eWAT. This effect requires mtCB1 activation and consequent regulation of the intramitochondrial cAMP-PKA pathway. Thus, CB1 receptors are functionally present at the mitochondrial level in eWAT adipocytes, adding another possible mechanism for peripheral regulation of energy metabolism.

NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice.

The nitric oxide (NO) signaling pathway in hypothalamic neurons plays a key role in the regulation of the secretion of gonadotropin-releasing hormone (GnRH), which is crucial for reproduction. We hypothesized that a disruption of neuronal NO synthase (NOS1) activity underlies some forms of hypogonadotropic hypogonadism. Whole-exome sequencing was performed on a cohort of 341 probands with congenital hypogonadotropic hypogonadism to identify ultrarare variants in NOS1. The activity of the identified NOS1 mutant proteins was assessed by their ability to promote nitrite and cGMP production in vitro. In addition, physiological and pharmacological characterization was carried out in a Nos1-deficient mouse model. We identified five heterozygous NOS1 loss-of-function mutations in six probands with congenital hypogonadotropic hypogonadism (2%), who displayed additional phenotypes including anosmia, hearing loss, and intellectual disability. NOS1 was found to be transiently expressed by GnRH neurons in the nose of both humans and mice, and Nos1 deficiency in mice resulted in dose-dependent defects in sexual maturation as well as in olfaction, hearing, and cognition. The pharmacological inhibition of NO production in postnatal mice revealed a critical time window during which Nos1 activity shaped minipuberty and sexual maturation. Inhaled NO treatment at minipuberty rescued both reproductive and behavioral phenotypes in Nos1-deficient mice. In summary, lack of NOS1 activity led to GnRH deficiency associated with sensory and intellectual comorbidities in humans and mice. NO treatment during minipuberty reversed deficits in sexual maturation, olfaction, and cognition in Nos1 mutant mice, suggesting a potential therapy for humans with NO deficiency.

GnRH replacement rescues cognition in Down syndrome.

At the present time, no viable treatment exists for cognitive and olfactory deficits in Down syndrome (DS). We show in a DS model (Ts65Dn mice) that these progressive nonreproductive neurological symptoms closely parallel a postpubertal decrease in hypothalamic as well as extrahypothalamic expression of a master molecule that controls reproduction-gonadotropin-releasing hormone (GnRH)-and appear related to an imbalance in a microRNA-gene network known to regulate GnRH neuron maturation together with altered hippocampal synaptic transmission. Epigenetic, cellular, chemogenetic, and pharmacological interventions that restore physiological GnRH levels abolish olfactory and cognitive defects in Ts65Dn mice, whereas pulsatile GnRH therapy improves cognition and brain connectivity in adult DS patients. GnRH thus plays a crucial role in olfaction and cognition, and pulsatile GnRH therapy holds promise to improve cognitive deficits in DS.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Kisspeptin Neurons in the Infundibular Nucleus of Ovariectomized Cats and Dogs Exhibit Unique Anatomical and Neurochemical Characteristics.

Neurons co-synthesizing kisspeptin (KP), neurokinin B (NKB), and dynorphin ("KNDy neurons") in the hypothalamic arcuate/infundibular nucleus (INF) form a crucial component of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) "pulse generator." The goal of our study was to characterize KP neuron distribution, neuropeptide phenotype and connectivity to GnRH cells in ovariectomized (OVX) dogs and cats with immunohistochemistry on formalin-fixed hypothalamic tissue sections. In both species, KP and NKB neurons occurred in the INF and the two cell populations overlapped substantially. Dynorphin was detected in large subsets of canine KP (56%) and NKB (37%) cells and feline KP (64%) and NKB (57%) cells; triple-labeled ("KNDy") somata formed ∼25% of all immunolabeled neurons. Substance P (SP) was present in 20% of KP and 29% of NKB neurons in OVX cats but not dogs, although 26% of KP and 24% of NKB neurons in a gonadally intact male dog also contained SP signal. Only in cats, cocaine- and amphetamine regulated transcript was also colocalized with KP (23%) and NKB (7%). In contrast with reports from mice, KP neurons did not express galanin in either carnivore. KP neurons innervated virtually all GnRH neurons in both species. Results of this anatomical study on OVX animals reveal species-specific features of canine and feline mediobasal hypothalamic KP neurons. Anatomical and neurochemical similarities to and differences from the homologous KP cells of more extensively studied rodent, domestic and primate species will enhance our understanding of obligate and facultative players in the molecular mechanisms underlying pulsatile GnRH/LH secretion.

Spatiotemporal expression in mouse brain of Kiaa2022, a gene disrupted in two patients with severe mental retardation.

We previously identified an inactivating disruption of the X-linked KIAA2022 gene by a chromosomal rearrangement in two male patients with severe mental retardation. In order to determine if KIAA2022 has a role during the development of the central nervous system, we have cloned its murine ortholog, Kiaa2022, determined its genomic structure and studied its expression during mouse development. We show that Kiaa2022 is preferentially expressed in the central nervous system and that the transcript is highly expressed in postmitotic neurons. The expression of Kiaa2022 is first detectable at E10.5 to reach a maximum at P3 where it is notably expressed in the hippocampus, the entorhinal cortex and strongly in the ventral premammillary nucleus. After P3, the expression of Kiaa2022 decreases and maintains very low levels thereafter. Our results show that Kiaa2022 is expressed in the developing brain and that it may play a role in postmitotic, maturing neurons.

Preclinical Assessment of Leptin Transport into the Cerebrospinal Fluid in Diet-Induced Obese Minipigs.

OBJECTIVE: A minipig model was employed to explore the changes in endogenous leptin transport into the central nervous system and in hypothalamic sensitivity to exogenous leptin when individuals are placed on high-fat diet (HFD) compared with standard diet. METHODS: Serum and cerebrospinal fluid (CSF) leptin concentrations during 10 weeks of HFD versus standard diet and exogenous leptin-induced STAT3 phosphorylation in the hypothalamus of minipigs were assessed, and the hypothalamic leptin-sensitive cells were characterized by immunofluorescence. RESULTS: The efficiency of the passage of endogenous blood-borne leptin into the CSF (measured as the log [CSF:serum leptin ratio]) decreased over time in minipigs fed a HFD (ß = -0.04 ± 0.005 per kilogram of weight gain in HFD; P < 0.0001), while it remained stable in minipigs fed a standard diet. However, the ability of peripherally administered leptin to activate its receptor in hypothalamic neurons was preserved in obese minipigs at 10 weeks of HFD. CONCLUSIONS: Together, these data are consistent with the existence of an early-onset tranport deficiency for endogenous circulating leptin into the brain in individuals developing obesity, preceding the acquisition of hypothalamic leptin resistance. Although additional studies are required to identify the underlying mechanisms, our study paves the way for the development of new preclinical pharmacological models targeting the restoration of the shuttling of peripheral leptin into the central nervous system to manage obesity.

The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism.

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.

Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B.

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.

Electrophysiology of Hypothalamic Magnocellular Neurons In vitro: A Rhythmic Drive in Organotypic Cultures and Acute Slices.

Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.

Reduction in adiposity affects the extent of afferent projections to growth hormone-releasing hormone and somatostatin neurons and the degree of colocalization of neuropeptides in growth hormone-releasing hormone and somatostatin cells of the ovine hypothalamus.

Various neuropeptides and neurotransmitters affect GH secretion by acting on GHRH and somatostatin (SRIF) cells. GH secretion is also affected by alteration in adiposity, which could be via modulation of GHRH and SRIF cells. We quantified colocalization of neuropeptides in GHRH and SRIF cells and afferent projections to these cells in lean (food restricted) and normally fed sheep (n=4/group). The number of GHRH-immunoreactive (IR) cells in the arcuate nucleus was higher in lean animals, but the number of SRIF-IR cells in the periventricular nucleus was similar in the two groups. A subpopulation of GHRH-IR cells colocalized neuropeptide Y in lean animals, but this was not seen in normally fed animals. GHRH/galanin (GAL) colocalization was higher in lean animals with no difference in numbers of GHRH/tyrosine hydroxylase or GHRH/GAL-like peptide cells. SRIF/enkephalin colocalization was lower in lean animals. The percentage of GHRH neurons receiving SRIF input was similar in lean and normally fed animals, but more GHRH cells received input from enkephalin afferents in normally fed animals. The percentage of SRIF cells receiving GHRH, neuropeptide Y, GAL, and orexin afferents was higher in lean animals. These findings provide an anatomical evidence of central mechanism(s) by which appetite-regulating peptides and dopamine could regulate GH secretion. Increased input to SRIF cells in lean animals may be inhibitory and permissive of increased GH. The appearance of NPY in GHRH cells of lean animals may be a mechanism for regulation of increasing GH secretion with reduced adiposity.

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.