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.

IntroductionGhrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction.MethodsThe effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors.Results and ConclusionsGhrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.

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.

An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7a-(acetylthio)-3-oxo-17a-pregn-4-ene-21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7a,17a)-17-hydroxy-3-oxo-7-propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR. Molecular Psychiatry (2005) 10, 790-798.

To determine whether the type 2 iodothyronine deiodinase (D2), the principal central nervous system enzyme converting T(4) to biologically active T(3), is regulated in tanycytes by immune activation, D2 activity was measured in the mediobasal hypothalamus (MBH) 4, 12, and 24 h after administration of bacterial lipopolysaccharide (LPS) and compared with D2 levels in the cortex and anterior pituitary of rats. In contrast to D2 activity in the cortex and anterior pituitary that showed a steady linear increase over 24 h, which was coincident with a decline in thyroid hormone and TSH levels, D2 activity peaked in the MBH 12 h after LPS administration. By in situ hybridization, the increased D2 mRNA synthesis induced by LPS was specifically localized to tanycytes lining the third ventricle. In vitro assays in HC11 and HEK-293 cells demonstrated that the p65 subunit of nuclear factor-kappaB markedly increased both rat and human D2 genes (dio2) as analyzed by promoter assays. No activation of human dio2 was observed when an 83-bp minimal promoter was used. We propose that LPS or LPS-induced cytokines directly induce D2 mRNA in tanycytes. The ensuing MBH-specific D2-mediated local thyrotoxicosis may suppress the hypothalamus-pituitary-thyroid axis by local feedback inhibition of hypophysiotropic TRH and/or TSH and contribute to the mechanism of central hypothyroidism associated with infection.

OBJECTIVE-Blockade of the CB1 receptor is one of the promising strategies for the treatment of obesity. Although antagonists suppress food intake and reduce body weight, the role of central versus peripheral CB1 activation on weight loss and related metabolic parameters remains to be elucidated. We therefore specifically assessed and compared the respective potential relevance of central nervous system (CNS) versus peripheral CB1 receptors in the regulation of energy homeostasis and lipid and glucose metabolism in diet-induced obese (DIO) rats.RESEARCH DESIGN AND METHODS-Both lean and DIO rats were used for our experiments. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR, and euglycemic-hyperinsulinemic clamps were used for insulin sensitivity and glucose metabolism studies.RESULTS-Specific CNS-CB1 blockade decreased body weight and food intake but, independent of those effects, had no beneficial influence on peripheral lipid and glucose metabolism. Peripheral treatment with CB1 antagonist (Rimonabant) also reduced food intake and body weight but, in addition, independently triggered lipid mobilization pathways in white adipose tissue and cellular glucose uptake. Insulin sensitivity and skeletal muscle glucose uptake were enhanced, while hepatic glucose production was decreased during peripheral infusion of the CB1 antagonist. However, these effects depended on the antagonistelicited reduction of food intake.CONCLUSIONS-Several relevant metabolic processes appear to independently benefit from peripheral blockade of CB1, while CNS-CB1 blockade alone predominantly affects food intake and body weight. Diabetes 57:2977-2991, 2008 T he incidence of obesity and the metabolic syndrome have grown to epidemic proportions, making increased research efforts toward discovery of novel anti-obesity therapies increasingly important. Endocannabinoids are key modulators of feeding behavior through the activation of the CB1 receptor (1,2), which is localized in the periphery as well as in many brain areas involved in the regulation of energy homeostasis and reward processes (3,4). Recent studies (5-11) have demonstrated that blocking the activity of the endogenous cannabinoid system may be a successful strategy for the treatment of obesity and the metabolic syndrome.It is well known that CB1 receptors in the hypothalamus might regulate food intake through the disinhibition of the release of melanin-concentrating hormone from lateral hypothalamic neurons (12) and the inhibition of the release and/or expression of corticotrophin-releasing hormone in the paraventricular nucleus (13). Both these effects are under the negative control of leptin, which is known to negatively control endocannabinoid tone in the hypothalamus (2). On the other hand, the effects of CB1 activation on ␣-melanocyte-stimulating hormone are controversial, since both inhibition and stimulation were reported in the study by Hentges et al. (14), and no downstream effects of ␣-melanocyte-stimulating hormone on endocannabinoi...

Mammalian reproduction is dependent upon intermittent delivery of luteinizing hormone-releasing hormone (LHRH) to the anterior pituitary. This mode of secretion is required to sensitize maximally the gonadotrophs to LHRH stimulation and to regulate gonadotropin gene expression. While LHRH secretion is pulsatile in nature, the origin of the pulse generator is unknown. In this report, we show that this oscillator could be located within the LHRH neuronal network. When immortalized LHRH neurons are placed into a perifusion system, LHRH is secreted into the medium in a pulsatile fashion under basal conditions. LHRH secretion and the number of LHRH pulses are reduced when calcium is removed from the medium. Perifusion also influences pro-LHRH processing, since the molar ratio of its processed products varies dramatically when the cells are transferred from a static system. Several different cellular mechanisms may underlie these changes in secretion and processing. Lucifer yellow experiments reveal that some cells are dye-coupled. Hence, these cells could be electrically coupled through gap junctions such that secretion from individual cells could be coordinated. Secretion could also be synchronized through the observed synapse-like contacts. These contacts could perform a negative-feedback role to regulate not only the amount of LHRH released but also the molecular forms secreted. The organization of LHRH neurons into interconnected clusters could serve to coordinate LHRH secretion from individual cells and, thereby, orchestrate functions in vivo as diverse as the onset of puberty, the timing of ovulation, and the duration of lactational infertility.Luteinizing hormone (LH)-releasing hormone (LHRH) is a major regulator of reproduction in mammals (1-3). While LHRH neuronal cell bodies are scattered throughout the anterior hypothalamic region, their nerve terminals converge on the median eminence. LHRH is secreted into the hypophysial portal circulation, where it is transported to the anterior pituitary to stimulate the release of LH and folliclestimulating hormone.In all mammalian species studied so far, secretion of LH into blood is episodic in nature (1,(4)(5)(6)(7)(8). Interestingly, LHRH is secreted into the hypophysial portal blood in a pulsatile manner (9-11), and LH pulses are preceded by LHRH release (1, 10, 11). In addition, LH pulsatile secretion is lost either after passive immunization with LHRH antiserum (12) or with administration of a LHRH antagonist (13). Lesions of either the medial basal hypothalamus or the arcuate nucleus also eradicate LH pulses (1). By comparison, continuous infusion of LHRH, which desensitizes the gonadotropes through down-regulation of the LHRH receptor (14), also abolishes episodic secretion of LH and reduces LH 03-and a-subunit mRNA levels (15, 16). While the relationship between LHRH and LH secretion is well established, it is unclear whether the generator for LHRH pulses is located within the LHRH neuronal network itself or within networks of other neighboring neuro...

Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.