Origin of thyrotropin-releasing hormone neurons that innervate the tuberomammillary nuclei.

Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons function as metabolic sensors that regulate the thyroid axis and energy homeostasis. Less is known about the role of other hypothalamic TRH neurons. As central administration of TRH decreases food intake and increases histamine in the tuberomammillary nuclei (TMN), and TMN histamine neurons are densely innervated by TRH fibers from an unknown origin, we mapped the location of TRH neurons that project to the TMN. The retrograde tracer, cholera toxin B subunit (CTB), was injected into the TMN E1-E2, E4-E5 subdivisions of adult Sprague-Dawley male rats. TMN projecting neurons were observed in the septum, preoptic area, bed nucleus of the stria terminalis (BNST), perifornical area, anterior paraventricular nucleus, peduncular and tuberal lateral hypothalamus (TuLH), suprachiasmatic nucleus and medial amygdala. However, CTB/pro-TRH178-199 double-labeled cells were only found in the TuLH. The specificity of the retrograde tract-tracing result was confirmed by administering the anterograde tracer, Phaseolus vulgaris leuco-agglutinin (PHAL) into the TuLH. Double-labeled PHAL-pro-TRH boutons were identified in all subdivisions of the TMN. TMN neurons double-labeled for histidine decarboxylase (Hdc)/PHAL, Hdc/Trh receptor (Trhr), and Hdc/Trh. Further confirmation of a TuLH-TRH neuronal projection to the TMN was established in a transgenic mouse that expresses Cre recombinase in TRH-producing cells following microinjection of a Cre recombinase-dependent AAV that expresses mCherry into the TuLH. We conclude that, in rodents, the TRH innervation of TMN originates in part from TRH neurons in the TuLH, and that this TRH population may contribute to regulate energy homeostasis through histamine Trhr-positive neurons of the TMN.

Cocaine- and amphetamine-regulated transcript (CART) is colocalized with the orexigenic neuropeptide Y and agouti-related protein and absent from the anorexigenic alpha-melanocyte-stimulating hormone neurons in the infundibular nucleus of the human hypothalamus.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered anorexigenic peptide. In rodents, CART inhibits food intake and is expressed in the anorexigenic alpha-MSH- but not in the orexigenic neuropeptide Y (NPY)- and agouti-related protein (AGRP)-synthesizing neurons of the arcuate nucleus. To understand whether CART is similarly expressed in feeding-related neuronal groups of the human hypothalamus as observed in rodents, colocalization of CART with alpha-MSH, NPY, AGRP, and melanin-concentrating hormone was studied using double-labeling immunofluorescence and confocal microscopy on human hypothalamic tissues obtained at autopsy. Unlike in rodents, we observed that CART is absent from the perikarya and axons of alpha-MSH-synthesizing neurons, but expressed in approximately one third of NPY/AGRP neurons in the human infundibular nucleus. In the lateral hypothalamus of the humans, colocalization of CART and melanin-concentrating hormone was observed, similar to that described in rodents. The anatomy of CART-containing neurons in the human infundibular nucleus differs markedly from that observed in the rodent brain, raising the question whether the colocalization of CART with orexigenic NPY and AGRP neurons is associated with an orexigenic role of CART in the human brain.

Distribution of ghrelin-immunoreactive neuronal networks in the human hypothalamus.

Ghrelin has been discovered as the endogenous ligand of the growth hormone secretagogue receptor (GHS-R). It stimulates growth hormone secretion and also potently increases food intake. To date, ghrelin is the only known peripheral orexigenic hormone. Recent studies have demonstrated that in addition to peripheral organs, ghrelin is also synthesized in the hypothalamus. In the present study, we examined the distribution of the ghrelin-immunoreactive (IR) elements in the human hypothalamus. Ghrelin-IR fibers were widely distributed throughout the hypothalamus. Based on the thickness of fibers, major subtypes of ghrelin-IR axons were observed: thick fibers with large varicosities and very fine axons with or without small varicosities. Dense networks of ghrelin-IR axons were observed in the hypothalamic suprachiasmatic, paraventricular, supraoptic, dorsomedial, ventromedial and infundibular nuclei and in the periventricular area. Ghrelin-IR axons also appeared in the external layer of the pituitary stalk. Ghrelin-IR cell bodies were not detected. Since hypothalamic regions innervated by ghrelin-IR axons also take part in the regulation of food intake and energy balance, the centrally synthesized ghrelin may play a major role in the central regulation of energy metabolism in humans.

Characterization of the nuclear factor-kappa B responsiveness of the human dio2 gene.

Type 2 iodothyronine deiodinase (D2) activates T4 by deiodination to T3, a process being the source of most T3 present in the brain. In the mediobasal hypothalamus, expression of the dio2 gene is potently activated by administration of bacterial lipopolysaccharide (LPS), which in turn mediates the modifications in thyroid homeostasis typically observed in patients with nonthyroidal illness syndrome. Here we show that LPS-induced D2 expression is also observed in human MSTO-211H cells that endogenously express D2. Exposure to LPS rapidly doubled D2 activity by a mechanism that was partially blocked by the nuclear factor-B (NF-B) inhibitor sulfasalazine. Next, the human dio2 5'-flanking region promoter assay was used in HC11 cells and the p65/NF-kappa B responsiveness mapped to the 3' approximately 600-bp region of hdio2 5'-flanking region, with an approximately 15-fold induction. Semiquantitative EMSA identified the strongest NF-B binding sites at the positions -683 bp (called no. 2) and -198 bp (no. 5) 5' to the transcriptional starting site. Despite the very similar NF-kappa B binding affinity of these two sites, site-directed mutagenesis and promoter assay indicated that only site no. 5 possessed transactivation potency in the presence of the p65 subunit of NF-kappa B. Other cytokine mediators such as signal transducer and activator of transcription-3 (STAT3) or signal transducer and activator of transcription-5 (STAT5) did not induce transcription of the dio2 gene. Our results indicate that inflammatory signals regulate D2 expression predominantly via the NF-kappa B pathway in a direct transcriptional manner and could contribute to the changes in thyroid economy observed in nonthyroidal illness syndrome during infection.

Interconnection between orexigenic neuropeptide Y- and anorexigenic alpha-melanocyte stimulating hormone-synthesizing neuronal systems of the human hypothalamus.

Peripheral feeding-related hormones such as leptin, insulin, and ghrelin exert their main central effects through neuropeptide Y- (NPY) synthesizing and alpha-melanocyte-stimulating hormone- (alpha-MSH) synthesizing neurons of the hypothalamic arcuate nucleus. In rodents, recent reports have described an asymmetric signaling between these neuron populations by showing that while NPY influences alpha-MSH-synthesizing neurons, the melanocortin-receptor agonist Melanotan II (MTII) does not modulate the electrophysiological properties of NPY neurons. The functional neuroanatomy of the relationship between these cell populations is unknown in humans. The aim of the current study was to analyze the putative relationship of the orexigenic NPY and anorexigenic alpha-MSH systems in the infundibular nucleus of the human hypothalamus, the analogue of the rodent arcuate nucleus. Double-labeling fluorescent immunocytochemistry for NPY and alpha-MSH was performed on postmortem sections of the human hypothalamus. The sections were analyzed by confocal laser microscopy. Both NPY- and alpha-MSH-immunoreactive (IR) neurons were embedded in dense, intermingling networks of NPY- and alpha-MSH-IR axons in the human infundibular nucleus. NPY-IR varicosities were observed in juxtaposition to all alpha-MSH-IR neurons. The mean number of NPY-IR axon varicosities on the surface of an alpha-MSH-IR neuron was approximately six. The majority of NPY-IR neurons were also contacted by alpha-MSH-IR varicosities, although, the number of such contacts was lower (two alpha-MSH-IR varicosities per NPY neuron). In summary, the present data demonstrate that these two antagonistic, feeding-related neuronal systems are interconnected in the infundibular nucleus, and the neuronal wiring possesses an asymmetric character in the human hypothalamus.