Different Hypothalamic Mechanisms Control Decreased Circulating Thyroid Hormone Levels in Infection and Fasting-Induced Non-Thyroidal Illness Syndrome in Male Thyroid Hormone Action Indicator Mice.

Background: Non-Thyroidal Illness Syndrome (NTIS) caused by infection or fasting is hallmarked by reduced circulating thyroid hormone (TH) levels. To better understand the role of local TH-action in the development of NTIS, we assessed tissue-specific changes of TH signaling in Thyroid Hormone Action Indicator (THAI) mice. Methods: NTIS was induced in young adult THAI mice by bacterial lipopolysaccharide (LPS)-administration or by 24 or 48 hours' fasting. Tissue-specific TH-action was assessed by the detection of changes of the Luciferase reporter of THAI mice with quantitative polymerase chain reaction along with tissue-specific examination of regulators of TH metabolism and signaling. Age dependence of revealed alterations of hypothalamic TH-action was also studied in 1-year-old male THAI mice. Results: LPS-treatment increased TH-action in the hypothalamic arcuate nucleus-median eminence (ARC-ME) region preceded by an increase of type 2 deiodinase (D2) expression in the same region and followed by the suppression of proTrh expression in the hypothalamic paraventricular nucleus (PVN). In contrast, LPS decreased both TH-action and D2 activity in the pituitary at both ages. Tshß expression and serum free thyroxine (fT4) and free triiodothyronine (fT3) levels decreased in LPS-treated young adults. Tshß expression and serum fT4 levels were not significantly affected by LPS treatment in aged animals. In contrast to LPS treatment, TH-action remained unchanged in the ARC-ME of 24 and 48 hours fasted animals accompanied with a modest decrease of proTrh expression in the PVN in the 24-hour group. Tshß expression and fT3 level were decreased in both fasted groups, but the fT4 decreased only in the 48 hours fasted animals. Conclusions: Although the hypothalamo-pituitary-thyroid (HPT) axis is inhibited both in LPS and fasting-induced NTIS, LPS achieves this by centrally inducing local hyperthyroidism in the ARC-ME region, while fasting acts without affecting hypothalamic TH signaling. Lack of downregulation of Tshß and fT4 in LPS-treated aged THAI mice suggests age-dependent alterations in the responsiveness of the HPT axis. The LPS-induced tissue-specific hypo-, eu-, and hyperthyroidism in different tissues of the same animal indicate that under certain conditions TH levels alone could be a poor marker of tissue TH signaling. In conclusion, decreased circulating TH levels in these two forms of NTIS are associated with different patterns of hypothalamic TH signaling.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans.

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Variable proopiomelanocortin expression in tanycytes of the adult rat hypothalamus and pituitary stalk.

It is generally believed that proopiomelanocortin (POMC) is expressed exclusively by neurons in the adult rodent brain. Unbeknownst to most researchers, however, Pomc in situ hybridization studies in the rat show specific labeling in the ventral wall of the hypothalamic third ventricle, which is formed by specialized ependymal cells, called tanycytes. Here we characterized this non-neuronal POMC expression in detail using in situ hybridization and immunohistochemical techniques, and report two unique characteristics. First, POMC mRNA and precursor protein expression in non-neuronal cells varies to a great degree as to the extent and abundance of expression. In brains with low-level expression, POMC mRNA and protein was largely confined to a population of tanycytes within the infundibular stalk/caudal median eminence, termed here γ tanycytes, and a subset of closely located ß and α2 tanycytes. In brains with high-level expression, POMC mRNA and protein was observed in the vast majority of α2, ß, and γ tanycytes. This variability was observed in both adult males and females; of 41 rats between 8 and 15 weeks of age, 17 had low-, 9 intermediate-, and 15 high-level POMC expression in tanycytes. Second, unlike other known POMC-expressing cells, tanycytes rarely contained detectable levels of adrenocorticotropin or α-melanocyte-stimulating hormone. The results indicate either a dynamic spatiotemporal pattern whereby low and high POMC syntheses in tanycytes occur periodically in each brain, or marked interindividual differences that may persist throughout adulthood. Future studies are required to examine these possibilities and elucidate the physiologic importance of POMC in tanycytes. J. Comp. Neurol. 525:411-441, 2017. © 2016 Wiley Periodicals, Inc.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Elucidation of the anatomy of a satiety network: Focus on connectivity of the parabrachial nucleus in the adult rat.

We hypothesized that brain regions showing neuronal activation after refeeding comprise major nodes in a satiety network, and tested this hypothesis with two sets of experiments. Detailed c-Fos mapping comparing fasted and refed rats was performed to identify candidate nodes of the satiety network. In addition to well-known feeding-related brain regions such as the arcuate, dorsomedial, and paraventricular hypothalamic nuclei, lateral hypothalamic area, parabrachial nucleus (PB), nucleus of the solitary tract and central amygdalar nucleus, other refeeding activated regions were also identified, such as the parastrial and parasubthalamic nuclei. To begin to understand the connectivity of the satiety network, the interconnectivity of PB with other refeeding-activated neuronal groups was studied following administration of anterograde or retrograde tracers into the PB. After allowing for tracer transport time, the animals were fasted and then refed before sacrifice. Refeeding-activated neurons that project to the PB were found in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamic area; arcuate, paraventricular, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; parasubthalamic nucleus; central amygdalar nucleus; area postrema; and nucleus of the solitary tract. Axons originating from the PB were observed to closely associate with refeeding-activated neurons in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamus; paraventricular, arcuate, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; central amygdalar nucleus; parasubthalamic nucleus; ventral posterior thalamic nucleus; area postrema; and nucleus of the solitary tract. These data indicate that the PB has bidirectional connections with most refeeding-activated neuronal groups, suggesting that short-loop feedback circuits exist in this satiety network. J. Comp. Neurol. 524:2803-2827, 2016. © 2016 Wiley Periodicals, Inc.

Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions.

TRH is a tripeptide amide that functions as a neurotransmitter but also serves as a neurohormone that has a critical role in the central regulation of the hypothalamic-pituitary-thyroid axis. Hypophysiotropic TRH neurons involved in this neuroendocrine process are located in the hypothalamic paraventricular nucleus and secrete TRH into the pericapillary space of the external zone of the median eminence for conveyance to anterior pituitary thyrotrophs. Under basal conditions, the activity of hypophysiotropic TRH neurons is regulated by the negative feedback effects of thyroid hormone to ensure stable, circulating, thyroid hormone concentrations, a mechanism that involves complex interactions between hypophysiotropic TRH neurons and the vascular system, cerebrospinal fluid, and specialized glial cells called tanycytes. Hypophysiotropic TRH neurons also integrate other humoral and neuronal inputs that can alter the setpoint for negative feedback regulation by thyroid hormone. This mechanism facilitates adaptation of the organism to changing environmental conditions, including the shortage of food and a cold environment. The thyroid axis is also affected by other adverse conditions such as infection, but the central mechanisms mediating suppression of hypophysiotropic TRH may be pathophysiological. In this review, we discuss current knowledge about the mechanisms that contribute to the regulation of hypophysiotropic TRH neurons under physiological and pathophysiological conditions.

CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

Coordination of hypothalamic and pituitary T3 production regulates TSH expression.

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

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.

Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.

Contribution of TNF-alpha and nuclear factor-kappaB signaling to type 2 iodothyronine deiodinase activation in the mediobasal hypothalamus after lipopolysaccharide administration.

To determine whether signaling through TNF and/or nuclear factor-kappaB contributes to bacterial lipopolysaccharide (LPS)-induced activation of type 2 iodothyronine deiodinase (D2) in tanycytes lining the floor and infralateral walls of the third ventricle, the effect of a TNF antagonist on D2 gene expression and LPS-induced Ikappa-Balpha expression in tanycytes were studied. Animals treated with soluble, rat, polyethylene glycol-conjugated TNF receptor type 1 (4 mg/kg body weight) before a single ip injection of LPS showed a significant reduction in circulating IL-6 levels but no effect on LPS-induced D2 mRNA in the majority of tanycytes with the exception of a subpopulation of alpha tanycytes in the wall of the third ventricle. LPS induced a rapid increase in Ikappa-Balpha mRNA in the pars tuberalis and a delayed response in alpha tanycytes but absent in all other tanycyte subsets. The LPS-induced increase in Ikappa-Balpha in the pars tuberalis was associated with increased TSHbeta gene expression in this tissue, but cAMP response element-binding protein (CREB) phosphorylation was observed only in a subset of alpha tanycytes. These data suggest that TNF and nuclear factor-kappaB signaling are not the primary, initiating mechanisms mediating the LPS-induced D2 response in tanycytes, but may contribute in part to sustaining the LPS-induced D2 response in a subset of alpha tanycytes. We hypothesize that in addition to TSH, other factors derived from the pars tuberalis may contribute to LPS-induced D2 activation in tanycytes.

Improved method for combination of immunocytochemistry and Nissl staining.

Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.

Induction of type 2 iodothyronine deiodinase in the mediobasal hypothalamus by bacterial lipopolysaccharide: role of corticosterone.

To determine whether endotoxin-induced activation of type 2 iodothyronine deiodinase (D2) in the mediobasal hypothalamus is dependent on circulating levels of corticosterone, the effect of bacterial lipopolysaccharide (LPS) on D2 gene expression was studied in adrenalectomized, corticosterone-clamped adult, male, Sprague Dawley rats. In sham-adrenalectomized animals, LPS (250 microg/100 g body weight) increased circulating levels of corticosterone and IL-6, as well as tanycyte D2 mRNA in the mediobasal hypothalamus. Adrenalectomized, corticosterone-clamped animals showed no significant rise in corticosterone after LPS, compared with saline-treated controls but increased IL-6 levels and tanycyte D2 mRNA similar to LPS-treated sham controls. To further clarify the potential role of corticosterone in the regulation of D2 gene expression by LPS, animals were administered high doses of corticosterone to attain levels similar to that observed in the LPS-treated group. No significant increase in D2 mRNA was observed in the mediobasal hypothalamus with the exception of a small subpopulation of cells in the lateral walls of the third ventricle. These data indicate that the LPS-induced increase in D2 mRNA in the mediobasal hypothalamus is largely independent of circulating corticosterone and indicate that mechanisms other than adrenal activation are involved in the regulation of most tanycyte D2-expressing cells by endotoxin.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Distribution of type 1 cannabinoid receptor (CB1)-immunoreactive axons in the mouse hypothalamus.

Type 1 cannabinoid receptor (CB1) is the principal receptor for endocannabinoids in the brain; it mainly occurs in preterminal/terminal axons and mediates retrograde neuronal signaling mechanisms. A large body of physiological and electrophysiological evidence indicates the critical role of CB1 in the regulation of hypothalamic functions. Conversely, the distribution of CB1-containing axons in the hypothalamus is essentially unknown. Therefore, we have analyzed the distribution and the ultrastructural characteristics of the CB1-immunoreactive (IR) axons in the mouse hypothalamus by using an antiserum against the C-terminal 31 amino acids of the mouse CB1. We found that CB1-IR axons innervated densely the majority of hypothalamic nuclei, except for the suprachiasmatic and lateral mammillary nuclei, in which only scattered CB1-IR fibers occurred. CB1-IR innervation of the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the external zone of the median eminence corroborated the important role of CB1 in the regulation of energy homeostasis and neuroendocrine functions. Ultrastructural studies to characterize the phenotype of CB1-IR fibers established that most CB1 immunoreactivity appeared in the preterminal and terminal portions of axons. The CB1-IR boutons formed axospinous, axodendritic, and axosomatic synapses. Analysis of labeled synapses in the paraventricular and arcuate nuclei detected approximately equal numbers of symmetric and asymmetric specializations. In conclusion, the study revealed the dense and differential CB1-IR innervation of most hypothalamic nuclei and the median eminence of the mouse brain. At the ultrastructural level, CB1-IR axons established communication with hypothalamic neurons via symmetric and asymmetric synapses indicating the occurrence of retrograde signaling by endocannabinoids in hypothalamic neuronal networks.

Infundibular tanycytes as modulators of neuroendocrine function: hypothetical role in the regulation of the thyroid and gonadal axis.

Tanycytes comprise a heterogeneous population of specialized cells of glial origin that line the floor and ventrolateral walls of the third ventricle between the rostral and caudal limits of the hypothalamic median eminence. While morphologic and ultrastructural features suggest a role as barrier cells, creating separate compartments between the cerebrospinal fluid, median eminence and hypothalamus, tanycytes likely have multiple other important functions that have yet to be fully elucidated. Possibilities to consider are a role in neuroendocrine regulation including modulation of the hypothalamic-pituitary-thyroid axis during fasting and infection, regulation of reproductive function, particularly in seasonal breeders, and in feeding.

Importance of melanocortin signaling in refeeding-induced neuronal activation and satiety.

To identify regions in the hypothalamus involved in refeeding and their regulation by alpha-MSH, adult rats were subjected to a 3-d fast, and 2 h after refeeding, the distribution of c-Fos-immunoreactive neurons was elucidated. Compared with fed and fasted animals, a significant increase (P < 0.001) in the number of c-Fos-immunoreactive cells was identified in refed animals in the supraoptic nucleus, magnocellular and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus (PVNv), and the dorsal and ventral subdivisions of the dorsomedial nucleus (DMNd and DMNv, respectively). Refeeding shifted the location of c-Fos-labeled neurons from the medial to lateral arcuate where c-Fos was induced in 88.7 +/- 2.2% of alpha-MSH-containing neurons. alpha-MSH-containing axons densely innervated the PVNv, DMNd, and DMNv and organized in close apposition to the majority of refeeding-activated c-Fos-positive neurons. To test whether the melanocortin system is involved in induction of c-Fos in these regions, the melanocortin 3/4 receptor antagonist, agouti-related protein (AGRP 83-132), was administered to fasting animals just before refeeding. Compared with artificial cerebrospinal fluid, a single intracerebroventricular bolus of agouti-related protein (5 microg/5 microl) not only significantly increased the total amount of food consumed within 2 h but also nearly abolished refeeding-induced c-Fos expression in the PVNv and DMNd and partially reduced c-Fos immunoreactivity in the DMNv. We conclude that refeeding activates a subset of neurons in the PVN and DMN as a result of increased melanocortin signaling and propose that one or more of these neuronal populations mediate the potent anorexic actions of alpha-MSH.

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.

The TRH neuron: a hypothalamic integrator of energy metabolism.

Thyrotropin-releasing hormone (TRH) has an important role in the regulation of energy homeostasis not only through effects on thyroid function orchestrated through hypophysiotropic neurons in the hypothalamic paraventricular nucleus (PVN), but also through central effects on feeding behavior, thermogenesis, locomotor activation and autonomic regulation. Hypophysiotropic TRH neurons are located in the medial and periventricular parvocellular subdivisions of the PVN and receive direct monosynaptic projections from two, separate, populations of leptin-responsive neurons in the hypothalamic arcuate nucleus containing either alpha-melanocyte-stimulating hormone (alpha-MSH) and cocaine- and amphetamine-regulated transcript (CART), peptides that promote weight loss and increase energy expenditure, or neuropeptide Y (NPY) and agouti-related protein (AGRP), peptides that promote weight gain and reduce energy expenditure. During fasting, the reduction in TRH mRNA in hypophysiotropic neurons mediated by suppression of alpha-MSH/CART simultaneously with an increase in NPY/AGRP gene expression in arcuate nucleus neurons contributes to the fall in circulating thyroid hormone levels, presumably by increasing the sensitivity of the TRH gene to negative feedback inhibition by thyroid hormone. Endotoxin administration, however, has the paradoxical effect of increasing circulating levels of leptin and melanocortin signaling and CART gene expression in arcuate nucleus neurons, but inhibiting TRH gene expression in hypophysiotropic neurons. This may be explained by an overriding inhibitory effect of endotoxin to increase type 2 iodothyroine deiodinase (D2) in a population of specialized glial cells, tanycytes, located in the base and infralateral walls of the third ventricle. By increasing the conversion of T4 into T3, tanycytes may increase local tissue concenetrations of thyroid hormone, and thereby induce a state of local tissue hyperthyroidism in the region of hypophysisotrophic TRH neurons. Other regions of the brain may also serve as metabolic sensors for hypophysiostropic TRH neurons including the ventrolateral medulla and dorsomedial nucleus of the hypothalamus that have direct monosynaptic projections to the PVN. TRH also exerts a number of effects within the central nervous system that may contribute to the regulation of energy homeostasis. Included are an increase in core body temperature mediated through neurons in the anterior hypothalamic-preoptic area that coordinate a variety of autonomic responses; arousal and locomotor activation through cholinergic and dopaminergic mechanisms on the septum and nucleus accumbens, respectively; and regulation of the cephalic phase of digestion. While the latter responses are largely mediated through cholinergic mechanisms via TRH neurons in the brainstem medullary raphe and dorsal motor nucleus of the vagus, effects of TRH on autonomic loci in the hypothalamic PVN may also be important. Contrary to the actions of T3 to increase appetite, TRH has central effects to reduce food intake in normal, fasting and stressed animals. The precise locus where TRH mediates this response is unknown. However, evidence that an anatomically separate population of nonhypophysiotropic TRH neurons in the anterior parvocellular subdivision of the PVN is integrated into the leptin regulatory control system by the same arcuate nucleus neuronal populations that innervate hypophysiotropic TRH neurons, raises the possibility that anterior parvocellular TRH neurons may be involved, possibly through interactions with the limbic nervous system.