The Musashi-1-type 2 deiodinase pathway regulates astrocyte proliferation.

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2 mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

IKKα deficiency disrupts the development of marginal zone and follicular B cells.

Only few genes have been confidently identified to be involved in the Follicular (FO) and Marginal Zone (MZ) B cell differentiation, migration, and retention in the periphery. Our group previously observed that IKKα kinase inactive mutant mice IKKαK44A/K44A have significantly lower number of MZ B cells whereas FO B cell numbers appeared relatively normal. Because kinase dead IKKα can retain some of its biological functions that may interfere in revealing its actual role in the MZ and FO B cell differentiation. Therefore, in the current study, we genetically deleted IKKα from the pro-B cell lineage that revealed novel functions of IKKα in the MZ and FO B lymphocyte development. The loss of IKKα produces a significant decline in the percentage of immature B lymphocytes, mature marginal zone B cells, and follicular B cells along with a severe disruption of splenic architecture of marginal and follicular zones. IKKα deficiency affect the recirculation of mature B cells through bone marrow. A transplant of IKKα knockout fetal liver cells into Rag-/- mice shows a significant reduction compared to control in the B cells recirculating through bone marrow. To reveal the genes important in the B cell migration, a high throughput gene expression analysis was performed on the IKKα deficient recirculating mature B cells (B220+IgMhi). That revealed significant changes in the expression of genes involved in the B lymphocyte survival, homing and migration. And several among those genes identified belong to G protein family. Taken together, this study demonstrates that IKKα forms a vial axis controlling the genes involved in MZ and FO B cell differentiation and migration.

GLP-1 Receptor Signaling Has Different Effects on the Perikarya and Axons of the Hypophysiotropic Thyrotropin-Releasing Hormone Synthesizing Neurons in Male Mice.

Background: Glucagon-like peptide 1 (GLP-1) is involved in the regulation of energy and glucose homeostasis. As GLP-1 has similar effects on the energy homeostasis as the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons that regulate the hypothalamic-pituitary-thyroid (HPT) axis, we raised the possibility that the TRH neurons are involved in the mediation of the effects of GLP-1. Therefore, the relationship and interaction of the GLP-1 system and the TRH neurons of the hypothalamic paraventricular nucleus (PVN) were studied. Methods: To examine the anatomical and functional relationship of TRH neurons and the GLP-1 system in the PVN, immunocytochemistry, in situ hybridization, in vitro patch-clamp electrophysiology, metabolic phenotyping, and explant experiments were performed. Results: Our data demonstrate that the TRH neurons of the PVN are innervated by GLP-1 producing neurons and express the GLP-1 receptor (GLP-1R). However, not only do the GLP-1-innervated TRH neurons express GLP-1R but the receptor is also present in the axons of the hypophysiotropic TRH neurons in the blood-brain barrier free median eminence (ME) suggesting that peripherally derived GLP-1 may also influence the TRH neurons. In vitro, GLP-1 increased the firing rate of TRH neurons and depolarized them. In addition, GLP-1 directly stimulated the GABAergic input of a population of TRH neurons. Furthermore, GLP-1 inhibited the release of TRH from the hypophysiotropic axons in the ME. In vivo, peripheral GLP-1R agonist administration markedly inhibited the food intake and the energy expenditure, but had no effect on the TRH expression in the PVN and resulted in lower circulating free T4 levels. Conclusions: Our results indicate that GLP-1R activation has a direct stimulatory effect on TRH neurons in the PVN, but the activation of GLP-1R may also inhibit TRH neurons by facilitating their inhibitory inputs or by inhibiting the axon terminals of these cells in the ME. The innervation of TRH neurons by GLP-1 neurons suggests that TRH neurons might be influenced by both circulating GLP-1 and by GLP-1 neurons of the nucleus tractus solitarii. The lack of GLP-1R agonist-induced regulation of TRH neurons in vivo suggests that the HPT axis does not mediate the GLP-1R agonist-induced weight loss.

Topography of the GLP-1/GLP-1 receptor system in the spinal cord of male mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are now commonly used to treat type 2 diabetes and obesity. GLP-1R signaling in the spinal cord has been suggested to account for the mild tachycardia caused by GLP-1R agonists, and may also be involved in the therapeutic effects of these drugs. However, the neuroanatomy of the GLP-1/GLP-1R system in the spinal cord is still poorly understood. Here we applied in situ hybridization and immunohistochemistry to characterize this system, and its relation to cholinergic neurons. GLP-1R transcript and protein were expressed in neuronal cell bodies across the gray matter, in matching distribution patterns. GLP-1R-immunolabeling was also robust in dendrites and axons, especially in laminae II-III in the dorsal horn. Cerebrospinal fluid-contacting neurons expressed GLP-1R protein at exceedingly high levels. Only small subpopulations of cholinergic neurons expressed GLP-1R, including a subset of sympathetic preganglionic neurons at the rostral tip of the intermediolateral nucleus. GLP-1 axons innervated all regions where GLP-1R neurons were distributed, except laminae II-III. Scattered preproglucagon (Gcg) mRNA-expressing neurons were identified in the cervical and lumbar enlargements. The results will facilitate further studies on how GLP-1 regulates the sympathetic system and other autonomic and somatic functions via the spinal cord.

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.

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.

Adult-born proopiomelanocortin neurons derived from Rax-expressing precursors mitigate the metabolic effects of congenital hypothalamic proopiomelanocortin deficiency.

OBJECTIVE: Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential regulators of energy balance. Selective loss of POMC production in these cells results in extreme obesity and metabolic comorbidities. Neurogenesis occurs in the adult hypothalamus, but it remains uncertain whether functional POMC neurons emerge in physiologically significant numbers during adulthood. Here, we tested whether Rax-expressing precursors generate POMC neurons in adult mice and rescue the metabolic phenotype caused by congenital hypothalamic POMC deficiency. METHODS: Initially, we identified hypothalamic Rax-expressing cell types using wild-type and Rax-CreERT2:Ai34D mice. Then we generated compound Rax-CreERT2:ArcPomcloxTB/loxTB mice in which endogenous hypothalamic Pomc expression is silenced, but can be restored by tamoxifen administration selectively in neurons derived from Rax+ progenitors. The number of POMC neurons generated by Rax+ progenitors in adult mice and their axonal projections was determined. The metabolic effects of these neurons were assessed by measuring food intake, bodyweight, and body composition, along with glucose and insulin levels. RESULTS: We found that Rax is expressed by tanycytes and a previously unrecognized cell type in the hypothalamic parenchyma of adult mice. Rax+ progenitors generated ~10% of the normal adult hypothalamic POMC neuron population within two weeks of tamoxifen treatment. The same rate and steady state of POMC neurogenesis persisted from young adult to aged mice. These new POMC neurons established terminal projections to brain regions that were involved in energy homeostasis. Mice with Rax+ progenitor-derived POMC neurons had reduced body fat mass, improved glucose tolerance, increased insulin sensitivity, and decreased bodyweight in proportion to the number of new POMC neurons. CONCLUSIONS: These data demonstrate that Rax+ progenitors generate POMC neurons in sufficient numbers during adulthood to mitigate the metabolic abnormalities of hypothalamic POMC-deficient mice. The findings suggest that adult hypothalamic neurogenesis is a robust phenomenon in mice that can significantly impact energy homeostasis.

Distributions of hypothalamic neuron populations coexpressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse.

The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFPVglut2 - or EGFPVgat -fluorescence, respectively. EGFPVglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFPVgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFPVgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFPVgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFPVgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFPVgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFPVgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine ß-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.

Type 1 cannabinoid receptor-containing axons innervate hypophysiotropic thyrotropin-releasing hormone-synthesizing neurons.

Hypophysiotropic TRH-synthesizing neurons of the hypothalamic paraventricular nucleus (PVN) have a critical role in the regulation of the energy homeostasis through control of the hypothalamic-pituitary-thyroid axis. Recently, endocannabinoids have been shown to exert inhibitory effects on TRH neurons via the type 1 cannabinoid receptor (CB1). To understand the anatomical basis for this regulatory mechanism, we determined whether CB1 is contained in axons innervating hypophysiotropic TRH neurons using a recently developed antiserum against the C-terminal portion of mouse CB1. CB1-immunoreactive axons densely innervated the parvicellular subdivisions of the PVN where the hypophysiotropic TRH neurons are located. By double-labeling immunocytochemistry, CB1-immunoreactive varicosities were observed in juxtaposition to the vast majority of TRH neurons in the PVN. At the ultrastructural level, CB1-immunoreactivity was observed in the preterminal portion of axons establishing both symmetric and asymmetric synaptic specializations with the perikarya and dendrites of TRH neurons in the PVN. These data demonstrate that CB1 is abundantly present in axons that are in synaptic association with hypophysiotropic TRH neurons, indicating an important role for endocannabinoids in the regulation of the hypothalamic-pituitary-thyroid axis. The presence of both symmetric and asymmetric type CB1 synapses on TRH neurons in the PVN suggests that endocannabinoids may influence both excitatory and inhibitory inputs of these neurons.

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.

Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network.

Hypothalamic POMC deficiency leads to obesity and metabolic deficiencies, largely due to the loss of melanocortin peptides. However, POMC neurons in the arcuate nucleus (ARC) are comprised of glutamatergic and GABAergic subpopulations. The developmental program, relative proportion and function of these two subpopulations are unresolved. To test whether glutamatergic POMC neurons serve a distinct role in maintaining energy homeostasis, we activated Pomc expression Cre- dependently in Vglut2-expressing neurons of mice with conditionally silenced Pomc alleles. The Vglut2-Pomc restored mice had normal ARC Pomc mRNA levels, POMC immunoreactivity, as well as body weight and body composition at age 12 weeks. Unexpectedly, the cumulative total of Vglut2+ glutamatergic- and Gad67+ GABAergic-Pomc neurons detected by in situ hybridization (ISH) exceeded 100% in both Vglut2- Pomc restored and control mice, indicating that a subpopulation of Pomc neurons must express both neuronal markers. Consistent with this hypothesis, triple ISH of C57BL/6J hypothalami revealed that 35% of ARC Pomc neurons were selectively Gad67+, 21% were selectively Vglut2+, and 38% expressed both Gad67 and Vglut2. The single Gad67+ and Vglut2+Pomc neurons were most prevalent in the rostral ARC, while the Vglut2/Gad67+ dual-phenotype cells predominated in the caudal ARC. A lineage trace using Ai9-tdTomato reporter mice to label fluorescently all Vglut2-expressing neurons showed equal numbers of tdTomato+ and tdTomato- POMC immunoreactive neurons. Together, these data suggest that POMC neurons exhibit developmental plasticity in their expression of glutamatergic and GABAergic markers, enabling re-establishment of normal energy homeostasis in the Vglut2-Pomc restored mice.

Prss56 expression in the rodent hypothalamus: Inverse correlation with pro-opiomelanocortin suggests oscillatory gene expression in adult rat tanycytes.

We recently reported that the number of hypothalamic tanycytes expressing pro-opiomelanocortin (Pomc) is highly variable among brains of adult rats. While its cause and significance remain unknown, identifying other variably expressed genes in tanycytes may help understand this curious phenomenon. In this in situ hybridization study, we report that the Prss56 gene, which encodes a trypsin-like serine protease and is expressed in neural stem/progenitor cells, shows a similarly variable mRNA expression in tanycytes of adult rats and correlates inversely with tanycyte Pomc mRNA. Prss56 was expressed in α1, ß1, subsets of α2, and some median eminence γ tanycytes, but virtually absent from ß2 tanycytes. Prss56 was also expressed in vimentin positive tanycyte-like cells in the parenchyma of the ventromedial and arcuate nuclei, and in thyrotropin beta subunit-expressing cells of the pars tuberalis of the pituitary. In contrast to adults, Prss56 expression was uniformly high in tanycytes in adolescent rats. In mice, Prss56-expressing tanycytes and parenchymal cells were also observed but fewer in number and without significant variations. The results identify Prss56 as a second gene that is expressed variably in tanycytes of adult rats. We propose that the variable, inversely correlating expression of Prss56 and Pomc reflect periodically oscillating gene expression in tanycytes rather than stable expression levels that vary between individual rats. A possible functional link between Prss56 and POMC, and Prss56 as a potential marker for migrating tanycytes are discussed.

YY1 Upregulates Checkpoint Receptors and Downregulates Type I Cytokines in Exhausted, Chronically Stimulated Human T Cells.

T cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-?). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28?p38MAPK/JNK?YY1? exhaustion.

Contribution of noradrenergic and adrenergic cell groups of the brainstem and agouti-related protein-synthesizing neurons of the arcuate nucleus to neuropeptide-y innervation of corticotropin-releasing hormone neurons in hypothalamic paraventricular nucleus of the rat.

CRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN) integrate neuronal and hormonal inputs and serve as a final common pathway to regulate the hypothalamic-pituitary-adrenal axis. One of the neuronal regulators of CRH neurons is neuropeptide Y (NPY) contained in axons that densely innervate CRH neurons. The three main sources of NPY innervation of the PVN are the hypothalamic arcuate nucleus and the noradrenergic and adrenergic neurons of the brainstem. To elucidate the origin of the NPY-immunoreactive (NPY-IR) innervation to hypophysiotropic CRH neurons, quadruple-labeling immunocytochemistry for CRH, NPY, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase was performed. Approximately 63% of NPY-IR varicosities on the surface of CRH neurons were catecholaminergic (22% noradrenergic and 41% adrenergic), and 37% of NPY-IR boutons were noncatecholaminergic. By triple-labeling immunofluorescence detection of NPY, CRH, and agouti-related protein, a marker of NPY axons projecting from the arcuate nucleus, the noncatecholaminergic, NPY-ergic axon population was shown to arise primarily from the arcuate nucleus. When NPY was administered chronically into the cerebral ventricle of fed animals, a dramatic reduction of CRH mRNA was observed in the PVN (NPY vs. control integrated density units, 23.9 +/- 2.7 vs. 77.09 +/- 15.9). We conclude that approximately two thirds of NPY-IR innervation to hypophysiotropic CRH neurons originates from catecholaminergic neurons of the brainstem, whereas the remaining one third arises from the arcuate nucleus. The catecholaminergic NPY innervation seems to modulate the activation of CRH neurons in association with glucoprivation and infection, whereas the NPY input from the arcuate nucleus may contribute to inhibition of CRH neurons during fasting.

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.

Expression patterns of WSB-1 and USP-33 underlie cell-specific posttranslational control of type 2 deiodinase in the rat brain.

The type 2 deiodinase (D2) activates thyroid hormone and constitutes an important source of 3,5,3',-triiodothyronine in the brain. D2 is inactivated via WSB-1 mediated ubiquitination but can be rescued from proteasomal degradation by USP-33 mediated deubiquitination. Using an in silico analysis of published array data, we found a significant positive correlation between the relative mRNA expression levels of WSB-1 and USP-33 in a set of 56 mouse tissues (r = 0.08; P < 0.04). Subsequently, we used in situ hybridization combined with immunocytochemistry in rat brain to show that in addition to neurons, WSB-1 and USP-33 are differently expressed in astrocytes and tanycytes, the two main D2 expressing cell types in this tissue. Tanycytes, which are thought to participate in the feedback regulation of TRH neurons express both WSB-1 and USP-33, indicating the potential for D2 ubiquitination and deubiquitination in these cells. Notably, only WSB-1 is expressed in glial fibrillary acidic protein-positive astrocytes throughout the brain. Although developmental and environmental signals are known to regulate the expression of WSB-1 and USP-33 in other tissues, our real-time PCR studies indicate that changes in thyroid status do not affect the expression of these genes in several rat brain regions, whereas in the mediobasal hypothalamus, changes in gene expression were minimal. In conclusion, the correlation between the relative mRNA levels of WSB-1 and USP-33 in numerous tissues that do not express D2 suggests that these ubiquitin-related enzymes share additional substrates besides D2. Furthermore, the data indicate that changes in WSB-1 and USP-33 expression are not part of the brain homeostatic response to hypothyroidism or hyperthyroidism.

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.

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.

Expression of vesicular glutamate transporter-2 in gonadotrope and thyrotrope cells of the rat pituitary. Regulation by estrogen and thyroid hormone status.

Immunocytochemical studies of the rat adenohypophysis identified a cell population that exhibits immunoreactivity for type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype. The in situ hybridization detection of VGLUT2 mRNA expression in adenohypophysial cells verified that VGLUT2 immunoreactivity is due to local synthesis of authentic VGLUT2. Dual-immunofluorescent studies of the hypophyses from male rats showed the presence of VGLUT2 in high percentages of LH (93.3 +/- 1.3%)-, FSH (44.7 +/- 3.9%)-, and TSH (70.0 +/- 5.6%)-immunoreactive cells and its much lower incidence in cells of the prolactin, GH, and ACTH phenotypes. Quantitative in situ hybridization studies have established that the administration of a single dose of 17-beta-estradiol (20 microg/kg; sc) to ovariectomized rats significantly elevated VGLUT2 mRNA in the adenohypophysis 16 h postinjection. Thyroid hormone dependence of VGLUT2 expression was addressed by the comparison of hybridization signals in animal models of hypo- and hyperthyroidism to those in euthyroid controls. Although hyperthyroidism had no effect on VGLUT2 mRNA, hypothyroidism increased adenohypophysial VGLUT2 mRNA levels. This coincided with a decreased ratio of VGLUT2-immunoreactive TSH cells, regarded as a sign of enhanced secretion. The presence of the glutamate marker VGLUT2 in gonadotrope and thyrotrope cells, and its up-regulation by estrogen or hypothyroidism, address the possibility that endocrine cells of the adenohypophysis may cosecrete glutamate with peptide hormones in an estrogen- and thyroid status-regulated manner. The exact roles of endogenous glutamate observed primarily in gonadotropes and thyrotropes, including its putative involvement in autocrine/paracrine regulatory mechanisms, will require clarification.

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.