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Tatiana L Fonseca,
Mayrin Correa-Medina,
Maira P O Campos,
Gabor Wittmann,
Joao P Werneck-de-Castro,
Rafael Arrojo E Drigo,
Magda Mora-Garzon,
Cintia Bagne Ueta,
Alejandro Caicedo, Csaba Fekete,
Balazs Gereben,
Ronald M Lechan,
Antonio C Bianco
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ABSTRACT: 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.
The Journal of clinical investigation 03/2013; · 15.39 Impact Factor
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ABSTRACT: Recent studies indicate that the effect of thyrotropin-releasing hormone (TRH) on the regulation of food intake may be mediated by histaminergic neurons. To elucidate the anatomical basis for a functional relationship between TRH- and histamine-synthesizing neuronal systems, double-labeling immunocytochemistry was performed on the tuberomammillary nucleus (TMN) of rats, the exclusive location of histaminergic neurons. TRH-immunoreactive (IR) innervation of the histaminergic neurons were detected in all five subnuclei (E1-5) of the TMN, but was most prominent in the E4 and E5 subnuclei where 100% of the histamine-IR neurons were contacted. The number of TRH-IR varicosities in contact with histamine-IR neurons was also greatest in the E4 and E5 subnuclei, averaging 27.0±1.2 in E4 and 7.9±0.5 in E5. Somewhat fewer histamine-IR neurons were juxtaposed by TRH-IR varicosities in E2 and E3 and contacted by 6.3±0.2 and 6.8±0.2 varicosities/innervated cell, respectively. The number of juxtapositions of TRH-IR axon varicosities with histamine-IR neurons was the lowest in the E1 subnucleus (85.7±0.9%; 4.0±0.2 varicosities/innervated cell). Ultrastructural analysis demonstrated that TRH-IR axons established both asymmetric and symmetric type synapses on the perikaryon and dendrites of the histamine-IR neurons, although the majority of synapses were asymmetric type. These data demonstrate that TRH neurons heavily innervate histaminergic neurons in all subdivisions of the TMN, with the densest innervation in the E4 and E5 subdivisions, and are likely to exert activating effects.
Brain research 10/2012; · 2.46 Impact Factor
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ABSTRACT: After fasting, satiety is observed within 2 h after reintroducing food, accompanied by activation of anorexigenic, pro-opiomelanocortin (POMC)-synthesising neurones in the arcuate nucleus (ARC), indicative of the critical role that α-melanocyte-stimulating hormone has in the regulation of meal size during refeeding. To determine whether refeeding-induced activation of POMC neurones in the arcuate is dependent upon the vagus nerve and/or ascending brainstem pathways, bilateral subdiaphragmatic vagotomy or transection of the afferent brainstem input to one side of the ARC was performed. One day after vagotomy or 2 weeks after brain surgery, animals were fasted and then refed for 2 h. Sections containing the ARC from vagotomised animals or animals with effective transection were immunostained for c-Fos and POMC to detect refeeding-induced activation of POMC neurones. Quantitative analyses of double-labelled preparations demonstrated that sham-operated and vagotomised animals markedly increased the number of c-Fos-immunoreactive (-IR) POMC neurones with refeeding. Furthermore, transection of the ascending brainstem pathway had no effect on diminishing c-Fos-immunoreactivity in POMC neurones on either side of the ARC, although it did diminish activation in a separate, subpopulation of neurones in the dorsomedial posterior ARC (dmpARC) on the transected side. We conclude that inputs mediated via the vagus nerve and/or arising from the brainstem do not have a primary role in refeeding-induced activation of POMC neurones in the ARC, and propose that these neurones may be activated solely by direct effects of circulating hormones/metabolites during refeeding. Activation of the dmpARC by refeeding indicates a previously unrecognised role for these neurones in appetite regulation in the rat.
Journal of Neuroendocrinology 06/2012; 24(11):1423-31. · 3.14 Impact Factor
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ABSTRACT: We previously demonstrated that refeeding after a prolonged fast activates a subset of neurons in the ventral parvocellular subdivision of the paraventricular nucleus (PVNv) as a result of increased melanocortin signaling. To determine whether these neurons contribute to satiety by projecting to the nucleus tractus solitarius (NTS), the retrogradely transported marker substance, cholera toxin-β (CTB), was injected into the dorsal vagal complex of rats that were subsequently fasted and refed for 2 h. By double-labeling immunohistochemistry, CTB accumulation was found in the cytoplasm of the majority of refeeding-activated c-Fos neurons in the ventral parvocellular subdivision of the hypothalamic paraventricular nucleus (PVNv). In addition, a large number of refeeding-activated c-Fos-expressing neurons were observed in the lateral parvocellular subdivision (PVNl) that also contained CTB and were innervated by axon terminals of proopiomelanocortin neurons. To visualize the location of neuronal activation within the NTS by melanocortin-activated PVN neurons, α-MSH was focally injected into the PVN, resulting in an increased number of c-Fos-containing neurons in the PVN and in the NTS, primarily in the medial and commissural parts. All refeeding-activated neurons in the PVNv and PVNl expressed the mRNA of the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), indicating their glutamatergic phenotype, but only rare neurons contained oxytocin. These data suggest that melanocortin-activated neurons in the PVNv and PVNl may contribute to refeeding-induced satiety through effects on the NTS and may alter the sensitivity of NTS neurons to vagal satiety inputs via glutamate excitation.
Endocrinology 06/2012; 153(8):3804-14. · 4.46 Impact Factor
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E Schéle, C Fekete,
P Egri,
T Füzesi,
M Palkovits,
É Keller,
Z Liposits,
B Gereben,
L Karlsson-Lindahl,
R Shao,
J-O Jansson
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ABSTRACT: Interleukin (IL)-6 deficient mice develop mature-onset obesity. Furthermore, i.c.v. administration of IL-6 increases energy expenditure, suggesting that IL-6 centrally regulates energy homeostasis. To investigate whether it would be possible for IL-6 to directly influence the energy homeostasis via hypothalamic regulation in humans and rodents, we mapped the distribution of the ligand binding IL-6 receptor α (IL-6Rα) in this brain region. In the human hypothalamus, IL-6Rα-immunoreactivity was detected in perikarya and first-order dendrites of neurones. The IL-6Rα-immunoreactive (-IR) neurones were observed posterior to the level of the interventricular foramen. There, IL-6Rα-IR neurones were located in the lateral hypothalamic, perifornical, dorsal and posterior hypothalamic areas, the hypothalamic dorsomedial nucleus and in the zona incerta. In the caudal part of the hypothalamus, the density of the IL-6Rα-IR neurones gradually increased. Double-labelling immunofluorescent studies demonstrated that IL-6Rα immunoreactivity was localised in the same neurones as the orexigenic neuropeptide, melanin-concentrating hormone (MCH). By contrast, IL-6Rα-immunoreactivity was not observed in the orexin B-IR neurones. To determine whether the observed expression of IL-6Rα is evolutionary conserved, we studied the co-localisation of IL-6Rα with MCH and orexin in the mouse hypothalamus, where IL-6Rα-immunoreactivity was present in numerous MCH-IR and orexin-IR neurones. Our data demonstrate that the MCH neurones of the human hypothalamus, as well as the MCH and orexin neurones of the mouse hypothalamus, contain IL-6Rα. This opens up the possibility that IL-6 influences the energy balance through the MCH neurones in humans, and both MCH and orexin neurones in mice.
Journal of Neuroendocrinology 02/2012; 24(6):930-43. · 3.14 Impact Factor
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Imre Kalló,
Petra Mohácsik,
Barbara Vida,
Anikó Zeöld,
Zsuzsanna Bardóczi,
Ann Marie Zavacki,
Erzsébet Farkas,
Andrea Kádár,
Erik Hrabovszky,
Rafael Arrojo E Drigo,
Liping Dong,
László Barna,
Miklós Palkovits,
Beáta A Borsay,
László Herczeg,
Ronald M Lechan,
Antonio C Bianco,
Zsolt Liposits, Csaba Fekete,
Balázs Gereben
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(6):e37860. · 4.09 Impact Factor
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ABSTRACT: 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.
The Journal of Comparative Neurology 09/2011; 520(5):1005-20. · 3.81 Impact Factor
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ABSTRACT: Cannabinoids suppress fertility via reducing hypothalamic GnRH output. γ-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl(2) in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs.
Endocrinology 10/2010; 151(12):5818-29. · 4.46 Impact Factor
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ABSTRACT: Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the hypothalamic paraventricular nucleus (PVN) in a partly overlapping distribution with non-hypophysiotropic TRH neurons. The distribution of hypophysiotropic TRH neurons in the rat PVN is well understood, but the localization of these neurons is unknown in mice. To determine the distribution and phenotype of hypophysiotropic TRH neurons in mice, double- and triple-labeling experiments were performed on sections of intact mice, and mice treated intravenously and intraperitoneally with the retrograde tracer Fluoro-Gold. TRH neurons were located in all parts of the PVN except the periventricular zone. Hypophysiotropic TRH neurons were observed only at the mid-level of the PVN, primarily in the compact part. In this part of the PVN, TRH neurons were intermingled with oxytocin and vasopressin neurons, but based on their size, the TRH neurons were parvocellular and did not contain magnocellular neuropeptides. Co-localization of TRH and cocaine- and amphetamine-regulated transcript (CART) were observed only in areas where hypophysiotropic TRH neurons were located. In accordance with the morphological observations, hypothyroidism increased TRH mRNA content of neurons only at the mid-level of the PVN. These data demonstrate that the distribution of hypophysiotropic TRH neurons in mice is vastly different from the pattern in rats, with a dominant occurrence of these neurosecretory cells in the compact part and adjacent regions at the mid-level of the PVN. Furthermore, our data demonstrate that the organization of the PVN is markedly different in mice and rats.
The Journal of Comparative Neurology 10/2010; 518(19):3948-61. · 3.81 Impact Factor
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ABSTRACT: 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.
Endocrinology 08/2010; 151(8):3827-35. · 4.46 Impact Factor
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ABSTRACT: Thyrotropin-releasing hormone (TRH) decreases food intake when administered intracerebroventricularly or into the ventromedial hypothalamus. However, it is unknown which population of TRH neurons exerts this anorexigenic function. In the rostral perifornical area, the pattern of TRH-expressing neurons is reminiscent of the distribution of neurons expressing urocortin3 (Ucn3) that also inhibits feeding when injected into the hypothalamic ventromedial nucleus (VMN). Since colocalization of TRH and Ucn3 may help to identify feeding-related TRH neurons, the putative coexpression of the two peptides was examined using fluorescent in situ hybridization combined with immunofluorescence. Almost all (95.5 +/- 0.2%) Ucn3-immunoreactive neurons in the perifornical area expressed pro-TRH mRNA, while 50.2 +/- 1.6% Ucn3 neurons were double-labeled in the bed nucleus of the stria terminalis (BNST). Only a few Ucn3/pro-TRH neurons were found outside these two areas. The distribution of axons containing both Ucn3 and TRH was examined by dual immunofluorescence. Ucn3/TRH fibers heavily innervated the VMN. In addition, high densities of double-labeled axons were observed in the lateral septal nucleus, posterior division of the BNST, medial amygdaloid nucleus, amygdalohippocampal area, and ventral hippocampus, forebrain areas associated with psychological stress and anxiety. We conclude that Ucn3 and TRH are coexpressed in a discrete, continuous population of neurons in the perifornical area and BNST, making Ucn3 a neurochemical marker to define a distinct subset of TRH neurons. The distribution of their axons suggests that Ucn3/TRH neurons may coordinate feeding and behavioral responses to stressful stimuli.
The Journal of Comparative Neurology 12/2009; 517(6):825-40. · 3.81 Impact Factor
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ABSTRACT: The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by approximately 50% (P < 0.05). A highly efficient TEB4 knockdown (>90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Delta18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 microM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.
Molecular and cellular biology 09/2009; 29(19):5339-47. · 6.06 Impact Factor
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ABSTRACT: Hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the paraventricular nucleus of the hypothalamus (PVN). These neurons are well-known to be stimulated by cold exposure through activation of ascending brainstem pathways, and are heavily innervated by catecholaminergic axons that contain dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), enzymes that generate noradrenaline and adrenaline, respectively. However, whether noradrenergic cell groups that lack PNMT contribute to the innervation of TRH neurons is not known. Therefore, triple-labeling immunofluorescence was performed using antibodies against DBH, PNMT and proTRH to determine the relative involvement of adrenaline-synthesizing and noradrenergic neurons in the innervation of TRH neurons in the PVN of rats. Using confocal microscopy, the number of PNMT/DBH (adrenaline-synthesizing) and single-labeled DBH (noradrenergic) boutons juxtaposed to proTRH neurons was quantified. Both noradrenergic and PNMT-containing varicosities were observed in close apposition to virtually all proTRH neurons. An average of 11.8+/-0.6 PNMT-containing and 7.4+/-1.0 noradrenergic boutons was present on the surface of proTRH cell bodies and proximal dendrites. Of all catecholaminergic axon-varicosities juxtaposed to proTRH neurons, 63.5+/-1.2% contained PNMT while the remaining 36.5+/-1.2% were immunopositive for DBH only. We conclude that both adrenaline-synthesizing and noradrenergic axons innervate hypophysiotropic TRH neurons, although there is a predominance of adrenaline-synthesizing fibers. Since adrenaline-synthesizing and noradrenergic cell groups of the brainstem may respond differently to various physiological stimuli, we hypothesize that the two cell groups are likely to mediate the effects of distinct stimuli toward the hypophysiotropic TRH neurons.
Brain research 08/2009; 1294:38-44. · 2.46 Impact Factor
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ABSTRACT: 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.
Journal of neuroscience methods 08/2009; 184(1):115-8. · 2.30 Impact Factor
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ABSTRACT: Cocaine- and amphetamine-regulated transcript (CART) peptides have been implicated in spinal pain transmission. A dense plexus of CART-immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. We demonstrated previously that the majority of these fibres originate from nociceptive primary afferents. Using tract tracing, multiple immunofluorescent labelling and electronmicroscopy we determined the proportion of peptidergic primary afferents expressing CART, looked for evidence for coexistence of CART with galanin in these afferents in lamina I and examined their targets. Almost all (97.9%) randomly selected calcitonin gene-related peptide (CGRP)-immunoreactive terminals were substance P (SP)-positive (+) and CART was detected in approximately half (48.6%) of them. Most (81.4%) of the CGRP/SPergic boutons were galanin+ and approximately half (49.0%) of these contained CART. Many (72.9%) of the CARTergic boutons which expressed CGRP were also immunoreactive for galanin, while only 8.6% of the CARTergic terminals were galanin+ without CGRP. Electron microscopy showed that most of the CART terminals formed asymmetrical synapses, mainly with dendrites. All different morphological and neurochemical subtypes of spinoparabrachial projection neurons in the lamina I received contacts from CART-immunoreactive nociceptive afferents. The innervation density from these boutons did not differ significantly between either the different neurochemical or the morphological subclasses of these cells. This suggests a nonselective innervation of lamina I projection neurons from a subpopulation of CGRP/SP afferents containing CART peptide. These results provide anatomical evidence for involvement of CART peptide in spinal pain transmission.
European Journal of Neuroscience 06/2009; 29(12):2375-87. · 3.63 Impact Factor
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ABSTRACT: The anterior parvocellular subdivision of the PVN (aPVN) contains nonhypophysiotropic thyrotropin-releasing hormone (TRH) neurons that are densely innervated by feeding-related neuronal groups of the hypothalamic arcuate nucleus. To determine how these TRH neurons are integrated within the brain, the major projection fields of this cell group were studied by anterograde and retrograde tract-tracing methods. Projection sites were identified by injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) into the aPVN, and subsequent double immunofluorescent staining was used to visualize axons containing both PHAL and pro-TRH. To distinguish between the projection sites of TRH neurons residing in the aPVN and the closely situated perifornical area, the retrograde tracer cholera toxin B subunit (CTB) was injected into regions where PHAL/pro-TRH-containing axons were densely accumulated. TRH neurons in the aPVN were found to project to the hypothalamic arcuate, dorsomedial and ventral premammillary nuclei, medial preoptic region, tuber cinereum area, paraventricular thalamic nucleus, bed nucleus of the stria terminalis, lateral septal nucleus, and central amygdaloid nucleus. Projection fields of perifornical TRH neurons were in partial overlap with those of the aPVN TRH cells. In addition, these neurons also innervated the hypothalamic ventromedial nucleus, the medial amygdaloid nucleus, and the amygdalohippocampal area. The data suggest that, through its efferent connections, aPVN TRH neurons may be involved in the regulation of energy homeostasis coordinately with effects on behavior, locomotor activity, and thermogenesis. In addition, the major differences in the projection fields of aPVN and perifornical TRH neurons suggest that these two TRH-synthesizing neuronal groups are functionally different.
The Journal of Comparative Neurology 03/2009; 515(3):313-30. · 3.81 Impact Factor
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ABSTRACT: Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.
Endocrinology 02/2009; 150(5):2283-91. · 4.46 Impact Factor
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ABSTRACT: 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.
Endocrinology 10/2008; 150(1):98-103. · 4.46 Impact Factor
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Ruben Nogueiras,
Christelle Veyrat-Durebex,
Paula M Suchanek,
Marcella Klein,
Johannes Tschöp,
Charles Caldwell,
Stephen C Woods,
Gabor Wittmann,
Masahiko Watanabe,
Zsolt Liposits, Csaba Fekete,
Ofer Reizes,
Francoise Rohner-Jeanrenaud,
Matthias H Tschöp
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ABSTRACT: 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.
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.
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 antagonist-elicited reduction of food intake.
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 09/2008; 57(11):2977-91. · 8.29 Impact Factor
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Mirjam Christ-Crain,
Blerina Kola,
Francesca Lolli, Csaba Fekete,
Dalma Seboek,
Gábor Wittmann,
Daniel Feltrin,
Susana C Igreja,
Sharon Ajodha,
Judith Harvey-White,
George Kunos,
Beat Müller,
Francois Pralong,
Gregory Aubert,
Giorgio Arnaldi,
Gilberta Giacchetti,
Marco Boscaro,
Ashley B Grossman,
Márta Korbonits
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ABSTRACT: 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.
The FASEB Journal 07/2008; 22(6):1672-83. · 5.71 Impact Factor
