Thyrotropin-releasing Hormone Neurons Research Articles

Galanin- but not galanin-like peptide-containing axon terminals innervate hypophysiotropic TRH-synthesizing neurons in the hypothalamic paraventricular nucleus

Galanin and galanin-like peptide (GALP) are both orexigenic peptides involved in the regulation of food intake and energy metabolism. To determine whether these peptides may directly influence the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, double-labeling immunocytochemistry was performed at light and electron microscopic levels using antisera against proTRH, galanin and GALP. Galanin-IR axons densely innervated all of the major parvocellular subdivisions of the PVN where proTRH neurons were identified. The periventricular and anterior parvocellular subdivisions exhibited a prominent network of galaninergic nerve fibers, while the density of fibers was less intense in the medial parvocellular subdivision. Galanin-immunoreactive (IR) axon varicosities were juxtaposed to the majority of TRH-synthesizing neurons in the anterior, medial and periventricular subdivisions of the PVN. Ultrastucturally, galanin-IR nerve terminals established symmetric type synapses with the perikarya of proTRH-IR neurons, suggesting an inhibitory nature of these contacts. In contrast, GALP immunoreactive fibers and nerve terminals concentrated primarily in the anterior parvocellular subdivision of the PVN and were found in association with only few proTRH-IR neurons in the periventricular and medial parvocellular subdivisions. In conclusion, the dense innervation of TRH neurons in all subdivisions of the PVN by galanin-IR axons indicates that galanin may be involved in the central regulation of the hypothalamic–pituitary–thyroid axis. In contrast, the relative paucity of GALP-containing axons in juxtapsoition to TRH neurons in the medial and periventricular parvocellular subdivisions of the PVN, the origin of hypophysiotropic TRH neurons, makes it unlikely that GALP similarly exerts direct regulatory effects on hypophysiotropic TRH neurons.

Central administration of cocaine- and amphetamine-regulated transcript increases phosphorylation of cAMP response element binding protein in corticotropin-releasing hormone-producing neurons but not in prothyrotropin-releasing hormone-producing neurons in the hypothalamic paraventricular nucleus

Cocaine- and amphetamine-regulated transcript (CART) has an important action on hypophysiotropic thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons to regulate the hypothalamic–pituitary–thyroid and adrenal axis, respectively. To elucidate the mechanisms by which CART mediates its effect on TRH and CRH neurons, we determined whether the exogenous administration of CART into the cerebrospinal fluid (CSF) phosphorylates the transcription factor, cyclic adenosine 5′-monophosphate response element binding protein (CREB), in the nucleus of TRH and CRH neurons. CART dramatically increased the percentage of phosphoCREB (PCREB) immunolabeled cell nuclei in the hypothalamic paraventricular nucleus (PVN) in fasted as well as fed rats at 10-min postinjection, particularly in the medial parvocellular subdivision of the PVN. Double immunolabelling with CRH antiserum revealed that CART increased the number of CRH neurons containing PCREB from 10.5±1.2 % to 87±1.2% ( P<0.001) in fasting animals and from 3.7±0.8% to 74±5.3% ( P<0.001) in fed animals. In contrast, no significant change was observed in the percentage of proTRH neurons colocalizing with PCREB either in the fasted (11.7±1.85%) or fed animals (4.2±2.2%) as compared to their respective vehicle controls (2.5±1.4% and 4.6±1%). Ultrastructural analysis revealed that CART establishes axosomatic and axodendritic contacts with CRH neurons in the PVN. These data demonstrate a selective effect of CART to phosphorylate CREB in CRH, but not TRH neurons in the PVN. Since CART is capable of increasing the gene expression of both CRH and TRH in hypophysiotropic neurons, and CART-containing axon terminals establish synaptic relationships with hypophysiotropic CRH and TRH neurons, we propose that CART may signal to the nucleus by more than one pathway.

Cocaine- and Amphetamine-Regulated Transcript Co-Contained in Thyrotropin-Releasing Hormone (TRH) Neurons of the Hypothalamic Paraventricular Nucleus Modulates TRH-Induced Prolactin Secretion

TRH synthesized in hypophysiotropic neurons of the hypothalamic paraventricular nucleus (PVN) stimulates the release of TSH and prolactin from the anterior pituitary gland. Recent data from our laboratories have demonstrated that TRH and cocaine- and amphetamine-regulated transcript (CART) are co-contained only in hypophysiotropic neurons in the PVN. To determine whether CART and TRH interact in the regulation of anterior pituitary function, we have studied the effects of CART on TRH-induced release of TSH and prolactin in anterior pituitary cell cultures, and the effects of hypo- and hyperthyroidism on CART mRNA in the PVN. Dispersed anterior lobe cells from male rats were treated with CART (10(-6), 10(-8), 10(-10), and 10(-12) m) or TRH (10(-7) m) alone and TRH (10(-7) m) combined with various concentrations of CART for 4 h at 37 C. The medium was assayed for prolactin and TSH by RIA. TRH resulted in a marked increase of both prolactin and TSH release, whereas CART had no effect on prolactin or TSH secretion. When the two peptides were used in combination, CART dose-dependently inhibited TRH-induced prolactin release but had no significant effect on TRH-induced TSH release. By semiquantitative analysis of in situ hybridization autoradiographs, CART mRNA was significantly elevated in hypothyroid animals, whereas a reduction in CART mRNA was observed in hyperthyroid animals compared with euthyroid controls. These data raise the possibility that CART expressed in hypophysiotropic TRH neurons has an important role in the modulation of TRH-induced prolactin secretion. Increased secretion of CART may be responsible for the reduced TRH-induced prolactin response during hypothyroidism.

Identification of the Downstream Targets of SIM1 and ARNT2, a Pair of Transcription Factors Essential for Neuroendocrine Cell Differentiation

SIM1 and ARNT2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors that control the differentiation of neuroendocrine lineages in the mouse hypothalamus. Heterozygous Sim1 mice also develop early onset obesity, possibly due to hypodevelopment of the hypothalamus. Although SIM1 and ARNT2 form heterodimers to direct the same molecular pathway, knowledge of this pathway is limited. To facilitate the identification of their downstream genes, we combined an inducible gene expression system in a neuronal cell line with microarray analysis to screen for their transcriptional targets. This method identified 268 potential target genes of SIM1/ARNT2 that displayed >1.7-fold induced expression. 15 of these genes were subjected to Northern analysis, and a high percentage of them were confirmed to be up-regulated. In vivo, several of these genes showed neuroendocrine hypothalamic expression correlating with that of Sim1. Furthermore, we found that expression of two of these potential targets, the Jak2 and thyroid hormone receptor beta2 genes, was lost in the neuroendocrine hypothalamus of the Sim1 mutant. The expression and predicted functions of many of these genes provide new insight into both the Sim1/Arnt2 action in neuroendocrine hypothalamus development and the molecular basis for the Sim1 haplo-insufficient obesity phenotype.

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9×10 6 cells in 35-mm dishes, with 10 μg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

Differential Responses of Thyrotropin-Releasing Hormone (TRH) Neurons to Cold Exposure or Suckling Indicate Functional Heterogeneity of the TRH System in the Paraventricular Nucleus of the Rat Hypothalamus

Thyrotropin-releasing hormone (TRH) is released from the median eminence upon neural stimulation such as cold or suckling exposure. Concomitant with the cold- or suckling-induced release of TRH is a rapid and transient increase in the expression of proTRH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. We employed two strategies to determine whether TRH neurons responding to cold exposure are different from those responding to suckling. First, we attempted to identify a marker of cellular activation in TRH neurons of the PVN. Cold induced c-fos expression in about 25% of TRH neurons of the PVN, but no induction was observed by suckling. Moreover, we explored the expression of a variety of immediate early genes including NGFI-A, fra-1 and c-jun, or CREB phosphorylation but found none to be induced by suckling. The number of cells expressing high levels of proTRH mRNA was counted and compared to total expressing cells. An increased number of cells expressing high levels of proTRH mRNA was observed when both stimuli were applied to the same animal, suggesting that different cells respond separately to each stimulus. We therefore analyzed the distribution of responsive TRH neurons as defined by the cellular level of proTRH mRNA. The proTRH mRNA signal was analyzed within three rostrocaudal zones of the PVN and within six mediolateral columns. Results showed that in response to cold, all areas of the PVN of the lactating rat present increased proTRH mRNA levels, including the anterior zone where few hypophysiotropic TRHergic cells are believed to reside. The distribution of the proTRH mRNA expressing cells in response to cold was quite comparable in female and in male rats. In contrast, the response after suckling was confined to the middle and caudal zones. Our results provide evidence of a functional specialization of TRH cells in the PVN.

BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.

Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.

Critical role for thyroid hormone receptor beta2 in the regulation of paraventricular thyrotropin-releasing hormone neurons.

Thyroid hormone thyroxine (T(4)) and tri-iodothyronine (T(3)) production is regulated by feedback inhibition of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) synthesis in the pituitary and hypothalamus when T(3) binds to thyroid hormone receptors (TRs) interacting with the promoters of the genes for the TSH subunit and TRH. All of the TR isoforms likely participate in the negative regulation of TSH production in vivo, but the identity of the specific TR isoforms that negatively regulate TRH production are less clear. To clarify the role of the TR-beta2 isoform in the regulation of TRH gene expression in the hypothalamic paraventricular nucleus, we examined preprothyrotropin-releasing hormone (prepro-TRH) expression in mice lacking the TR-beta2 isoform under basal conditions, after the induction of hypothyroidism with propylthiouracil, and in response to T(3) administration. Prepro-TRH expression was increased in hypothyroid wild-type mice and markedly suppressed after T(3) administration. In contrast, basal TRH expression was increased in TR-beta2-null mice to levels seen in hypothyroid wild-type mice and did not change significantly in response to induction of hypothyroidism or T(3) treatment. However, the suppression of TRH mRNA expression in response to leptin reduction during fasting was preserved in TR-beta2-null mice. Thus TR-beta2 is the key TR isoform responsible for T(3)-mediated negative-feedback regulation by hypophysiotropic TRH neurons.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling.

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptin's effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

Association of Cocaine- and Amphetamine-Regulated Transcript-Immunoreactive Elements with Thyrotropin-Releasing Hormone-Synthesizing Neurons in the Hypothalamic Paraventricular Nucleus and Its Role in the Regulation of the Hypothalamic–Pituitary–Thyroid Axis during Fasting

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

Acute ethanol administration induces changes in TRH and proenkephalin expression in hypothalamic and limbic regions of rat brain

Thyrotropin releasing hormone (TRH) present in several brain areas has been proposed as a neuromodulator. Its administration produces opposite effects to those observed with acute ethanol consumption. Opioid peptides, in contrast, have been proposed to mediate some of the effects of alcohol intoxication. We measured TRH content and the levels of its mRNA in hypothalamic and limbic zones 1–24 h after acute ethanol injection. We report here fast and transient changes in the content of TRH and its mRNA in these areas. The levels of proenkephalin mRNA varied differently from those of proTRH mRNA, depending on the time and region studied. Wistar rats were administered one dose of ethanol (intraperitoneal, 3 g/kg body weight) and brains dissected in hypothalamus, hippocampus, amygdala, n. accumbens and frontal cortex, for TRH quantification by radioimmunoassay or for proTRH mRNA measurement by RT-PCR. After 1 h injection, TRH levels were increased in hippocampus and decreased in n. accumbens; after 4 h, it decreased in the hypothalamus, frontal cortex and amygdala, recovering to control values in all regions at 24 h. ProTRH mRNA levels increased at 1 h post-injection in total hypothalamus and hippocampus, while they decreased in the frontal cortex. The effect of ethanol was also studied in primary culture of hypothalamic cells; a fast and transient increase in proTRH mRNA was observed at 1 h of incubation (0.001% final ethanol concentration). Changes in the mRNA levels of proTRH and proenkephalin were quantified by in situ hybridization in rats administered ethanol intragastrically (2.5 g/kg). Opposite alterations were observed for these two mRNAs in hippocampus and frontal cortex, while in n. accumbens and the paraventricular nucleus of the hypothalamus, both mRNA levels were increased but with different kinetics. These results give support for TRH and enkephalin neurons as targets of ethanol and, as possible mediators of some of its observed behavioral effects.

Neuropeptide Y Innervation and Neuropeptide-Y-Y1-Receptor-Expressing Neurons in the Paraventricular Hypothalamic Nucleus of the Mouse

The paraventricular hypothalamic nucleus (PVH) serves as integrator and link between the neuroendocrine and autonomic nervous systems. Neuropeptide-Y (NPY)-producing neurons in the arcuate nucleus project to the PVH, where neurons expressing NPY Y1 receptor (Y1R) have been demonstrated. This projection has been suggested to be involved in the regulation of parameters related to energy metabolism, e.g. food intake and thermoregulation. The present study aimed at characterizing this pathway and chemically defining Y1R-expressing neurons by means of immunohistochemistry. The densely distributed NPY-immunoreactive (ir) terminals in the PVH co-stained for agouti gene-related protein (AGRP) mainly in the medial parvocellular regions, indicating an origin in the arcuate nucleus. This was in contrast to noradrenergic/adrenergic terminals in the PVH, which were less frequently seen to contain NPY-like immunoreactivity. Furthermore, AGRP-ir terminals were seen forming abundant close appositions on Y1R-ir cell bodies. Double staining revealed co-existence of Y1R-like immunoreactivity and immunoreactivities for thyrotropin-releasing hormone (TRH) and, to a minor extent, cocaine- and amphetamine-regulated transcript peptide in parvocellular neurons. No Y1R-like immunoreactivity was noted in parvocellular neurons expressing corticotropin-releasing hormone or in magnocellular neurons expressing vasopressin or oxytocin. The present results suggest that the arcuatoparaventricular NPY projection targets the TRH neurons preferentially via the Y1R, whereas the NPYergic regulation of corticotropinergic and magnocellular neurons may be relayed through other subtypes of NPY receptors. This study further defines the link between NPY-induced feeding and the hypothalamus-pituitary-thyroid axis.

Hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons: histochemical relationship to thyrotropin-releasing hormone, melanin-concentrating hormone, orexin/hypocretin and neuropeptide Y

Recent demonstrations of the feeding-inhibitory properties of putative peptides derived from cocaine- and amphetamine-regulated transcript (CART) raise the question of interactions between CART peptides and other messenger molecules implicated in the control of food intake. The present study investigated the histochemical relationship of CART to the neuropeptides thyrotropin-releasing hormone (TRH), melanin-concentrating hormone (MCH), orexin/hypocretin and neuropeptide Y (NPY) in the hypothalamus. Double-label in situ hybridization showed that CART to a great extent is co-expressed with TRH in hypothalamic paraventricular nucleus neurons. This technique was also used to demonstrate that MCH, but not orexin/hypocretin, mRNA colocalized with CART in neurons of the dorsomedial hypothalamic nucleus/lateral hypothalamic area. CART-peptide immunoreactive cell bodies in this region, as well as in the arcuate nucleus and the medial posterodorsal nucleus of the amygdala, were all seen to have close appositions formed by NPY-immunoreactive nerve terminals. Lastly, in a study of mice treated with the neurotoxin, monosodium glutamate, which targets the arcuate nucleus, a near-total ablation of CART peptide immunoreactive cell bodies in this nucleus was accompanied by decreased terminal staining for CART peptide in the paraventricular hypothalamic nucleus, the arcuate nucleus itself and in the dorsomedial hypothalamic nucleus. These findings further define the position of hypothalamic CART neurons within the hierarchy of brain circuitries regulating energy balance, demonstrating the presence of CART peptide in several cell populations that form putative down-stream targets of NPY terminals, including hypophysiotropic TRH neurons and lateral hypothalamic MCH neurons.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on spatial memory and on cholinergic and peptidergic neurons in rats with ibotenate-induced lesions of the nucleus basalis magnocellularis

We conducted behavioral and neurochemical studies of a novel prolyl endopeptidase inhibitor, ( S)2-[[( S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]- N-(phenylmethyl)-1-pyrrolidine-carboxamide (JTP-4819), in rats with lesions of the nucleus basalis magnocellularis (NBM-lesioned rats) induced by ibotenate. Administration of JTP-4819 (1 and 3 mg/kg, p.o.), on and after the 8th day, significantly shortened the escape latency in the Morris water maze as compared to the vehicle-treated group. JTP-4819 also significantly increased the path length in the quadrant with the platform removed in the spatial probe trial. Neurochemical studies of brains removed after the Morris water maze task showed that choline acetyltransferase activity in the cerebral cortex, but not the hippocampus, was significantly reduced by NBM lesioning, while there were no changes of muscarinic M1 receptor binding activity detected using [ 3H]pirenzepine. JTP-4819 had almost no effect on these cholinergic parameters in NBM-lesioned rats. Substance P-like immunoreactivity (LI), thyrotropin-releasing hormone (TRH)-LI, and arginine-vasopressin-LI were not significantly changed in the cerebral cortex and hippocampus of NBM-lesioned rats as compared to sham-operated rats. However, these neuropeptide levels were significantly increased in both brain regions by repeated administration of JTP-4819 (1, 3 and/or 10 mg/kg, p.o.). These results suggest that JTP-4819 ameliorated memory impairment due to NBM lesioning by potentiating SP, TRH and AVPergic neurons secondary to PEP inhibition.

Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin.

Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Delta337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non-triodothyronine (T3)-suppressible serum TSH and pituitary TSH-beta mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.

Localization of mRNA Encoding Thyrotropin-Releasing Hormone Precursor in the Brain of Sockeye Salmon

Abstract The distribution of mRNA encoding thyrotropin-releasing hormone (TRH) precursor in the brain of sockeye salmon was studied by in situ hybridization histochemistry using digoxigenin-labeled riboprobes as a basis to investigate its physiological functions in the salmon brain. Since seasonal variation in TRH gene expression was expected in relation to smolting or maturation, fish were sampled in February and October. In both groups, TRH mRNA was widely distributed in discrete brain regions including the internal cellular layer (ICL) of the olfactory bulb, postcommissural nucleus of area ventralis telencephali (Vp), nucleus preopticus parvocellularis anterioris (PPa), nucleus preopticus magnocellularis, dorsal zone of periventricular hypothalamus (Hd), torus semicircularis, and also the motor nucleus of vagus nerve in the medulla oblongata. TRH neurons in ICL and Hd are round and small with diameters of 5–10 μm. In contrast, TRH neurons in the ventral telencephalon and the preoptic area are medium-si...

Segregation of the intra- and extrahypothalamic neuropeptide Y and catecholaminergic inputs on paraventricular neurons, including those producing thyrotropin-releasing hormone

In fasting, declining circulating thyroid hormone levels coincide with suppressed thyrotropin-releasing hormone (TRH) mRNA and peptide levels and elevated NPY release and binding in the parvicellular paraventricular nucleus (PVN). It is suggested that NPY, in parallel with triggering feeding behavior, interrupts normal thyroid feedback in food deprivation. To gain further insights into the involvement of NPY in the regulation of TRH cells, this study sought to elucidate the source of the NPY innervation of TRH neurons. The median forebrain bundle (MFB) that carries the ascending NPY fibers from the brain stem catecholaminergic nuclei was unilaterally transected. Animals were sacrificed 2 and 5 days after surgery and double immunocytochemistry for NPY and TRH or tyrosine hydroxylase (TH) and TRH was performed on sections from the PVN. Two days after the surgery, light microscopic examination revealed no changes in the numbers of NPY boutons making putative contacts with TRH cell bodies and proximal dendrites. On the other hand, under the electron microscope, NPY- and TH-immunoreactive fibers containing autophagous cytolysosomes, an early sign of catecholaminergic fiber degeneration, were found to establish asymmetric synapses on distal dendrites and dendritic spines of TRH-immunoreactive cells. However, the same electron microscopic analysis did not reveal any degenerating NPY-immunolabeled fibers in synaptic contact with TRH cell bodies and proximal dendrites. Five days after the surgery, when NPY and TH immunoreactivities were no longer detected in the ipsilateral MFB, no decrease in the numbers of NPY and TH boutons on TRH cell bodies and proximal dendrites could be detected, when compared to the contralateral side. Electron microscopy revealed fibers with Wallerian degeneration establishing asymmetric synapses exclusively on the distal dendrites and spines of TRH neurons. In conclusion, this study demonstrated that the NPY and catecholaminergic input on PVN TRH cells are of mixed origin. The cell bodies and proximal dendrites of TRH neurons receive a robust, putative inhibitory NPY input from the hypothalamus. The distal dendrites and dendritic spines of the TRH cells also receive a putative stimulatory NPY input from the brain stem catecholaminergic neurons. It is suggested that because of its proximal location and abundance, NPY of hypothalamic origin exerts a tonic inhibition on PVN TRH cells that interrupts negative thyroid feedback during food deprivation. Furthermore, it is likely that a general inhibition and not stimulation of parvicellular PVN activity may underlie the triggering of feeding behavior by hypothalamic NPY.

The Arcuate Nucleus Is the Major Source for Neuropeptide Y-Innervation of Thyrotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus

The Arcuate Nucleus Is the Major Source for Neuropeptide Y-Innervation of Thyrotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.

Glucocorticoids stimulate TRH and c- fos/c- jun gene co-expression in cultured hypothalamic neurons

To explore whether the protooncogenes, c- fos/c- jun, might be involved in regulating the effect of glucocorticoids on thyrotropin-releasing hormone in fetal rat diencephalic neurons, their localization and transcriptional activity were investigated using double-labeled in situ hybridization, Northern blot and nuclear run-on assays. The results showed that TRH mRNA was coexpressed with both c- jun and c- fos in the same neurons. Treatment with dexamethasone, a synthetic glucocorticoid, at 10 −8 M, enhanced transcriptional activity resulting in an increase in both cell number and intensity of all three mRNAs. The existence of c- fos/c- jun in thyrotropin-releasing hormone neurons and the increased transcriptional activity following dexamethasone treatment suggests that these protooncogenes could mediate the effect of glucocorticoids on thyrotropin-releasing hormone gene expression.