An amygdala circuit that suppresses social engagement.

Innate social behaviours, such as mating and fighting, are fundamental to animal reproduction and survival1. However, social engagements can also put an individual at risk2. Little is known about the neural mechanisms that enable appropriate risk assessment and the suppression of hazardous social interactions. Here we identify the posteromedial nucleus of the cortical amygdala (COApm) as a locus required for the suppression of male mating when a female mouse is unhealthy. Using anatomical tracing, functional imaging and circuit-level epistatic analyses, we show that suppression of mating with an unhealthy female is mediated by the COApm projections onto the glutamatergic population of the medial amygdalar nucleus (MEA). We further show that the role of the COApm-to-MEA connection in regulating male mating behaviour relies on the neuromodulator thyrotropin-releasing hormone (TRH). TRH is expressed in the COApm, whereas the TRH receptor (TRHR) is found in the postsynaptic MEA glutamatergic neurons. Manipulating neural activity of TRH-expressing neurons in the COApm modulated male mating behaviour. In the MEA, activation of the TRHR pathway by ligand infusion inhibited mating even towards healthy female mice, whereas genetic ablation of TRHR facilitated mating with unhealthy individuals. In summary, we reveal a neural pathway that relies on the neuromodulator TRH to modulate social interactions according to the health status of the reciprocating individual. Individuals must balance the cost of social interactions relative to the benefit, as deficits in the ability to select healthy mates may lead to the spread of disease.

The Antagonist pGlu-ßGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor.

After we identified pGlu-ßGlu-Pro-NH2 as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH's binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-ßGlu-Pro-NH2. The allosteric binding of this TRH antagonist is more robust than TRH's binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.

Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans.

In vertebrates thyrotropin-releasing hormone (TRH) is a highly conserved neuropeptide that exerts the hormonal control of thyroid-stimulating hormone (TSH) levels as well as neuromodulatory functions. However, a functional equivalent in protostomian animals remains unknown, although TRH receptors are conserved in proto- and deuterostomians. Here we identify a TRH-like neuropeptide precursor in Caenorhabditis elegans that belongs to a bilaterian family of TRH precursors. Using CRISPR/Cas9 and RNAi reverse genetics, we show that TRH-like neuropeptides, through the activation of their receptor TRHR-1, promote growth in Celegans TRH-like peptides from pharyngeal motor neurons are required for normal body size, and knockdown of their receptor in pharyngeal muscle cells reduces growth. Mutants deficient for TRH signaling have no defects in pharyngeal pumping or isthmus peristalsis rates, but their growth defect depends on the bacterial diet. In addition to the decrease in growth, trh-1 mutants have a reduced number of offspring. Our study suggests that TRH is an evolutionarily ancient neuropeptide, having its origin before the divergence of protostomes and deuterostomes, and may ancestrally have been involved in the control of postembryonic growth and reproduction.

P38/TRHr-Dependent Regulation of TPO in Thyroid Cells Contributes to the Hypothyroidism of Triclosan-Treated Rats.

BACKGROUND/AIMS: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs). METHODS: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474), or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. RESULTS: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3) and thyroxine (T4). Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr) was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO) was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3) and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1) were also induced by triclosan. CONCLUSION: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians.

Correction and Republication: Effect of Di-(2-ethylhexyl) phthalate on the hypothalamus-pituitary-thyroid axis in adolescent rat.

Di-(2-ethylhexyl) phthalate (DEHP) is extensively used in many personal care and consumer products, which has resulted in widespread human exposure. Limited studies have suggested that exposure to DEHP may affect thyroid function, but little is known about the effect and mechanisms of DEHP exposure on the hypothalamic-pituitary-thyroid axis (HPTA). The present study was conducted to elucidate the potential mechanisms in which DEHP disrupts the function of the HPTA. Wistar rats were administered DEHP by gavage at 0, 5, 50, and 500 mg/kg/day for 28 days and then sacrificed within 24 h following the last dose. Hormones of HPTA was quantified with radioimmunoassay and enzyme-linked immunosorbent assay, protein levels of thyrotropin-releasing hormone receptor (TRHR) and thyroid-stimulating hormone receptor (TSHR) were analyzed by Western blot and immunohistochemistry, expression levels of TRHR and TSHR mRNA were measured by quantitative real-time PCR. Rats treated with DEHP resulted in increased bodyweight, on the HPTA, down-regulated the protein levels of TRH in the hypothalamus, up-regulated the protein and mRNA levels of TRHR in the pituitary, down-regulated mRNA expression of TSHR in the thyroid, while the difference of TSH in various dose groups was not statistically significant and T3, T4, FT3, FT4 levels in serum were decreased compared with control. DEHP could interfere with the balance of HPTA of adolescent rats, and increase the body weight, down-regulate the homeostasis of thyroid related hormones and receptors expression levels.

Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development.

UNLABELLED: Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. SIGNIFICANCE STATEMENT: The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is significantly reduced due to a delayed maturation of S-cone to OFF cone bipolar signaling. These results provide evidence that the retina uses multiple strategies for computing DS responses across different stimulus conditions.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

CREB/TRH pathway in the central nervous system regulates energy expenditure in response to deprivation of an essential amino acid.

BACKGROUND: In the central nervous system (CNS), thyrotropin-releasing hormone (TRH) has an important role in regulating energy balance. We previously showed that dietary deprivation of leucine in mice increases energy expenditure through CNS-dependent regulation. However, the involvement of central TRH in this regulation has not been reported. METHODS: Male C57J/B6 mice were maintained on a control or leucine-deficient diet for 7 days. Leucine-deprived mice were either third intracerebroventricular (i.c.v.) injected with a TRH antibody followed by intraperitoneal (i.p.) injection of triiodothyronine (T3) or i.c.v. administrated with an adenovirus of shCREB (cAMP-response element binding protein) followed by i.c.v. injection of TRH. Food intake and body weight were monitored daily. Oxygen consumption, physical activity and rectal temperature were assessed after the treatment. After being killed, the hypothalamus and the brown adipose tissue were collected and the expression of related genes and proteins related was analyzed. In other experiments, control or leucine-deficient medium incubated primary cultured neurons were either infected with adenovirus-mediated short hairpin RNA targeting extracellular signal-regulated kinases 1 and 2 (Ad-shERK1/2) or transfected with plasmid-overexpressing protein phosphatase 1 regulatory subunit 3C (PPP1R3C). RESULTS: I.c.v. administration of anti-TRH antibodies significantly reduced leucine deprivation-stimulated energy expenditure. Furthermore, the effects of i.c.v. TRH antibodies were reversed by i.p. injection of T3 during leucine deprivation. Moreover, i.c.v. injection of Ad-shCREB (adenovirus-mediated short hairpin RNA targeting CREB) significantly suppressed leucine deprivation-stimulated energy expenditure via modulation of TRH expression. Lastly, TRH expression was regulated by CREB, which was phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing protein Ser/Thr phosphatase type 1 (PP1) under leucine deprivation in vitro. CONCLUSIONS: Our data indicate a novel role for TRH in regulating energy expenditure via T3 during leucine deprivation. Furthermore, our findings reveal that TRH expression is activated by CREB, which is phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing PP1. Collectively, our studies provide novel insights into the regulation of energy homeostasis by the CNS in response to an essential amino-acid deprivation.

Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gß proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K+ channels.

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca(2+) levels has been shown to rely on G(q/11) and PLC activation, the molecular mechanisms involved in the regulation of ERG K(+) channels by TRH are still partially unknown. We have analysed the effects of ßγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH(3) cells ß-ARK/CT, Gα(t), and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the ßγ scavengers affected the TRH-induced shift in V (1/2). Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH(3) cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH(3) cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca(2+) and ERG responses in GH(3) cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH(3) and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.

Genome-wide association and replication studies identified TRHR as an important gene for lean body mass.

Low lean body mass (LBM) is related to a series of health problems, such as osteoporotic fracture and sarcopenia. Here we report a genome-wide association (GWA) study on LBM variation, by using Affymetrix 500K single-nucleotide polymorphism (SNP) arrays. In the GWA scan, we tested 379,319 eligible SNPs in 1,000 unrelated US whites and found that two SNPs, rs16892496 (p = 7.55 x 10(-8)) and rs7832552 (p = 7.58 x 10(-8)), within the thyrotropin-releasing hormone receptor (TRHR) gene were significantly associated with LBM. Subjects carrying unfavorable genotypes at rs16892496 and rs7832552 had, on average, 2.70 and 2.55 kg lower LBM, respectively, compared to those with alternative genotypes. We replicated the significant associations in three independent samples: (1) 1488 unrelated US whites, (2) 2955 Chinese unrelated subjects, and (3) 593 nuclear families comprising 1972 US whites. Meta-analyses of the GWA scan and the replication studies yielded p values of 5.53 x 10(-9) for rs16892496 and 3.88 x 10(-10) for rs7832552. In addition, we found significant interactions between rs16892496 and polymorphisms of several other genes involved in the hypothalamic-pituitary-thyroid and the growth hormone-insulin-like growth factor-I axes. Results of this study, together with the functional relevance of TRHR in muscle metabolism, support the TRHR gene as an important gene for LBM variation.

Pyroglutamyl peptidase II inhibition enhances the analeptic effect of thyrotropin-releasing hormone in the rat medial septum.

Thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH(2)) has multiple, but transient, homeostatic functions in the brain. It is hydrolyzed in vitro by pyroglutamyl peptidase II (PPII), a narrow specificity ectoenzyme with a preferential localization in the brain, but evidence that PPII controls TRH communication in the brain in vivo is scarce. We therefore studied in male Wistar rats the distribution of PPII mRNA in the septum and the consequence of PPII inhibition on the analeptic effect of TRH injected into the medial septum. Twelve to 14% of cell profiles expressed PPII mRNA in the medial septum-diagonal band of Broca; in this region the specific activity of PPII was relatively high. Twenty to 35% of PPII mRNA-labeled profiles were positive for TRH-receptor 1 (TRH-R1) mRNA. The intramedial septum injection of TRH reduced, in a dose-dependent manner, the duration of ethanol-induced loss of righting reflex (LORR). Injection of the PPII inhibitor pGlu-Asn-Pro-7-amido-4-methylcoumarin into the medial septum enhanced the effect of TRH. The injection of a phosphinic TRH analog, a higher-affinity inhibitor of PPII, diminished the duration of LORR by itself. In contrast, the intraseptal injection of pGlu-Asp-Pro-NH(2), a peptide that did not inhibit PPII activity, or an inhibitor of prolyl oligopeptidase did not change the duration of LORR. We conclude that in the medial septum PPII activity may limit TRH action, presumably by reducing the concentration of TRH in the extracellular fluid around cells coexpressing PPII and TRH-R1.

Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

Thyrotropin-releasing hormone induces Ca2+ increase in a subset of vagal nodose ganglion neurons.

Thyrotropin-releasing hormone (TRH) plays a central role in metabolic homeostasis, and single-cell sequencing has recently demonstrated that vagal sensory neurons in the nodose ganglion express thyrotropin-releasing hormone receptor 1 (TRHR1). Here, in situ hybridization validated the presence of TRHR1 in nodose ganglion (NG) neurons and immunohistochemistry showed that the receptor is expressed at the protein level. However, it has yet to be demonstrated whether TRHR1 is functionally active in NG neurons. Using NG explants transduced with a genetically encoded Ca2+ indicator (GECI), we show that TRH increases Ca2+ in a subset of NG neurons. TRH-induced Ca2+ transients were briefer compared to those induced by CCK-8, 2-Me-5-HT and ATP. Blocking Na+ channels with TTX or Na+ substitution did not affect the TRH-induced Ca2+ increase, but blocking Gq signaling with YM-254890 abolished the TRH-induced response. Field potential recordings from the vagus nerve in vitro showed an increase in response to TRH, suggesting that TRH signaling produces action potentials in NG neurons. These observations indicate that TRH activates a small group of NG neurons, involving Gq pathways, and we hypothesize that these neurons may play a role in gut-brain signaling.

ß-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-Releasing Hormone and Taltirelin.

In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of ß-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/ß-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in ß-arrestin2-deficient cells suggest that the ß-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.

Sex-dependent and -independent regulation of thyrotropin-releasing hormone expression in the hypothalamic dorsomedial nucleus by negative energy balance, exercise, and chronic stress.

The dorsomedial nucleus of the hypothalamus (DMH) is part of the brain circuits that modulate organism responses to the circadian cycle, energy balance, and psychological stress. A large group of thyrotropin-releasing hormone (Trh) neurons is localized in the DMH; they comprise about one third of the DMH neurons that project to the lateral hypothalamus area (LH). We tested their response to various paradigms. In male Wistar rats, food restriction during adulthood, or chronic variable stress (CVS) during adolescence down-regulated adult DMH Trh mRNA levels compared to those in sedentary animals fed ad libitum; two weeks of voluntary wheel running during adulthood enhanced DMH Trh mRNA levels compared to pair-fed rats. Except for their magnitude, female responses to exercise were like those in male rats; in contrast, in female rats CVS did not change DMH Trh mRNA levels. A very strong negative correlation between DMH Trh mRNA levels and serum corticosterone concentration in rats of either sex was lost in CVS rats. CVS canceled the response to food restriction, but not that to exercise in either sex. TRH receptor 1 (Trhr) cells were numerous along the rostro-caudal extent of the medial LH. In either sex, fasting during adulthood reduced DMH Trh mRNA levels, and increased LH Trhr mRNA levels, suggesting fasting may inhibit the activity of TRHDMH->LH neurons. Thus, in Wistar rats DMH Trh mRNA levels are regulated by negative energy balance, exercise and chronic variable stress through sex-dependent and -independent pathways.

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

Reversal of hemorrhagic shock in rats using the metabolically stable thyrotropin-releasing hormone analog taltirelin hydrate.

We investigated the effect of taltirelin hydrate ((−)-N-[(S)-hexahydro-1-methyl- 2,6-dioxo-4-pyrimidinyl-carbonyl]-L-histidyl-L-prolinamide tetrahydrate; taltirelin), a metabolically stable thyrotropin-releasing hormone (TRH) analog, on circulatory function, respiratory function, and viable time after bleeding in urethane-anesthetized rats. Massive volume-controlled bleeding caused marked reductions in mean arterial pressure (MAP) and respiratory rate (RR). The vital signs of control rats were lost within an average of 23 min after bleeding. Intravenous administration of taltirelin (0.03−0.3 mg/kg) and TRH (1 and 3 mg/kg) immediately after bleeding accelerated recovery of MAP and RR, and prolonged viable time in a dose-dependent manner. The potency of taltirelin in accelerating MAP and RR recovery and prolonging viable time was higher when compared with that of TRH. In addition, recovery of MAP and RR and the extension of viable time by taltirelin were inhibited by preintraperitoneal administration of atropine sulfate, which is a centrally acting muscarinic antagonist, but not by that of atropine methylbromide, which is a peripherally acting muscarinic antagonist. Taltirelin also recovered decreased arterial pH, bicarbonate ions, and base excess, and prevented a decrease in arterial oxygen saturation. In conclusion, the anti-shock effect of taltirelin was more potent than that of TRH. Taltirelin activity was mediated by the central muscarinic cholinergic system. In addition, taltirelin also corrected metabolic acidosis. These results suggest that taltirelin could be useful in the treatment of hypovolemic shock.

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon (Oncorhynchus nerka).

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon (Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina.