Thyrotropin-releasing hormone receptor 1-deficient mice display increased depression and anxiety-like behavior

TRH is a neuropeptide with a variety of hormonal and neurotransmitter/neuromodulator functions. In particular, TRH has pronounced acute antidepressant effects in both humans and animals and has been implicated in the mediation of the effects of other antidepressive therapies. Two G protein-coupled receptors, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified. Here we report the generation and phenotypic characterization of mice deficient in TRH-R1. The TRH-R1 knockout mice have central hypothyroidism and mild hyperglycemia but exhibit normal growth and development and normal body weight and food intake. Behaviorally, the TRH-R1 knockout mice display increased anxiety and depression levels while behaving normally in a number of other aspects examined. These results provide the first direct evidence that the endogenous TRH system is involved in mood regulation, and this function is carried out through TRH-R1-mediated neural pathways.     

Shank mutant mice as an animal model of autism

In this review, we focus on the role of the Shank family of proteins in autism. In recent years, autism research has been flourishing. With genetic, molecular, imaging and electrophysiological studies being supported by behavioural studies using animal models, there is real hope that we may soon understand the fundamental pathology of autism. There is also genuine potential to develop a molecular-level pharmacological treatment that may be able to deal with the most severe symptoms of autism, and clinical trials are already underway. The Shank family of proteins has been strongly implicated as a contributing factor in autism in certain individuals and sits at the core of the alleged autistic pathway. Here, we analyse studies that relate Shank to autism and discuss what light this sheds on the possible causes of autism.     

Hypermetabolism in mice caused by the central action of an unliganded thyroid hormone receptor alpha1

Thyroid hormone, via its nuclear receptors TRalpha and TRbeta, controls metabolism by acting locally in peripheral tissues and centrally by regulating sympathetic signaling. We have defined aporeceptor regulation of metabolism by using mice heterozygous for a mutant TRalpha1 with low affinity to T3. The animals were hypermetabolic, showing strongly reduced fat depots, hyperphagia and resistance to diet-induced obesity accompanied by induction of genes involved in glucose handling and fatty acid metabolism in liver and adipose tissues. Increased lipid mobilization and beta-oxidation occurred in adipose tissues, whereas blockade of sympathetic signaling to brown adipose tissue normalized the metabolic phenotype despite a continued perturbed hormone signaling in this cell type. The results define a novel and important role for the TRalpha1 aporeceptor in governing metabolic homeostasis. Furthermore, the data demonstrate that a nuclear hormone receptor affecting sympathetic signaling can override its autonomous effects in peripheral tissues.     

BDNF-restricted knockout mice as an animal model for aggression

Mice with global deletion of one brain-derived neurotrophic factor (BDNF) allele or with forebrain-restricted deletion of both alleles show elevated aggression, but this phenotype is accompanied by other behavioral changes, including increases in anxiety and deficits in cognition. Here we performed behavioral characterization of conditional BDNF knockout mice generated using a Cre recombinase driver line, KA1-Cre, which expresses Cre in few areas of brain: highly at hippocampal area CA3 and moderately in dentate gyrus, cerebellum and facial nerve nucleus. The mutant animals exhibited elevated conspecific aggression and social dominance, but did not show changes in anxiety-like behaviors assessed using the elevated plus maze and open field test. There were no changes in depression-like behaviors tested in the forced swim test, but small increase in immobility in the tail suspension test. In cognitive tasks, mutants showed normal social recognition and normal spatial and fear memory, but exhibited a deficit in object recognition. Thus, this knockout can serve as a robust model for BDNF-dependent aggression and object recognition deficiency.     

Corticotropin-releasing hormone receptor 2-deficient mice have reduced intestinal inflammatory responses

Corticotropin-releasing hormone (CRH) and urocortins (Ucn) bind with various affinities to two G-protein-coupled receptors, CRHR1 and CRHR2, which are expressed in brain and in peripheral tissues, including immune cells. CRHR2-deficient mice display anxiety-like behavior, hypersensitivity to stress, altered feeding behavior and metabolism, and cardiovascular abnormalities. However, the phenotype of these mice in inflammatory responses has not been determined. In the present study we found that compared with wild-type CRHR2-null mice developed substantially reduced intestinal inflammation and had lower intestinal mRNA expression of the potent chemoattractants keratinocyte chemokine and monocyte chemoattractant protein 1 following intraluminal exposure to Clostridium difficile toxin A, a potent enterotoxin that mediates antibiotic-associated diarrhea and colitis in humans. This effect was recapitulated by administration of astressin 2B, a selective CRHR2 antagonist, before toxin A exposure. Moreover, Ab array analysis revealed reduced expression of several inflammatory chemokines, including keratinocyte chemokine and monocyte chemoattractant protein 1 in toxin A-exposed mice pretreated with astressin 2B. Real-time RT-PCR of wild-type mouse intestine showed that only UcnII, but not other Ucn, was significantly up-regulated by ileal administration of toxin A at 4 h compared with buffer exposure. We also found that human colonic epithelial HT-29 cells express CRHR2alpha mRNA, whereas expression of beta and gamma spliced variants was minimal. Moreover, treatment of HT-29 cells with UcnII, which binds exclusively to CRHR2, stimulated expression of IL-8 and monocyte chemoattractant protein 1. Taken together, these results provide direct evidence that CRHR2 mediates intestinal inflammatory responses via release of proinflammatory mediators at the colonocyte level.     

autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.     

Generation and characterization of neuregulin-2-deficient mice

The neuregulins (NRGs) are a family of four structurally related growth factors that are expressed in the developing and adult brain. NRG-1 is essential for normal heart formation and has been implicated in the development and maintenance of both neurons and glia. NRG-2 was identified on the basis of its homology to NRG-1 and, like NRG-1, is expressed predominantly by neurons in the central nervous system. We have generated mice with the active domain of NRG-2 deleted in an effort to characterize the biological function of NRG-2 in vivo. In contrast to the NRG-1 knockout animals, NRG-2 knockouts have no apparent heart defects and survive embryogenesis. Mutant mice display early growth retardation and reduced reproductive capacity. No obvious histological differences were observed in the major sites of NRG-2 expression. Our results indicate that in vivo NRG-2 activity differs substantially from that of NRG-1 and that it is not essential for normal development in utero.     

Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications

TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, we utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of [3H]methyl TRH [( 3H]MeTRH) to the TRH receptor. We found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal-cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data we hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. We further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.     

BALB/c小鼠作为单纯疱疹病毒1型感染模型的免疫学分析

在单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)小鼠感染及其相关研究中,临床病理和免疫学指标对其分析具有重要技术意义。本研究观察了HSV-1在不同条件下感染BALB/c小鼠后的多个免疫学指标,包括外周血单核细胞(peripheral blood mononuclear cell,PBMC)群体中树突细胞比例及功能、血清中和抗体水平、PBMC中HSV-1抗原特异性T细胞水平,以及潜伏感染期小鼠神经组织中CD8^＋ T细胞浸润情况。结果显示,HSV-1毒株Mckrae、17＋以角膜及滴鼻途径感染3周龄及6周龄BALB/c小鼠后,小鼠PBMC中树突细胞数量增加,并显示出刺激病毒抗原特异性T细胞增殖的能力。病毒感染后35 d,小鼠PBMC中未检测到白细胞介素4(interleukin 4,IL-4)^＋抗原特异性T细胞,但能检测到低水平的γ干扰素(interferon γ,IFN-γ)^＋抗原特异性T细胞;小鼠血清中未检测到或仅能检测到低水平的中和抗体。HSV-1以皮下及足垫注射途径感染BALB/c小鼠90 d后,足垫感染途径较皮下感染诱导出更高水平的血清中和抗体,PBMC中可检测到IL-4^＋及IFN-γ^＋抗原特异性T细胞,但不同毒株及小鼠周龄之间出现T细胞反应程度差异。组织病理学结果表明,各组小鼠三叉神经组织中均有CD8^＋ T细胞浸润。这些结果提示,不同HSV-1毒株以不同途径感染不同周龄BALB/c小鼠后,均可刺激树突细胞成熟及呈递病毒抗原,但血清中和抗体及PBMC中病毒抗原特异性T细胞水平在不同毒株、感染途径及小鼠周龄之间有差异。     

Requirement of thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice

We recently reported that TRH-deficient mice showed characteristic tertiary hypothyroidism. In the present study, we investigated how this tertiary hypothyroidism occurred particularly in pre- and postnatal stages. Immunohistochemical analysis revealed a number of TSH-immunopositive cells in the TRH-/- pituitary on embryonic day 17.5 and at birth. The mutant pituitary at birth in pups born from TRH-deficient dams also showed no apparent morphological changes, indicating no requirement of either maternal or embryonic TRH for the development of pituitary thyrotrophs. In contrast, apparent decreases in number and level of staining of TSH-immunopositive cells were observed after postnatal day 10 in mutant pituitary. Similar decreases were observed in the 8-week-old mutant pituitary, while no apparent changes were observed in other pituitary hormone-producing cells, and prolonged TRH administration completely reversed this effect. Consistent with these morphological results, TRH-/- mice showed normal thyroid hormone levels at birth, but the subsequent postnatal increase was depressed, resulting in hypothyroidism. As expected, TSH content in the TRH-/- pituitary showed a marked reduction to only 40% of that in the wild type. Despite hypothyroidism in the mutant mice, both the pituitary TSHbeta and alpha mRNA levels were lower than those of the wild-type pituitary. These phenotypic changes were specific to the pituitary thyrotrophs. These findings indicated that 1) TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs, including the normal feedback regulation of the TSH gene by thyroid hormone; 2) neither maternal nor embryonic TRH is required for normal development of the fetal pituitary thyrotroph; and 3) TRH-deficient mice do not exhibit hypothyroidism at birth. Moreover, reflecting its name, TRH has more critical effects on the pituitary thyrotrophs than on other pituitary hormone-producing cells.     

In vivo autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone $\text{in vivo}$ after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.     

Effect of central thyrotropin-releasing hormone on pancreatic blood flow in rats

Central neuropeptides play a role in physiological regulation through the autonomic nervous system. Thyrotropin-releasing hormone (TRH) is a neuropeptide distributed throughout the central nervous system and acts as a neurotransmitter to regulate gastric and hepatic functions through vagal-cholinergic pathways. In this study, the central effect of TRH on pancreatic blood flow was investigated in urethane-anesthetized rats. Pancreatic blood flow was determined by laser Doppler flowmetery. After measurement of basal blood flow, a stable TRH analog, RX 77368 (1-50 ng) or saline was injected intracisternally. Pancreatic blood flow was observed for 120 min thereafter. In some experiments, pretreatment with atropine methyl nitrate (0.15 mg/kg, i.p.), NG-nitro-L-arginine-methyl ester (10 mg/kg, i.v.), or 6-hydroxydopamine (6-OHDA;180 mg/kg, i.p.), or subdiaphragmatic vagotomy was performed. Intracisternal injection of TRH analog dose-dependently increased pancreatic blood flow with a peak response occurring 30 min after injection. The stimulatory effect of TRH analog on pancreatic blood flow was blocked by vagotomy, atropine, and NG-nitro-L-arginine-methyl ester, but not by 6-hydroxydopamine. Intravenous administration of the TRH analog did not influence pancreatic blood flow in the same animal model. These results indicate that TRH acts in the central nervous system to stimulate pancreatic blood flow through vagal-cholinergic and nitric oxide-dependent pathways.     

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH₂ where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3′-amino-1H-1′,2′,4′-triazolyl) built on N of aminoglycine or N^β of ,β-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants >1 μM.     

Germinal center-independent affinity maturation in tumor necrosis factor receptor 1-deficient mice

Germinal centers (GCs) have been identified as site at which the somatic mutation of immunoglobulins occurs.However, somatic mutations in immunoglobulins have also been observed in animals that normally do not harbor germinal centers. This clearly indicates that somatic mutations can occur in the absence of germinal centers.We therefore attempted to determine whether or not GCs exist in TNFR1-deficient mice, and are essential for the somatic mutation of immunoglobulins, using (4-hydroxy-3-nitropheny)acetyl-ovalbumin (NP-OVA). Both wild-type and TNFR1-deficient mice were immunized with NPOVA, and then examined with regard to the existence of GCs. No typical B-cell follicles were detected in the TNFR1-deficient mice. Cell proliferation was detected throughout all splenic tissue types, and no in vivo immunecomplex retention was observed in the TNFR1-deficient mice. All of these data strongly suggest that no GCs were formed in the TNFR1-deficient mice. Although TNFR1-deficient mice are unable to form GCs, serological analyses indicated that affinity maturation had been achieved in both the wild-type and TNFR1-deficient mice. We therefore isolated and sequenced several DNA clones from wild-type and the TNFR1-deficient mice. Eight out of 12 wild-type clones, and 11 out of 14 clones of the TNFR-1-deficient mice contained mutations at the CDR1 site. Thus, the wild-type and TNFR1-deficient mice were not extremely different with regard to types and rates of somatic mutation. Also, high-affinity antibodies were detected in both types of mice. Collectively, our data appear to show that affinity maturation may occur in TNFR1-deficient mice, which completely lack GCs.     

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.     

Regulated dimerization of the thyrotropin-releasing hormone receptor affects receptor trafficking but not signaling

To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.     

Rat ear reattachment as an animal model

The external ear of the rat is an excellent model for practicing microsurgical dissection and for the refinement of microvascular anastomoses, techniques that are crucial for microvascular en bloc tissue transfer and replantation. Preparation of the rat ear for replantation requires familiarity with the vascular anatomy and gentle tissue handling with atraumatic dissection of arterial and venous pedicles, steps similarly crucial in raising free flaps for microvascular transfer. The strategy of performing accurate reduction and stabilization of the tubal cartilage prior to vessel repairs, anastomosing the more deeply seated external carotid artery prior to the more superficial posterior facial vein, is as critical to rat ear replantation as for digital reattachment. In addition, the rat ear as compared to other animal models such as the rabbit ear or canine hindlimbs is much less expensive. Compared to the rat hindlimb model, rat ears are much easier to observe, which is a distinct advantage when used as a model for long-term study of replantation, revascularization, or transplantation.     

Evaluation of early gastric mucosal permeability induced by central thyrotropin-releasing hormone administration

Accumulating evidence suggests that central thyrotropin-releasing hormone (TRH) administration induces gastric erosion 4 h after administration through the vagal nerves. However, early changes in the gastric mucosa during these 4 h have not been described. To assess early changes in the gastric mucosa after intracisternal injection of a stable TRH analog, pGlu-His-(3,3'-dimethyl)-ProNH2 (RX-77368), we measured the blood-to-lumen 51Cr-labeled EDTA clearance and examined the effects of vagotomy, atropine, omeprazole, and hydrochloric acid (HCl) on RX-77368-induced mucosal permeability. A cytoprotective dose of RX-77368 (1.5 ng) did not increase mucosal permeability. However, higher doses significantly increased mucosal permeability. Permeability peaked within 20 min and gradually returned to control levels in response to a 15-ng dose (submaximal dose). Increased mucosal permeability was not recovered after a 150-ng dose (ulcerogenic dose). This increase in permeability was inhibited by vagotomy or atropine. Intragastric perfusion with HCl did not change the RX-77368 (15 ng)-induced increase in permeability, but completely inhibited the recovery of permeability after the peak. Pretreatment with omeprazole did not change the RX-77368 (15 ng)-induced increase in permeability, but quickened the recovery of permeability after the peak. These data indicate that the RX-77368-induced increase in permeability is mediated via the vagal-cholinergic pathway and is not a secondary change in RX-77368-induced acid secretion. Inhibited recovery of permeability on exposure to an ulcerogenic RX-77368 dose or on exposure to HCl plus a submaximal dose of RX-77368 may be crucial for the induction of gastric mucosal lesions by central RX-77368 administration.     

Synthesis and central nervous system actions of thyrotropin-releasing hormone analogs containing a 1-substituted 2-oxoimidazolidine moiety

A series of thyrotropin-releasing hormone (TRH) analogs in which the pyroglutamic acid residue was replaced by (S)-2-oxoimidazolidine-4-carboxylic acid (Oic-OH) and the related derivatives was prepared, and the central nervous system (CNS) actions were examined. Of these, 1-benzyl-Oic-His-Pro-NH2 (2c) showed the most potent activities, which were 1.5-8 times greater than those of TRH. Moreover, the thyrotropin (TSH)-releasing activity of 2c was about 1/16 times weaker than that of TRH.     

Narcolepsy: cholinergic receptor changes in an animal model

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.