Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture.

A single bout of low-frequency electroacupuncture (EA) causing muscle contractions increases whole-body glucose uptake in insulin-resistant rats. We explored the underlying mechanism of this finding and whether it can be translated into clinical settings. Changes in glucose infusion rate (GIR) were measured by euglycemic-hyperinsulinemic clamp during and after 45 min of low-frequency EA in 21 overweight/obese women with polycystic ovary syndrome (PCOS) and 21 controls matched for age, weight, and body mass index (experiment 1) and in rats receiving autonomic receptor blockers (experiment 2). GIR was higher after EA in controls and women with PCOS. Plasma serotonin levels and homovanillic acid, markers of vagal activity, decreased in both controls and patients with PCOS. Adipose tissue expression of pro-nerve growth factor (proNGF) decreased, and the mature NGF/proNGF ratio increased after EA in PCOS, but not in controls, suggesting increased sympathetic-driven adipose tissue metabolism. Administration of α-/ß-adrenergic receptor blockers in rats blocked the increase in GIR in response to EA. Muscarinic and dopamine receptor antagonist also blocked the response but with slower onset. In conclusion, a single bout of EA increases whole-body glucose uptake by activation of the sympathetic and partly the parasympathetic nervous systems, which could have important clinical implications for the treatment of insulin resistance.-Benrick, A., Kokosar, M., Hu, M., Larsson, M., Maliqueo, M., Marcondes, R. R., Soligo, M., Protto, V., Jerlhag, E., Sazonova, A., Behre, C. J., Højlund, K., Thorén, P., Stener-Victorin, E. Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture.

Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block.

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200-239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

Pathology of natural highly pathogenic avian influenza H5N1 infection in wild tufted ducks (Aythya fuligula).

Highly pathogenic avian influenza (HPAI) subtype H5N1 is an infectious systemic viral disease that results in high morbidity and mortality in poultry, and has been reported in a wide range of wild bird species during the last few years. An outbreak of HPAI H5N1 occurred in wild birds in Sweden in 2006 that affected several duck species, geese, swans, gulls, and raptors. Tufted ducks (Aythya fuligula) accounted for the largest number of positive cases and, therefore, were selected for more in-depth histologic and immunohistochemical evaluations. The main histologic lesions associated with the presence of avian influenza antigen were found in the brain, pancreas, and upper respiratory tract. Other tissues in which influenza antigen was variably found included liver, lung, adrenal glands, kidneys, and peripheral nerve ganglia. The current study describes the pathology and viral tissue targeting of H5N1 by using histology, polymerase chain reaction, and immunohistochemistry, and highlights the range and variation in the presentation of the natural disease in tufted ducks.

Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005.

BACKGROUND: Although the important role of the non-structural 1 (NS) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Europe is incomplete. In this study we determined the subtypes and prevalence of influenza A viruses present in mallards in Northern Europe and further analysed the NS gene of these isolates in order to obtain a more detailed knowledge about the genetic variation of NS gene of influenza A virus in their natural hosts. RESULTS: A total number of 45 influenza A viruses of different subtypes were studied. Eleven haemagglutinin- and nine neuraminidase subtypes in twelve combinations were found among the isolated viruses. Each NS gene reported here consisted of 890 nucleotides; there were no deletions or insertions. Phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present at the same time in the same geographic location in the mallard populations in Northern Europe. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. The degree of variation within the alleles is very low. In our study allele A viruses displays a maximum of 5% amino acid divergence while allele B viruses display only 2% amino acid divergence. All the viruses isolated from mallards in Northern Europe possessed the typical avian ESEV amino acid sequence at the C-terminal end of the NS1 protein. CONCLUSION: Our finding indicates the existence of a large reservoir of different influenza A viruses in mallards population in Northern Europe. Although our phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present in the mallards populations in Northern Europe, allele B viruses appear to be less common in natural host species than allele A, comprising only about 13% of the isolates sequenced in this study.

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006.

BACKGROUND: The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized. RESULTS: The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described. CONCLUSION: The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen.

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.

Evaluation of different sampling methods and results of real-time PCR for detection of feline herpes virus-1, Chlamydophila felis and Mycoplasma felis in cats.

OBJECTIVE: To investigate how different sampling techniques affect detection of DNA from feline herpes virus Type 1 (FHV-1), Chlamydophila felis and Mycoplasma felis and to study the correlation between positive test results and clinical signs in cats. ANIMALS: Fifty-one cats; 24 with ocular signs and 27 healthy control cats. PROCEDURES: Samples were collected from all cats using cotton swabs, conjunctival and corneal biopsies, and corneal scrapings. Samples were analyzed for presence of FHV-1, C. felis, M. felis, and feline DNA, defined by 28S rDNA, by using real-time PCR. RESULTS: In affected cats, FHV-1 was detected in only one cat; C. felis and M. felis were not detected in any affected cats. None of the three organisms was detected in any control cats. Feline DNA was demonstrated in all conjunctival samples, in 82% of corneal swabs, 92% of corneal scrapings, and 100% of keratectomy samples. CONCLUSIONS: Because of the generally low detection rate for FHV-1, C. felis, and M. felis DNA in this study, differences regarding sampling technique could not be determined and correlation between positive test results and degree of clinical signs could not be made. Detection of feline DNA in most samples irrespective of sampling technique, suggests a low prevalence of FHV-1, C. felis and M. felis in this population of cats.

Development of a real-time RT-PCR assay for improved detection of Borna disease virus.

Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L genes were designed by aligning the sequences of known BDV strains. After optimisation, the sensitivity and specificity of the rRT-PCR were established. The detection limit was set to 10-100 viral genomic copies per reaction and the assay detects the BDV strains V and He/80, as well as the most divergent BDV strain known so far, No/98. Furthermore, the system detected feline BDV variants in five naturally infected cats and a feline isolate used in experimental infection of cats. This rRT-PCR assay will be a powerful tool in further studies of BDV, including epidemiological screening and diagnosis.

Validation of a real-time PCR assay for the detection of bovine herpesvirus 1 in bovine semen.

A real-time polymerase chain reaction (PCR) assay was developed for detection of the presence of bovine herpesvirus type 1 (BoHV-1) in extended bovine semen. The assay detects a region encoding a highly conserved glycoprotein B gene. The real-time PCR assay was validated for specificity, sensitivity and repeatability using spiked semen and semen from naturally infected animals. The real-time PCR was very rapid, highly repeatable and more sensitive (lower detection limits) than conventional virus isolation method for the detection of BoHV-1 in extended semen. The specificity of the assay is as expected. The assay had an analytical sensitivity of 0.38 TCID(50) virus spiked into negative semen. The second real-time PCR system for the detection of the bovine growth hormone (bGH) gene was applied as an internal control for the DNA extraction and PCR. The bGH PCR can be performed separately to BoHV-1 PCR, or in a duplex format. The real-time PCR assay is intended for use in international trade. The complete validation dossier based on this study and an international inter-laboratory ring trial has been accredited by the Office International des Epizooties (OIE) and has been recommended to be adopted as a prescribed test for international trade.

Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) modulates the activity of matrix metalloproteinase (MMP) 9, an important mediator of vascular remodeling and plaque instability in atherosclerosis. This study aimed to analyze the expression of NGAL in atherosclerotic plaques and myocardial infarction (MI). METHODS AND RESULTS: Atherosclerotic apolipoprotein E (apoE)(-/-) x low-density lipoprotein receptor (LDLR)(-/-) and C57BL/6J control mice were exposed to brief hypoxic stress (10 minutes of 10% oxygen). Expression of the mouse NGAL homolog (24p3) and MMP-9 was analyzed 48 hours later by quantitative RT-PCR, immunohistochemistry, and zymography. Hypoxic stress increased NGAL/24p3 mRNA in the cardiac vasculature. NGAL/24p3 was also increased in atherosclerotic plaques of apolipoprotein E(-/-) x LDLR(-/-) mice compared with C57BL/6J mice. Mice developing MI exhibited the highest plaque mRNA expression of NGAL/24p3 and MMP-9. Zymography revealed strong proteolytic activity in areas rich in 24p3 and MMP-9 protein. Immunohistochemistry performed on human carotid endarterectomy specimens and control tissue from the internal mammary artery showed colocalization of MMP-9 and NGAL with macrophages in the atherosclerotic plaques. CONCLUSIONS: NGAL/24p3 is increased in atherosclerotic plaques and MI. Colocalization with MMP-9 in areas with high-proteolytic activity suggests a role for NGAL/24p3 in modulating the MMP-9-mediated remodeling of plaques and infarcted hearts.

Delta FosB regulates wheel running.

DeltaFosB is a transcription factor that accumulates in a region-specific manner in the brain after chronic perturbations. For example, repeated administration of drugs of abuse increases levels of DeltaFosB in the striatum. In the present study, we analyzed the effect of spontaneous wheel running, as a model for a natural rewarding behavior, on levels of DeltaFosB in striatal regions. Moreover, mice that inducibly overexpress DeltaFosB in specific subpopulations of striatal neurons were used to study the possible role of DeltaFosB on running behavior. Lewis rats given ad libitum access to running wheels for 30 d covered what would correspond to approximately 10 km/d and showed increased levels of DeltaFosB in the nucleus accumbens compared with rats exposed to locked running wheels. Mice that overexpress DeltaFosB selectively in striatal dynorphin-containing neurons increased their daily running compared with control littermates, whereas mice that overexpress DeltaFosB predominantly in striatal enkephalin-containing neurons ran considerably less than controls. Data from the present study demonstrate that like drugs of abuse, voluntary running increases levels of DeltaFosB in brain reward pathways. Furthermore, overexpression of DeltaFosB in a distinct striatal output neuronal population increases running behavior. Because previous work has shown that DeltaFosB overexpression within this same neuronal population increases the rewarding properties of drugs of abuse, results of the present study suggest that DeltaFosB may play a key role in controlling both natural and drug-induced reward.

Running increases ethanol preference.

Wheel running performed by rats is reinforcing, rewarding and possibly addictive. In this study we analyzed if wheel running could affect ethanol preference. Lewis rats, known to be both addiction-prone and to develop an excessive wheel running behavior, were given access to ethanol in a two-bottle free-choice paradigm. The animals reached a high and stable ethanol intake after 5 weeks. In the next phase, rats were subjected to ethanol withdrawal for 1, 2 or 4 weeks with or without access to running wheels. Finally animals were again given access to ethanol in the same two-bottle free-choice paradigm, combined with access to running wheels. The rats that ran in running wheels during 1 or 2, but not 4, weeks of ethanol withdrawal increased both ethanol intake and preference as compared with the control group that did not have access to the wheels. Previous studies have demonstrated that low doses of morphine increases ethanol preference. Here we show that also running potentiates ethanol intake and preference. Thus, running which shares many of the reinforcing properties with addictive drugs appears to potentiate rats to an increased preference for ethanol. Our results describe a behavioral interaction where running increases ethanol consumption.

Investigations into shaking mink syndrome: an encephalomyelitis of unknown cause in farmed mink (Mustela vison) kits in Scandinavia.

An apparently novel neurological disease clinically characterized by shaking, tremors, seizures, staggering gait, and ataxia was first observed in farmed mink kits in Denmark in 2000 and subsequently in Sweden, Denmark, and Finland in 2001, and again in Denmark in 2002. Lymphoplasmacytic encephalomyelitis was found in the affected kits. The lesions were most severe in the brainstem and cerebellum and consisted of neuronal degeneration and necrosis, neuronophagia, focal and diffuse gliosis, perivascular cuffs formed by lymphocytes, plasma cells and macrophages, and segmental loss of Purkinje cells. Testing was conducted to determine the cause of the disease, including general virological investigations (virus culture, negative-staining electron microscopy, immunoelectron microscopy, polymerase chain reaction for herpesviruses, adenoviruses, pestiviruses, and coronaviruses), tests for specific viral diseases (canine distemper, Borna disease, Louping ill, West Nile virus infection, tick-borne encephalitis, Aleutian disease), tests for protozoa (Toxoplasma gondii, Neospora caninum, Encephalitozoon cuniculi), bacteria (general culture, listeria, Clamydophila psittaci), and intracerebral inoculation of neonatal mice. The results of all these investigations were negative. One group of 3 mink kits inoculated intracerebrally with brain homogenate of affected mink developed clinical signs and histological lesions similar to those observed in naturally infected mink. Based on the histopathological features, it is postulated that the disease is caused by a yet unidentified virus.

Advances in viral disease diagnostic and molecular epidemiological technologies.

The early and rapid detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Genetic characterization of the NS gene indicates co-circulation of two sub-lineages of highly pathogenic avian influenza virus of H5N1 subtype in Northern Europe in 2006.

The non-structural (NS) gene of highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI-H5N1) isolated in Baltic Sea area of Sweden in 2006 was studied. The phylogenetic analysis data demonstrated that two distinct sub-lineages of HPAI-H5N1 were circulating during the outbreak in Northern Europe in Spring 2006. Sub-lineage I viruses fell into the same clade as viruses found in Denmark and Germany and formed a sub-clade which also included viruses isolated in the Russian Federation in late 2005. Sub-lineage II viruses formed a sub-clade closely related to European, Middle Eastern and African isolates reported in 2006. Analysis of the inferred amino acid sequences of the NS1 protein showed a deletion of five amino acids at positions 80-84. No viruses represented in this study contained Glu92 in the NS1 and all isolates contained the avian-like ESKV amino acid sequences at the NS1 C-terminal end. Sub-lineage I isolates contained unique substitutions V194I in NS1 and G63E in Nuclear export protein (NEP).

Influence of the adenosine A1 receptor on blood pressure regulation and renin release.

The present study was performed to investigate the role of adenosine A1 receptors in regulating blood pressure in conscious mice. Adenosine A1-receptor knockout (A1R-/-) mice and their wild-type (A1R+/+) littermates were placed on standardized normal-salt (NS), high-salt (HS), or salt-deficient (SD) diets for a minimum of 10 days before telemetric blood pressure and urinary excretion measurements in metabolic cages. On the NS diet, daytime and nighttime mean arterial blood pressure (MAP) was 7-10 mmHg higher in A1R-/- than in A1R+/+ mice. HS diet did not affect the MAP in A1R-/- mice, but the daytime and nighttime MAP of the A1R+/+ mice increased by approximately 10 mmHg, to the same level as that in the A1R-/-. On the SD diet, day- and nighttime MAP decreased by approximately 6 mmHg in both A1R-/- and A1R+/+ mice, although the MAP remained higher in A1R-/- than in A1R+/+ mice. Although plasma renin levels decreased with increased salt intake in both genotypes, the A1R-/- mice had an approximately twofold higher plasma renin concentration on all diets compared with A1R+/+ mice. Sodium excretion was elevated in the A1R-/- compared with the A1R+/+ mice on the NS diet. There was no difference in sodium excretion between the two genotypes on the HS diet. Even on the SD diet, A1R-/- mice had an increased sodium excretion compared with A1R+/+ mice. An abolished tubuloglomerular feedback response and reduced tubular reabsorption can account for the elevated salt excretion found in A1R-/- animals. The elevated plasma renin concentrations found in the A1R-/- mice could also result in increased blood pressure. Our results confirm that adenosine, acting through the adenosine A1 receptor, plays an important role in regulating blood pressure, renin release, and sodium excretion.

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice.

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.

Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy.

The platelet-derived growth factors are implicated in development of fibrotic reactions and disease in several organs. We have overexpressed platelet-derived growth factor-C in the heart using the alpha-myosin heavy chain promoter and created a transgenic mouse that exhibits cardiac fibrosis followed by hypertrophy with sex-dependent phenotypes. The transgenic mice developed several pathological changes including cardiac fibroblast proliferation and deposition of collagen, hypertrophy, vascular defects, and the presence of Anitschkow cells in the adult myocardium. Male mice developed a hypertrophic phenotype, whereas female mice were more severely affected and developed dilated cardiomyopathy, leading to heart failure and sudden death. The vascular defects initially included dilation of microvessels and vascular leakage. Subsequently, a marked loss of microvessels, formation of large vascular sac-like structures, and an increased density of smooth muscle-coated vessels were observed in the myocardium. In part, the observed vascular changes may be because of an up-regulation of vascular endothelial growth factor in cardiac fibroblasts of the transgenic hearts. This unique animal model reveals that a potent mitogen for cardiac fibroblasts result in an expansion of the interstitium that induce a secondary sex-dependent hypertrophic response in the cardiomyocytes.

Decreased ethanol preference and wheel running in Nurr1-deficient mice.

Nurr1 (Nr4a2) is a transcription factor expressed in dopamine cells from early development and throughout life. Null mutants for Nurr1 lack the ventral midbrain dopamine neurons and die soon after birth. Animals with a heterozygous deletion are viable and display no apparent abnormality. We have investigated the impact of heterozygous deletion of Nurr1 on ethanol consumption in adult mice as a model for drug-induced reward and on wheel running as a model for natural reward. Interestingly, Nurr1 heterozygous mice never developed high ethanol consumption nor did they develop as much running behaviour as did the wild-type animals. Thus, Nurr1 appears to have a key role for the reinforcing properties of ethanol and running that underlies the development of excessive reward-seeking behaviours characteristic for addiction. Quantitative trait loci mapping using C57Bl/6 and DBA/2 mice describe a locus for ethanol preference on chromosome 2, wherein Nurr1 is located. We found two dinucleotide repeats in the Nurr1 promoter that were longer in mice with low preference for ethanol (DBA/2 and 129/Sv) than in mice with high preference for ethanol (C57Bl/6J and C57Bl/6NIH). These sequential data are compatible with Nurr1 as a candidate gene responsible for the quantitative trait loci for ethanol preference on mouse chromosome 2. Together, our data thus imply involvement of Nurr1 in the transition to a state of high ethanol consumption as well as in the development of a high amount of wheel running in mice.

Retardation of post-natal development caused by a negatively acting thyroid hormone receptor alpha1.

Most patients with the syndrome resistance to thyroid hormone (RTH) express a mutant thyroid hormone receptor beta (TRbeta) with transdominant negative transcriptional effects. Since no patient with a mutant TRalpha has been identified, we introduced a point mutation into the mouse thyroid hormone receptor (TRalpha1) locus originally found in the TRbeta gene, that reduces ligand binding 10-fold. Heterozygous 2- to 3-week- old mice exhibit a severe retardation of post-natal development and growth, but only a minor reduction in serum thyroxine levels. Homozygous mice died before 3 weeks of age. Adult heterozygotes overcome most of these defects except for cardiac function abnormalities, suggesting that other factors compensate for the receptor defect. However, the additional deletion of the TRbeta gene in this mouse strain caused a 10-fold increase in serum thyroxine, restored hormonal regulation of target genes for TRs, and rescued the growth retardation. The data demonstrate a novel array of effects mediated by a dominant negative TRalpha1, and may provide important clues for identification of a potentially unrecognized human disorder and its treatment.