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Sample records for heterozygous knockout mice

  1. Leukemogenesis in heterozygous PU.1 knockout mice.

    PubMed

    Genik, Paula C; Vyazunova, Irina; Steffen, Leta S; Bacher, Jeffery W; Bielefeldt-Ohmann, Helle; McKercher, Scott; Ullrich, Robert L; Fallgren, Christina M; Weil, Michael M; Ray, F Andrew

    2014-09-01

    Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic background of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss.

  2. Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

    PubMed

    Yokoi, Fumiaki; Chen, Huan-Xin; Dang, Mai Tu; Cheetham, Chad C; Campbell, Susan L; Roper, Steven N; Sweatt, J David; Li, Yuqing

    2015-01-01

    DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA(ΔE)). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA(ΔE) does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia. PMID:25799505

  3. Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior.

    PubMed

    Fentress, H M; Klar, R; Krueger, J J; Sabb, T; Redmon, S N; Wallace, N M; Shirey-Rice, J K; Hahn, M K

    2013-11-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET(+/-) ), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET(+/-) mouse establishes an activated state of existing surface NET proteins. The NET(+/-) mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET(+/-) mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET(+/-) mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders.

  4. Norepinephrine Transporter Heterozygous Knockout Mice Exhibit Altered Transport and Behavior

    PubMed Central

    Fentress, HM; Klar, R; Krueger, JK; Sabb, T; Redmon, SN; Wallace, NM; Shirey-Rice, JK; Hahn, MK

    2013-01-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically-driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET+/−), demonstrating that they display an ~50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity, assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET+/− mouse establishes an activated state of existing, surface NET proteins. NET+/− mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris Water Maze. These data suggest recovery of near basal activity in NET+/− mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET+/− mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  5. Tendon fascicle gliding in wild type, heterozygous, and lubricin knockout mice.

    PubMed

    Kohrs, Ross T; Zhao, Chunfeng; Sun, Yu-Long; Jay, Gregory D; Zhang, Ling; Warman, Matthew L; An, Kai-Nan; Amadio, Peter C

    2011-03-01

    The objective of this study was to investigate the role of lubricin in the lubrication of tendon fascicles. Lubricin, a glycoprotein, lubricates cartilage and tendon surfaces, but the function of lubricin within the tendon fascicle is unclear. We developed a novel method to assess the gliding resistance of a single fascicle in a mouse tail model and used it to test the hypothesis that gliding resistance would be increased in lubricin knockout mice. Thirty-six mouse tails were used from 12 wild type, 12 heterozygous, and 12 lubricin knockout mice. A 15 mm long fascicle segment was pulled proximally after being divided distally. The peak resistance during fascicle pullout and the fascicle perimeter were measured. Lubricin expression was evaluated by immunohistochemistry. The peak gliding resistance in the lubricin knockout mice was significantly higher than in the wild type (p < 0.05). Fascicles from heterozygous mice were intermediate in value, but not significantly different from either wild type or lubricin knockout fascicles in peak gliding resistance. No significant difference was found in fascicle perimeter among the three groups. No correlation was observed between fascicle perimeter and gliding resistance. While lubricin was detected by immunostaining on the fascicle surface in wild type and heterozygous mice, lubricin was not detectable in the tendons of knockout mice. We conclude that the absence of lubricin is associated with increased interfascicular friction and that lubricin may play an important role in interfascicular lubrication.

  6. The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice.

    PubMed

    Reuvers, John; Thoreson, Andrew R; Zhao, Chunfeng; Zhang, Ling; Jay, Gregory D; An, Kai-Nan; Warman, Matthew L; Amadio, Peter C

    2011-10-01

    The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity. A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out ⁻/⁻, heterozygous ⁺/⁻ and wild-type ⁺/⁺. A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties. There was no significant difference in the Young's modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice. Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated.

  7. The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice

    PubMed Central

    Reuvers, John; Thoreson, Andrew R.; Zhao, Chunfeng; Zhang, Ling; Jay, Gregory D.; An, Kai-Nan; Warman, Matthew L.; Amadio, Peter C.

    2014-01-01

    The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity. A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out (−/−), heterozygous (+/−) and wild-type (+/+). A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties. There was no significant difference in the Young’s modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice. Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated. PMID:21821131

  8. Adenomatous polyposis coli heterozygous knockout mice display hypoactivity and age-dependent working memory deficits

    PubMed Central

    Koshimizu, Hisatsugu; Fukui, Yasuyuki; Takao, Keizo; Ohira, Koji; Tanda, Koichi; Nakanishi, Kazuo; Toyama, Keiko; Oshima, Masanobu; Taketo, Makoto Mark; Miyakawa, Tsuyoshi

    2011-01-01

    A tumor suppressor gene, Adenomatous polyposis coli (Apc), is expressed in the nervous system from embryonic to adulthood stages, and transmits the Wnt signaling pathway in which schizophrenia susceptibility genes, including T-cell factor 4 (TCF4) and calcineurin (CN), are involved. However, the functions of Apc in the nervous system are largely unknown. In this study, as the first evaluation of Apc function in the nervous system, we have investigated the behavioral significance of the Apc gene, applying a battery of behavioral tests to Apc heterozygous knockout (Apc+/−) mice. Apc+/− mice showed no significant impairment in neurological reflexes or sensory and motor abilities. In various tests, including light/dark transition, open-field, social interaction, eight-arm radial maze, and fear conditioning tests, Apc+/− mice exhibited hypoactivity. In the eight-arm radial maze, Apc+/− mice 6–7 weeks of age displayed almost normal performance, whereas those 11–12 weeks of age showed a severe performance deficit in working memory, suggesting that Apc is involved in working memory performance in an age-dependent manner. The possibility that anemia, which Apc+/− mice develop by 17 weeks of age, impairs working memory performance, however, cannot be excluded. Our results suggest that Apc plays a role in the regulation of locomotor activity and presumably working memory performance. PMID:22347851

  9. Age-Dependent Deficits in Fear Learning in Heterozygous BDNF Knock-Out Mice

    ERIC Educational Resources Information Center

    Endres, Thomas; Lessmann, Volkmar

    2012-01-01

    Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF…

  10. In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype.

    PubMed

    Gineste, C; De Winter, J M; Kohl, C; Witt, C C; Giannesini, B; Brohm, K; Le Fur, Y; Gretz, N; Vilmen, C; Pecchi, E; Jubeau, M; Cozzone, P J; Stienen, G J M; Granzier, H; Labeit, S; Ottenheijm, C A C; Bendahan, D; Gondin, J

    2013-04-01

    Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele. PMID:23375831

  11. Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration.

    PubMed

    Studer, P; da Silva, C G; Revuelta Cervantes, J M; Mele, A; Csizmadia, E; Siracuse, J J; Damrauer, S M; Peterson, C R; Candinas, D; Stroka, D M; Ma, A; Bhasin, M; Ferran, C

    2015-12-01

    Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these

  12. X-linked Christianson syndrome: heterozygous female Slc9a6 knockout mice develop mosaic neuropathological changes and related behavioral abnormalities

    PubMed Central

    Sikora, Jakub; Leddy, Jennifer; Gulinello, Maria; Walkley, Steven U.

    2016-01-01

    ABSTRACT Christianson syndrome (CS) is an X-linked neurodevelopmental and neurological disorder characterized in males by core symptoms that include non-verbal status, intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and hyperkinesis. CS is caused by mutations in the SLC9A6 gene, which encodes a multipass transmembrane sodium (potassium)-hydrogen exchanger 6 (NHE6) protein, functional in early recycling endosomes. The extent and variability of the CS phenotype in female heterozygotes, who presumably express the wild-type and mutant SLC9A6 alleles mosaically as a result of X-chromosome inactivation (XCI), have not yet been systematically characterized. Slc9a6 knockout mice (Slc9a6 KO) were generated by insertion of the bacterial lacZ/β-galactosidase (β-Gal) reporter into exon 6 of the X-linked gene. Mutant Slc9a6 KO male mice have been shown to develop late endosomal/lysosomal dysfunction associated with glycolipid accumulation in selected neuronal populations and patterned degeneration of Purkinje cells (PCs). In heterozygous female Slc9a6 KO mice, β-Gal serves as a transcriptional/XCI reporter and thus facilitates testing of effects of mosaic expression of the mutant allele on penetrance of the abnormal phenotype. Using β-Gal, we demonstrated mosaic expression of the mutant Slc9a6 allele and mosaically distributed lysosomal glycolipid accumulation and PC pathology in the brains of heterozygous Slc9a6 KO female mice. At the behavioral level, we showed that heterozygous female mice suffer from visuospatial memory and motor coordination deficits similar to but less severe than those observed in X-chromosome hemizygous mutant males. Our studies in heterozygous Slc9a6 KO female mice provide important clues for understanding the likely phenotypic range of Christianson syndrome among females heterozygous for SLC9A6 mutations and might improve diagnostic practice and genetic counseling by helping to characterize this presumably

  13. Hyperglycemia Induced by Glucokinase Deficiency Accelerates Atherosclerosis Development and Impairs Lesion Regression in Combined Heterozygous Glucokinase and the Apolipoprotein E-Knockout Mice

    PubMed Central

    Adingupu, Damilola D.; Andréasson, Anne-Christine; Ahnmark, Andrea

    2016-01-01

    Aim. Models combining diabetes and atherosclerosis are important in evaluating the cardiovascular (CV) effects and safety of antidiabetes drugs in the development of treatments targeting CV complications. Our aim was to evaluate if crossing the heterozygous glucokinase knockout mouse (GK+/−) and hyperlipidemic mouse deficient in apolipoprotein E (ApoE−/−) will generate a disease model exhibiting a diabetic and macrovascular phenotype. Methods. The effects of defective glucokinase on the glucose metabolism and on the progression and regression of atherosclerosis on high-fat diets were studied in both genders of GK+/−ApoE−/− and ApoE−/− mice. Coronary vascular function of the female GK+/−ApoE−/− and ApoE−/− mice was also investigated. Results. GK+/−ApoE−/− mice show a stable hyperglycemia which was increased on Western diet. In oral glucose tolerance test, GK+/−ApoE−/− mice showed significant glucose intolerance and impaired glucose-stimulated insulin secretion. Plasma lipids were comparable with ApoE−/− mice; nevertheless the GK+/−ApoE−/− mice showed slightly increased atherosclerosis development. Conclusions. The GK+/−ApoE−/− mice showed a stable and reproducible hyperglycemia, accelerated atherosclerotic lesion progression, and no lesion regression after lipid lowering. This novel model provides a promising tool for drug discovery, enabling the evaluation of compound effects against both diabetic and cardiovascular endpoints simultaneously in one animal model. PMID:27774459

  14. GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice.

    PubMed

    Park, Jong-In; Powers, James F; Tischler, Arthur S; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2005-02-01

    Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.

  15. Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes.

    PubMed

    Kyzar, Evan J; Pham, Mimi; Roth, Andrew; Cachat, Jonathan; Green, Jeremy; Gaikwad, Siddharth; Kalueff, Allan V

    2012-12-01

    Serotonin transporter (SERT) and brain-derived neurotrophic factor (BDNF) are key modulators of molecular signaling, cognition and behavior. Although SERT and BDNF mutant mouse phenotypes have been extensively characterized, little is known about their self-grooming behavior. Grooming represents an important behavioral domain sensitive to environmental stimuli and is increasingly used as a model for repetitive behavioral syndromes, such as autism and attention deficit/hyperactivity disorder. The present study used heterozygous ((+/-)) SERT and BDNF male mutant mice on a C57BL/6J background and assessed their spontaneous self-grooming behavior applying both manual and automated techniques. Overall, SERT(+/-) mice displayed a general increase in grooming behavior, as indicated by more grooming bouts and more transitions between specific grooming stages. SERT(+/-) mice also aborted more grooming bouts, but showed generally unaltered activity levels in the observation chamber. In contrast, BDNF(+/-) mice displayed a global reduction in grooming activity, with fewer bouts and transitions between specific grooming stages, altered grooming syntax, as well as hypolocomotion and increased turning behavior. Finally, grooming data collected by manual and automated methods (HomeCageScan) significantly correlated in our experiments, confirming the utility of automated high-throughput quantification of grooming behaviors in various genetic mouse models with increased or decreased grooming phenotypes. Taken together, these findings indicate that mouse self-grooming behavior is a reliable behavioral biomarker of genetic deficits in SERT and BDNF pathways, and can be reliably measured using automated behavior-recognition technology.

  16. Comparative analyses of lung transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins and in foxf1 heterozygous knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority...

  17. Xpa knockout mice.

    PubMed

    de Vries, A; van Steeg, H

    1996-10-01

    The xeroderma pigmentosum group A correcting (XPA) gene encodes a DNA binding zinc-finger protein that recognizes DNA damage. As such the XPA protein participates in the initial step of the process of nucleotide excision repair. The multicomponent nucleotide excision repair pathway is one of the most thoroughly studied mechanisms that defends both eukaryotic and prokaryotic cells against the deleterious effects of UV-B and several chemical components. In the absence of nucleotide excision repair common cellular processes like transcription and replication are disturbed by persisting (unrepaired) DNA lesions (adducts), which may lead to the accumulation of gene mutations and ultimately to cancer. Xeroderma pigmentosum patients have a > 2000 fold increased risk to develop skin cancer at sun-exposed areas. Here we describe that XPA-deficient transgenic mice show features that mimic the phenotype found in humans. Furthermore, the possible use of Xpa- and other nucleotide excision repair deficient mice in cancer research will be outlined in more detail. PMID:9110400

  18. Heterozygous knockout of the Bmi-1 gene causes an early onset of phenotypes associated with brain aging.

    PubMed

    Gu, Minxia; Shen, Lihua; Bai, Lei; Gao, Junying; Marshall, Charles; Wu, Ting; Ding, Jiong; Miao, Dengshun; Xiao, Ming

    2014-02-01

    Previous studies reported that the polycomb group gene Bmi-1 is downregulated in the aging brain. The aim of this study was to investigate whether decreased Bmi-1 expression accelerates brain aging by analyzing the brain phenotype of adult Bmi-1 heterozygous knockout (Bmi-1(+/-)) mice. An 8-month-old Bmi-1(+/-) brains demonstrated mild oxidative stress, revealed by significant increases in hydroxy radical and nitrotyrosine, and nonsignificant increases in reactive oxygen species and malonaldehyde compared with the wild-type littermates. Bmi-1(+/-) hippocampus had high apoptotic percentage and lipofuscin deposition in pyramidal neurons associated with upregulation of cyclin-dependent kinase inhibitors p19, p27, and p53 and downregulation of anti-apoptotic protein Bcl-2. Mild activation of astrocytes was also observed in Bmi-1(+/-) hippocampus. Furthermore, Bmi-1(+/-) mice showed mild spatial memory impairment in the Morris Water Maze test. These results demonstrate that heterozygous Bmi-1 gene knockout causes an early onset of age-related brain changes, suggesting that Bmi-1 has a role in regulating brain aging.

  19. Knockout of Foxp2 disrupts vocal development in mice.

    PubMed

    Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

    2016-03-16

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control.

  20. Lingual deficits in neurotrophin double knockout mice.

    PubMed

    Nosrat, Irina V; Agerman, Karin; Marinescu, Andrea; Ernfors, Patrik; Nosrat, Christopher A

    2004-12-01

    Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF(-/-) and wild-type mice. Taste papillae morphology was severely distorted in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF(-/-) and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF(-/-) and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF(-/-) xNT-3(-/-) mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development. PMID:16217617

  1. FAK-heterozygous mice display enhanced tumour angiogenesis

    PubMed Central

    Kostourou, Vassiliki; Lechertier, Tanguy; Reynolds, Louise E.; Lees, Delphine M.; Baker, Marianne; Jones, Dylan T.; Tavora, Bernardo; Ramjaun, Antoine R.; Birdsey, Graeme M.; Robinson, Stephen D.; Parsons, Maddy; Randi, Anna M.; Hart, Ian R; Hodivala-Dilke, Kairbaan

    2013-01-01

    Genetic ablation of endothelial Focal Adhesion Kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularisation. Here we show that reduced stromal-FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumor growth in vivo. Our results highlight a potential novel role for FAK as a non-linear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis. PMID:23799510

  2. Knockout of Foxp2 disrupts vocal development in mice

    PubMed Central

    Castellucci, Gregg A.; McGinley, Matthew J.; McCormick, David A.

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/−) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/− mice. In comparison to their WT littermates, Foxp2+/− mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/− song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene’s role in general vocal motor control. PMID:26980647

  3. Vulnerability to mild predator stress in serotonin transporter knockout mice.

    PubMed

    Adamec, Robert; Burton, Paul; Blundell, Jacqueline; Murphy, Dennis L; Holmes, Andrew

    2006-06-01

    Effect of predator stress on rat and mouse anxiety-like behavior may model aspects of post traumatic stress disorder (PTSD). A single cat exposure of wild type (C57, CFW) mice can produce lasting anxiety-like effects in the elevated plus maze, light/dark box tests and startle. In addition, female but not male C57 mice are made more anxious in the plus maze by exposure to predator odors alone, suggesting differential vulnerability to predator stressors of differing intensity. There is a link between genetic variation in the serotonin (5-HT) transporter (SERT) and anxiety in humans. This prompted the generation of SERT knockout mice [see Holmes A, Murphy DL, Crawley, JN. Biol Psychiatry 2003;54(10):953-9]. Present work used these mice to determine if there was a link between vulnerability to the anxiogenic effects of predator odors and abnormalities of 5-HT transmission induced by a life long reduction in 5-HT reuptake. Wild type (WT, C57 background), heterozygous (SERT +/-, HET) mice and homozygous knockout (SERT -/-, KO) were assigned to handled control groups or groups exposed for 10 min to a large testing room rich in cat odor. One week after handling or room exposure, anxiety testing took place in the dark phase of the light/dark cycle, in red light. Predator odor exposure was selectively anxiogenic in the plus maze and light/dark box tests in SERT -/- mice. Exposure to predator odor did not potentiate startle. Findings suggest a role for abnormalities in 5-HT transmission in vulnerability to some of the lasting anxiogenic effects of species relevant stressors and possibly in vulnerability to PTSD. PMID:16546269

  4. Characterization of the heterozygous glucokinase knockout mouse as a translational disease model for glucose control in type 2 diabetes

    PubMed Central

    Baker, D J; Atkinson, A M; Wilkinson, G P; Coope, G J; Charles, A D; Leighton, B

    2014-01-01

    Background and Purpose The global heterozygous glucokinase (GK) knockout (gkwt/del) male mouse, fed on a high-fat (60% by energy) diet, has provided a robust and reproducible model of hyperglycaemia. This model could be highly relevant to some facets of human type 2 diabetes (T2D). We aimed to investigate the ability of standard therapeutic agents to lower blood glucose at translational doses, and to explore the glucose-lowering potential of novel glucokinase activators (GKAs) in this model. Experimental Approach We measured the ability of insulin, metformin, glipizide, exendin-4 and sitagliptin, after acute or repeat dose administration, to lower free-feeding glucose levels in gkwt/del mice. Further, we measured the ability of novel GKAs, GKA23, GKA71 and AZD6370 to control glucose either alone or in combination with some standard agents. Key Results A single dose of insulin (1 unit·kg−1), metformin (150, 300 mg·kg−1), glipizide (0.1, 0.3 mg·kg−1), exendin-4 (2, 20 μg·kg−1) and GKAs reduced free-feeding glucose levels. Sitagliptin (10 mg·kg−1), metformin (300 mg·kg−1) and AZD6370 (30, 400 mg·kg−1) reduced glucose excursions on repeat dosing. At a supra-therapeutic dose (400 mg·kg−1), AZD6370 also lowered basal levels of glucose without inducing hypoglycaemia. Conclusion and Implications Standard glucose-lowering therapeutic agents demonstrated significant acute glucose lowering in male gkwt/del mice at doses corresponding to therapeutic free drug levels in man, suggesting the potential of these mice as a translatable model of human T2D. Novel GKAs also lowered glucose in this mouse model. PMID:24772483

  5. Delayed Wound Healing in CXCR2 Knockout Mice

    PubMed Central

    Devalaraja, Radhika M.; Nanney, Lillian B.; Qian, Qinghua; Du, Jianguo; Yu, Yingchun; Devalaraja, Madhav N.; Richmond, Ann

    2009-01-01

    Previous studies demonstrated that the CXC chemokine, MGSA/GRO-α and its receptor, CXCR2, are expressed during wound healing by keratinocytes and endothelial cells at areas where epithelialization and neovascularization occur. The process of wound healing is dependent on leukocyte recruitment, keratinocyte proliferation and migration, and angiogenesis. These processes may be mediated in part by CXC chemokines, such as interleukin-8 and MGSA/GRO-α. To examine further the significance of CXC chemokines in wound healing, full excisional wounds were created on CXCR2 wild-type (+/+), heterozygous (+/−), or knockout (−/−) mice. Wounds were histologically analyzed for neutrophil and monocyte infiltration, neovascularization and epithelialization at days 3, 5, 7, and 10 postwounding. The CXCR2−/− mice exhibited defective neutrophil recruitment, an altered temporal pattern of monocyte recruitment, and altered secretion of interleukin-1β. Significant delays in wound healing parameters, including epithelialization and decreased neovascularization, were also observed in CXCR2−/− mice. In vitro wounding experiments with cultures of keratinocytes established from −/− and +/+ mice revealed a retardation in wound closure in CXCR2−/− keratinocytes, suggesting a role for this receptor on keratinocytes in epithelial resurfacing that is independent of neutrophil recruitment. These in vitro and in vivo studies further establish a pathophysiologic role for CXCR2 during cutaneous wound repair. PMID:10951241

  6. Disruption of NBS1 gene leads to early embryonic lethality in homozygous null mice and induces specific cancer in heterozygous mice

    SciTech Connect

    Kurimasa, Akihiro; Burma, Sandeep; Henrie, Melinda; Ouyang, Honghai; Osaki, Mitsuhiko; Ito, Hisao; Nagasawa, Hatsumi; Little, John B.; Oshimura, Mitsuo; Li, Gloria C.; Chen, David J.

    2002-04-15

    Nijmegen breakage syndrome (NBS) is a rare autosomal recessive chromosome instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition, with cellular features similar to that of ataxia telangiectasia (AT). NBS results from mutations in the mammalian gene Nbs1 that codes for a 95-kDa protein called nibrin, NBS1, or p95. To establish an animal model for NBS, we attempted to generate NBS1 knockout mice. However, NBS1 gene knockouts were lethal at an early embryonic stage. NBS1 homozygous(-/-) blastocyst cells cultured in vitro showed retarded growth and subsequently underwent growth arrest within 5 days of culture. Apoptosis, assayed by TUNEL staining, was observed in NBSI homozygous(-/-) blastocyst cells cultured for four days. NBSI heterozygous(+/-) mice were normal, and exhibited no specific phenotype for at least one year. However, fibroblast cells from NBSI heterozygous(+/-) mice displayed an enhanced frequency of spontaneous transformation to anchorage-independent growth as compared to NBS1 wild-type(+/+) cells. Furthermore, heterozygous(+/-) mice exhibited a high incidence of hepatocellular carcinoma after one year compared to wild-type mice, even though no significant differences in the incidence of other tumors such as lung adenocarcinoma and lymphoma were observed. Taken together, these results strongly suggest that NBS1 heterozygosity and reduced NBSI expression induces formation of specific tumors in mice.

  7. Enhanced Tumor Formation in Mice Heterozygous for Blm Mutation

    NASA Astrophysics Data System (ADS)

    Heppner Goss, Kathleen; Risinger, Mary A.; Kordich, Jennifer J.; Sanz, Maureen M.; Straughen, Joel E.; Slovek, Lisa E.; Capobianco, Anthony J.; German, James; Boivin, Gregory P.; Groden, Joanna

    2002-09-01

    Persons with the autosomal recessive disorder Bloom syndrome are predisposed to cancers of many types due to loss-of-function mutations in the BLM gene, which encodes a recQ-like helicase. Here we show that mice heterozygous for a targeted null mutation of Blm, the murine homolog of BLM, develop lymphoma earlier than wild-type littermates in response to challenge with murine leukemia virus and develop twice the number of intestinal tumors when crossed with mice carrying a mutation in the Apctumor suppressor. These observations indicate that Blm is a modifier of tumor formation in the mouse and that Blm haploinsufficiency is associated with tumor predisposition, a finding with important implications for cancer risk in humans.

  8. Urea Transporter Physiology Studied in Knockout Mice

    PubMed Central

    Li, Xuechen; Chen, Guangping; Yang, Baoxue

    2012-01-01

    In mammals, there are two types of urea transporters; urea transporter (UT)-A and UT-B. The UT-A transporters are mainly expressed in kidney epithelial cells while UT-B demonstrates a broader distribution in kidney, heart, brain, testis, urinary tract, and other tissues. Over the past few years, multiple urea transporter knockout mouse models have been generated enabling us to explore the physiological roles of the different urea transporters. In the kidney, deletion of UT-A1/UT-A3 results in polyuria and a severe urine concentrating defect, indicating that intrarenal recycling of urea plays a crucial role in the overall capacity to concentrate urine. Since UT-B has a wide tissue distribution, multiple phenotypic abnormalities have been found in UT-B null mice, such as defective urine concentration, exacerbated heart blockage with aging, depression-like behavior, and earlier male sexual maturation. This review summarizes the new insights of urea transporter functions in different organs, gleaned from studies of urea transporter knockout mice, and explores some of the potential pharmacological prospects of urea transporters. PMID:22745630

  9. Generation of knockout mice using engineered nucleases.

    PubMed

    Sung, Young Hoon; Jin, Young; Kim, Seokjoong; Lee, Han-Woong

    2014-08-15

    The use of engineered nucleases in one-cell stage mouse embryos is emerging as an efficient alternative to conventional gene targeting in mouse embryonic stem (ES) cells. These nucleases are designed or reprogrammed to specifically induce double strand breaks (DSBs) at a desired genomic locus, and efficiently introduce mutations by both error-prone and error-free DNA repair mechanisms. Since these mutations frequently result in the loss or alteration of gene function by inserting, deleting, or substituting nucleotide sequences, engineered nucleases are enabling us to efficiently generate gene knockout and knockin mice. Three kinds of engineered endonucleases have been developed and successfully applied to the generation of mutant mice: zinc-finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs). Based on recent advances, here we provide experimentally validated, detailed guidelines for generating non-homologous end-joining (NHEJ)-mediated mutant mice by microinjecting TALENs and RGENs into the cytoplasm or the pronucleus of one-cell stage mouse embryos.

  10. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference.

  11. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference. PMID:27142766

  12. Production of a mouse strain with impaired glucose tolerance by systemic heterozygous knockout of the glucokinase gene and its feasibility as a prediabetes model.

    PubMed

    Saito, Mikako; Kaneda, Asako; Sugiyama, Tae; Iida, Ryousuke; Otokuni, Keiko; Kaburagi, Misako; Matsuoka, Hideaki

    2015-01-01

    Exon II of glucokinase (Gk) was deleted to produce a systemic heterozygous Gk knockout (Gk(+/-)) mouse. The relative expression levels of Gk in the heart, lung, liver, stomach, and pancreas in Gk(+/-) mice ranged from 0.41-0.68 versus that in wild (Gk(+/+)) mice. On the other hand, its expression levels in the brain, adipose tissue, and muscle ranged from 0.95-1.03, and its expression levels in the spleen and kidney were nearly zero. Gk knockout caused no remarkable off-target effect on the expression of 7 diabetes causing genes (Shp, Hnf1a, Hnf1b, Irs1, Irs2, Kir6.2, and Pdx1) in 10 organs. The glucose tolerance test was conducted to determine the blood glucose concentrations just after fasting for 24 h (FBG) and at 2 h after high-glucose application (GTT2h). The FBG-GTT2h plots obtained with the wild strain fed the control diet (CD), Gk(+/-) strain fed the CD, and Gk(+/-) strain fed the HFD were distributed in separate areas in the FBG-GTT2h diagram. The respective areas could be defined as the normal state, prediabetes state, and diabetes state, respectively. Based on the results, the criteria for prediabetes could be defined for the Gk(+/-) strain developed in this study. PMID:25765873

  13. Production of a mouse strain with impaired glucose tolerance by systemic heterozygous knockout of the glucokinase gene and its feasibility as a prediabetes model

    PubMed Central

    SAITO, Mikako; KANEDA, Asako; SUGIYAMA, Tae; IIDA, Ryousuke; OTOKUNI, Keiko; KABURAGI, Misako; MATSUOKA, Hideaki

    2015-01-01

    Exon II of glucokinase (Gk) was deleted to produce a systemic heterozygous Gk knockout (Gk+/−) mouse. The relative expression levels of Gk in the heart, lung, liver, stomach, and pancreas in Gk+/− mice ranged from 0.41–0.68 versus that in wild (Gk+/+) mice. On the other hand, its expression levels in the brain, adipose tissue, and muscle ranged from 0.95–1.03, and its expression levels in the spleen and kidney were nearly zero. Gk knockout caused no remarkable off-target effect on the expression of 7 diabetes causing genes (Shp, Hnf1a, Hnf1b, Irs1, Irs2, Kir6.2, and Pdx1) in 10 organs. The glucose tolerance test was conducted to determine the blood glucose concentrations just after fasting for 24 h (FBG) and at 2 h after high-glucose application (GTT2h). The FBG-GTT2h plots obtained with the wild strain fed the control diet (CD), Gk+/− strain fed the CD, and Gk+/− strain fed the HFD were distributed in separate areas in the FBG-GTT2h diagram. The respective areas could be defined as the normal state, prediabetes state, and diabetes state, respectively. Based on the results, the criteria for prediabetes could be defined for the Gk+/− strain developed in this study. PMID:25765873

  14. Methylphenidate restores novel object recognition in DARPP-32 knockout mice.

    PubMed

    Heyser, Charles J; McNaughton, Caitlyn H; Vishnevetsky, Donna; Fienberg, Allen A

    2013-09-15

    Previously, we have shown that Dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32) knockout mice required significantly more trials to reach criterion than wild-type mice in an operant reversal-learning task. The present study was conducted to examine adult male and female DARPP-32 knockout mice and wild-type controls in a novel object recognition test. Wild-type and knockout mice exhibited comparable behavior during the initial exploration trials. As expected, wild-type mice exhibited preferential exploration of the novel object during the substitution test, demonstrating recognition memory. In contrast, knockout mice did not show preferential exploration of the novel object, instead exhibiting an increase in exploration of all objects during the test trial. Given that the removal of DARPP-32 is an intracellular manipulation, it seemed possible to pharmacologically restore some cellular activity and behavior by stimulating dopamine receptors. Therefore, a second experiment was conducted examining the effect of methylphenidate. The results show that methylphenidate increased horizontal activity in both wild-type and knockout mice, though this increase was blunted in knockout mice. Pretreatment with methylphenidate significantly impaired novel object recognition in wild-type mice. In contrast, pretreatment with methylphenidate restored the behavior of DARPP-32 knockout mice to that observed in wild-type mice given saline. These results provide additional evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory. These results also indicate that the behavioral deficits in DARPP-32 knockout mice may be restored by the administration of methylphenidate.

  15. Mice heterozygous for the ATM gene are more sensitive to heavy ions exposure than are wildtypes

    NASA Astrophysics Data System (ADS)

    Worgul, B.; Smilenov, L.; Brenner, D.; Vazquez, M.; Hall, E.

    Previous studies have shown that the eyes of atm heterozygous mice exposed to Low LET radiation (X-rays) are more susceptible to the development of cataracts than are those of wildtype mice. The findings, as well as others, run counter to the assumption underpinning current radiation safety guidelines, that individuals are all equally sensitive to the biological effects of radiation. A question, highly relevant to human space activities is whether or not, in similar fashion there may exist a genetic predisposition to High LET radiation damage. Again the lens and, its primary radiopathy, cataract, were used to assay for the effects of ATM deficiency in a late-responding tissue. Together with those of wildtypes, the eyes of AT heterozygous knockout mice were exposed to 325 mGy of 1 GEV/amu 56Fe ions at the AGS facility of Brookhaven National Laboratory. The fluence was equivalent to 1 ion per nuclear area. As was the case in the earlier X-ray studies all irradiations were done on the 28th day after birth. Controls consisted of wildtype irradiated as well as unirradiated wildtype and heterozygotes. Ten mice from each group were examined weekly by conventional slitlamp biomicroscopy for a total of 35 weeks. The time required for prevalence to reach 50% (T50) as an endpoint for each stage indicated that not only cataract onset but also progression were accelerated in the mice haplo-deficient for the atm gene. For example the T50 for definitive cataract onset (stage 1) in the atm heterozygotes was 10 weeks whereas 17 weeks were required for the wildtypes. Similarly at the conclusion of the experiment (35 weeks), 40% of the lenses of allele-deficient mice had progressed to stage 3 (near fully opaque and obviously visually debilitating), while only one lens (5%) from the wildtype irradiated eyes achieved that stage. The data show that heterozygosity for the atm gene predisposes the eye to the cataractogenic influence of heavy ions and suggest that AT heterozygotes in the

  16. Increased hepatotoxicity of acetaminophen in Hsp70i knockout mice

    SciTech Connect

    Tolson, J. Keith; Dix, David J.; Voellmy, Richard W.; Roberts, Stephen M. . E-mail: smr@ufl.edu

    2006-01-15

    The effect of the inducible forms of 70 kDa heat shock protein (Hsp70i) on acetaminophen (APAP) hepatotoxicity was assessed in an Hsp70i knockout mouse model. Absence of the Hsp70i protein in liver was verified by monitoring Hsp levels in knockout and control mice after heat stress (41.5 {sup o}C water bath immersion for 30 min). Hsp70i knockout mice were more susceptible to APAP-induced hepatotoxicity than controls, as indicated by elevated serum alanine aminotransferase activities 24 and 48 h after the APAP dose. Increased APAP hepatotoxicity in knockout mice was verified by morphological evaluation of liver sections. The difference in toxic response to APAP between knockout and control strain mice could not be attributed to differences in APAP bioactivation, assessed by measurement of CYP2E1 and glutathione S-transferase activities, hepatic nonprotein sulfhydryl content, or covalent binding of reactive APAP metabolites to proteins. Pretreatment with transient hyperthermia to produce a general upregulation of Hsps resulted in decreased APAP hepatotoxicity in both the knockout and control strains. Among thermally-pretreated mice, hepatotoxicity of APAP was greater in the knockouts compared with the control strain. These observations suggest that increased Hsp70i expression in response to APAP acts to limit the extent of tissue injury. Results further suggest that other factors related to heat stress can also contribute to protection against APAP toxicity.

  17. Characteristics of aldehyde dehydrogenase 2 (Aldh2) knockout mice.

    PubMed

    Yu, Hsu-Sheng; Oyama, Tsunehiro; Isse, Toyohi; Kitakawa, Kyoko; Ogawa, Masanori; Pham, Thi-Thu-Phuong; Kawamoto, Toshihiro

    2009-11-01

    Acetaldehyde is an intermediate of ethanol oxidation. It covalently binds to DNA, and is known as a carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2), and Aldh2 knockout mice appear to be a valid animal model for humans with inactive ALDH2. This review gives an overview of published studies on Aldh2 knockout mice, which were treated with ethanol or acetaldehyde. According to these studies, it was found that Aldh2 -/- mice (Aldh2 knockout mice) are more susceptible to ethanol and acetaldehyde-induced toxicity than Aldh2 +/+ mice (wild type mice). When mice were fed with ethanol, the mortality was increased. When they were exposed to atmospheres containing acetaldehyde, the Aldh2 -/- mice showed more severe toxic symptoms, like weight loss and higher blood acetaldehyde levels, as compared with the Aldh2 +/+ mice. Thus, ethanol and acetaldehyde treatment affects Aldh2 knockout mice more than wild type mice. Based on these findings, it is suggested that ethanol consumption and acetaldehyde inhalation are inferred to pose a higher risk to ALDH2-inactive humans. These results also support that ALDH2-deficient humans who habitually consume alcohol have a higher rate of cancer than humans with functional ALDH2. PMID:19874182

  18. COMPARISON OF CITRUS COUMARINS ON CARCINOGEN-DETOXIFYING ENZYMES IN NRF2 KNOCKOUT MICE

    PubMed Central

    Prince, Misty; Li, Yan; Childers, Asper; Itoh, Ken; Yamamoto, Masayuki; Kleiner, Heather E.

    2009-01-01

    Naturally occurring coumarins possess anti-carcinogenic activities in part by inducing carcinogen-detoxifying enzymes glutathione S-transferase (GST) and/or NAD(P)H quinone oxidoreductase (NQO1). Our goal was to determine whether citrus coumarins induce hepatic GST and/or NQO1 via activation of Nrf2 and the antioxidant response element. First, HepG2 cells stably transfected with the ARE and a green fluorescent protein (GFP) reporter were treated with increasing concentrations of coumarins and compared to positive controls. tert-butylhydroquinone (TBHQ) and oltipraz increased GFP fluorescence, as did coumarin, limettin, auraptene, imperatorin, and 7,8-benzoflavone, suggesting that they activate the ARE, whereas isopimpinellin did not increase GFP fluorescence. Next, the effects of orally-administered coumarins and oltipraz on hepatic GST and NQO1 activities were compared in Nrf2 knockout mice or Nrf2 heterozygous mice exhibiting the wild-type phenotype. Oltipraz, auraptene, imperatorin, isopimpinellin, and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. This effect was abrogated in Nrf2(−/−) mice dosed with oltipraz, attenuated in mice Nrf2(−/−) mice treated with auraptene and imperatorin, and still significant in Nrf2(−/−) mice treated with isopimpinellin. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2(−/−) mice. These results strongly suggest that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism. Although structurally similar, isopimpinellin did not appear to activate HepG2-ARE-GFP and the Nrf2 knockout mouse study suggests that isopimpinellin may induce GST and NQO1 via additional mechanisms. PMID:19150646

  19. Unique nuclear vacuoles in the motor neurons of conditional ADAR2-knockout mice.

    PubMed

    Sasaki, Shoichi; Takenari Yamashita; Hideyama, Takuto; Kwak, Shin

    2014-03-01

    A reduction in adenosine deaminase acting on RNA 2 (ADAR2) activity causes the death of spinal motor neurons specifically via the GluA2 Q/R site-RNA editing failure in sporadic amyotrophic lateral sclerosis (ALS). We studied, over time, the spinal cords of ADAR2-knockout mice, which are the mechanistic model mice for sporadic ALS, using homozygous ADAR2(flox/flox)/VAChT-Cre.Fast (AR2), homozygous ADAR2(flox/flox)/VAChT-Cre.Slow (AR2Slow), and heterozygous ADAR2(flox/+)/VAChT-Cre.Fast (AR2H) mice. The conditional ADAR2-knockout mice were divided into 3 groups by stage: presymptomatic (AR2H mice), early symptomatic (AR2 mice, AR2H mice) and late symptomatic (AR2Slow mice). Light-microscopically, some motor neurons in AR2 and AR2H mice (presymptomatic) showed simple neuronal atrophy and astrogliosis, and AR2H (early symptomatic) and AR2Slow mice often showed vacuoles predominantly in motor neurons. The number of vacuole-bearing anterior horn neurons decreased with the loss of anterior horn neurons in AR2H mice after 40 weeks of age. Electron-microscopically, in AR2 mice, while the cytoplasm of normal-looking motor neurons was almost always normal-appearing, the interior of dendrites was frequently loose and disorganized. In AR2H and AR2Slow mice, large vacuoles without a limiting membrane were observed in the anterior horns, preferentially in the nuclei of motor neurons, astrocytes and oligodendrocytes. Nuclear vacuoles were not observed in AR2res (ADAR2(flox/flox)/VAChT-Cre.Fast/GluR-B(R/R)) mice, in which motor neurons express edited GluA2 in the absence of ADAR2. These findings suggest that ADAR2-reduction is associated with progressive deterioration of nuclear architecture, resulting in vacuolated nuclei due to a Ca(2+)-permeable AMPA receptor-mediated mechanism.

  20. Comprehensive behavioral analysis of RNG105 (Caprin1) heterozygous mice: Reduced social interaction and attenuated response to novelty

    PubMed Central

    Ohashi, Rie; Takao, Keizo; Miyakawa, Tsuyoshi; Shiina, Nobuyuki

    2016-01-01

    RNG105 (also known as Caprin1) is a major RNA-binding protein in neuronal RNA granules, and is responsible for mRNA transport to dendrites and neuronal network formation. A recent study reported that a heterozygous mutation in the Rng105 gene was found in an autism spectrum disorder (ASD) patient, but it remains unclear whether there is a causal relation between RNG105 deficiency and ASD. Here, we subjected Rng105+/− mice to a comprehensive behavioral test battery, and revealed the influence of RNG105 deficiency on mouse behavior. Rng105+/− mice exhibited a reduced sociality in a home cage and a weak preference for social novelty. Consistently, the Rng105+/− mice also showed a weak preference for novel objects and novel place patterns. Furthermore, although the Rng105+/− mice exhibited normal memory acquisition, they tended to have relative difficulty in reversal learning in the spatial reference tasks. These findings suggest that the RNG105 heterozygous knockout leads to a reduction in sociality, response to novelty and flexibility in learning, which are implicated in ASD-like behavior. PMID:26865403

  1. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

  2. Impaired learning and motor behavior in heterozygous Pafah1b1 (Lis1) mutant mice.

    PubMed

    Paylor, R; Hirotsune, S; Gambello, M J; Yuva-Paylor, L; Crawley, J N; Wynshaw-Boris, A

    1999-01-01

    Heterozygous mutation or deletion of Pafab1b1 (LIS1) in humans is associated with syndromes with type 1 lissencephaly, a severe brain developmental disorder resulting from abnormal neuronal migration. We have created Lis1 heterozygous mutant mice by gene targeting. Heterozygous mutant mice are viable and fertile, but display global organizational brain defects as a result of impaired neuronal migration. To assess the functional impact of the mutation, Lis1 heterozygous mice and their wild-type littermates were evaluated on a wide variety of behavioral tests. Lis1 mutant mice displayed abnormal hindpaw clutching responses and were impaired on a rotarod test. Lis1 heterozygous mice were also impaired in the spatial learning version of the Morris water task. Impaired motor behavior and spatial learning and memory in Lis1 mutant mice indicates that impaired neuronal migration can have functional effects on complex behavioral responses. The behavioral findings also support the use of the Lis1 mutant mice as a model from human type 1 lissencephaly.

  3. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    NASA Technical Reports Server (NTRS)

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

  4. Knockout Mice Challenge our Concepts of Glucose Homeostasis and the Pathogenesis of Diabetes

    PubMed Central

    2003-01-01

    A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes. PMID:15061645

  5. Physical, Heritable and Age-Related Factors as Modifiers of Radiation Cancer Risk in Patched Heterozygous Mice

    SciTech Connect

    Pazzaglia, Simonetta Pasquali, Emanuela M.Sc.; Tanori, Mirella; Mancuso, Mariateresa; Leonardi, Simona; Di Majo, Vincenzo; Rebessi, Simonetta; Saran, Anna

    2009-03-15

    Purpose: To address the tumorigenic potential of exposure to low/intermediate doses of ionizing radiation and to identify biological factors influencing tumor response in a mouse model highly susceptible to radiogenic cancer. Methods and Materials: Newborn Ptc1 heterozygous mice were exposed to X-ray doses of 100, 250, and 500 mGy, and tumor development was monitored for their lifetime. Additional groups were irradiated with the same doses and sacrificed at fixed times for determination of short-term endpoints, such as apoptosis and early preneoplastic lesions in cerebellum. Finally, groups of Ptc1 heterozygous mice were bred on the C57BL/6 background to study the influence of common variant genes on radiation response. Results: We have identified a significant effect of low-intermediate doses of radiation (250 and 500 mGy) in shortening mean survival and inducing early and more progressed stages of tumor development in the cerebellum of Ptc1{sup +/-} mice. In addition, we show that age at exposure and heritable factors are potent modifiers of radiation-related cancer risk. Conclusions: The Ptc1 knockout mouse model offers a highly sensitive system that may potentially help to improve understanding and quantification of risk at low doses, such as doses experienced in occupational and medical exposures, and clarify the complex interactions between genetic and environmental factors underlying cancer susceptibility.

  6. Heterozygous L1-deficient mice express an autism-like phenotype.

    PubMed

    Sauce, Bruno; Wass, Christopher; Netrakanti, Meera; Saylor, Joshua; Schachner, Melitta; Matzel, Louis D

    2015-10-01

    The L1CAM (L1) gene encodes a cell adhesion molecule that contributes to several important processes in the developing and adult nervous system, including neuronal migration, survival, and plasticity. In humans and mice, mutations in the X chromosome-linked gene L1 cause severe neurological defects in males. L1 heterozygous female mice with one functional copy of the L1 gene show complex morphological features that are different from L1 fully-deficient and wild-type littermate mice. However, almost no information is available on the behavior of L1 heterozygous mice and humans. Here, we investigated the behavior of heterozygous female mice in which the L1 gene is constitutively inactivated. These mice were compared to wild-type littermate females. Animals were assessed in five categories of behavioral tests: five tests for anxiety/stress/exploration, four tests for motor abilities, two tests for spatial learning, three tests for social behavior, and three tests for repetitive behavior. We found that L1 heterozygous mice express an autism-like phenotype, comprised of reduced social behaviors and excessive self-grooming (a repetitive behavior also typical in animal models of autism). L1 heterozygous mice also exhibited an increase in sensitivity to light, assessed by a reluctance to enter the lighted areas of novel environments. However, levels of anxiety, stress, motor abilities, and spatial learning in L1 heterozygous mice were similar to those of wild-type mice. These observations raise the possibility that using molecules known to trigger L1 functions may become valuable in the treatment of autism in humans. PMID:26079769

  7. Heterozygous L1-deficient mice express an autism-like phenotype.

    PubMed

    Sauce, Bruno; Wass, Christopher; Netrakanti, Meera; Saylor, Joshua; Schachner, Melitta; Matzel, Louis D

    2015-10-01

    The L1CAM (L1) gene encodes a cell adhesion molecule that contributes to several important processes in the developing and adult nervous system, including neuronal migration, survival, and plasticity. In humans and mice, mutations in the X chromosome-linked gene L1 cause severe neurological defects in males. L1 heterozygous female mice with one functional copy of the L1 gene show complex morphological features that are different from L1 fully-deficient and wild-type littermate mice. However, almost no information is available on the behavior of L1 heterozygous mice and humans. Here, we investigated the behavior of heterozygous female mice in which the L1 gene is constitutively inactivated. These mice were compared to wild-type littermate females. Animals were assessed in five categories of behavioral tests: five tests for anxiety/stress/exploration, four tests for motor abilities, two tests for spatial learning, three tests for social behavior, and three tests for repetitive behavior. We found that L1 heterozygous mice express an autism-like phenotype, comprised of reduced social behaviors and excessive self-grooming (a repetitive behavior also typical in animal models of autism). L1 heterozygous mice also exhibited an increase in sensitivity to light, assessed by a reluctance to enter the lighted areas of novel environments. However, levels of anxiety, stress, motor abilities, and spatial learning in L1 heterozygous mice were similar to those of wild-type mice. These observations raise the possibility that using molecules known to trigger L1 functions may become valuable in the treatment of autism in humans.

  8. Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice.

    PubMed

    Dai, Ying; Kiselak, Tomas; Clark, Joanne; Clore, Elizabeth; Zheng, Kangni; Cheng, Allen; Kujoth, Gregory C; Prolla, Tomas A; Maratos-Flier, Eleftheria; Simon, David K

    2013-07-01

    The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.

  9. Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice

    PubMed Central

    Dai, Ying; Kiselak, Tomas; Clark, Joanne; Clore, Elizabeth; Zheng, Kangni; Cheng, Allen; Kujoth, Gregory C.; Prolla, Tomas A.; Maratos-Flier, Eleftheria; Simon, David K.

    2013-01-01

    The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits. PMID:23542163

  10. Rapamycin extends lifespan and delays tumorigenesis in heterozygous p53+/- mice.

    PubMed

    Komarova, Elena A; Antoch, Marina P; Novototskaya, Liliya R; Chernova, Olga B; Paszkiewicz, Geraldine; Leontieva, Olga V; Blagosklonny, Mikhail V; Gudkov, Andrei V

    2012-10-01

    TOR (Target of Rapamycin) pathway accelerates cellular and organismal aging. Similar to rapamycin, p53 can inhibit the mTOR pathway in some mammalian cells. Mice lacking one copy of p53 (p53+/- mice) have an increased cancer incidence and a shorter lifespan. We hypothesize that rapamycin can delay cancer in heterozygous p53+/- mice. Here we show that rapamycin (given in a drinking water) extended the mean lifespan of p53+/- mice by 10% and when treatment started early in life (at the age less than 5 months) by 28%. In addition, rapamycin decreased the incidence of spontaneous tumors. This observation may have applications in management of Li-Fraumeni syndrome patients characterized by heterozygous mutations in the p53 gene.

  11. Induction of functional Brm protein from Brm knockout mice

    PubMed Central

    Thompson, Kenneth W.; Marquez, Stefanie B.; Lu, Li; Reisman, David

    2015-01-01

    Once the knockout of the Brm gene was found to be nontumorigenic in mice, the study of BRM's involvement in cancer seemed less important compared with that of its homolog, Brg1. This has likely contributed to the disparity that has been observed in the publication ratio between BRG1 and BRM. We show that a previously published Brm knockout mouse is an incomplete knockout whereby a truncated isoform of Brm is detected in normal tissue and in tumors. We show that this truncated Brm isoform has functionality comparable to wild type Brm. By immunohistochemistry (IHC), this truncated Brm is undetectable in normal lung tissue and is minimal to very low in Brmnull tumors. However, it is significant in a subset (~40%) of Brg1/Brm double knockout (DKO) tumors that robustly express this truncated BRM, which in part stems from an increase in Brm mRNA levels. Thus, it is likely that this mutant mouse model does not accurately reflect the role that Brm plays in cancer development. We suggest that the construction of a completely new mouse Brm knockout, where Brm is functionally absent, is needed to determine whether or not Brm is actually tumorigenic and if Brm might be a tumor suppressor. PMID:26097869

  12. Behavioral characterization of P311 knockout mice

    PubMed Central

    Taylor, Gregory A.; Rodriguiz, Ramona M.; Greene, Robert I.; Daniell, Xiaoju; Henry, Stanley C.; Crooks, Kristy R.; Kotloski, Robert; Tessarollo, Lino; Phillips, Lindsey E.; Wetsel, William C.

    2013-01-01

    P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses. PMID:18616608

  13. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    PubMed Central

    2014-01-01

    Glycosaminoglycans (GAGs) are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs. PMID:25126564

  14. Evidence for increased tissue androgen sensitivity in neurturin knockout mice.

    PubMed

    Simanainen, Ulla; Gao, Yan Ru Ellen; Desai, Reena; Jimenez, Mark; Spaliviero, Jennifer; Keast, Janet R; Handelsman, David J

    2013-01-01

    Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family and signals through GDNF family receptor alpha 2 (GFRα2). We hypothesised that epithelial atrophy reported in the reproductive organs of Ntn (Nrtn)- and Gfrα2 (Gfra2)-deficient mice could be due to NTN affecting the hormonal environment. To investigate this, we compared the reproductive organs of Ntn- and Gfrα2-deficient male mice in parallel with an analysis of their circulating reproductive hormone levels. There were no significant structural changes within the organs of the knockout mice; however, serum and intratesticular testosterone and serum LH levels were very low. To reconcile these observations, we tested androgen sensitivity by creating a dihydrotestosterone (DHT) clamp (castration plus DHT implant) to create fixed circulating levels of androgens, allowing the evaluation of androgen-sensitive endpoints. At the same serum DHT levels, serum LH levels were lower and prostate and seminal vesicle weights were higher in the Ntn knockout (NTNKO) mice than in the wild-type mice, suggesting an increased response to androgens in the accessory glands and hypothalamus and pituitary of the NTNKO mice. Testicular and pituitary responsiveness was unaffected in the NTNKO males, as determined by the response to the human chorionic gonadotrophin or GNRH analogue, leuprolide, respectively. In conclusion, our results suggest that NTN inactivation enhances androgen sensitivity in reproductive and neuroendocrine tissues, revealing a novel mechanism to influence reproductive function and the activity of other androgen-dependent tissues.

  15. Decreased lung compliance and air trapping in heterozygous SP-B-deficient mice.

    PubMed

    Clark, J C; Weaver, T E; Iwamoto, H S; Ikegami, M; Jobe, A H; Hull, W M; Whitsett, J A

    1997-01-01

    Genetic ablation of the murine SP-B gene in transgenic mice caused lethal perinatal respiratory distress in homozygous offspring, whereas heterozygous SP-B (+/-) mice survived postnatally. In adult SP-B(+/-) mice, surfactant protein B mRNA and the alveolar lavage SP-B protein were reduced by 50% compared with wild-type littermates, consistent with the inactivation of a single SP-B allele. Expression of SP-A, SP-C, and SP-D proteins was not affected in SP-B(+/-) mice. Heterozygous SP-B(+/-) mice reached maturity in numbers expected by Mendelian inheritance of a recessive gene. Lung morphology and both intracellular and extracellular phospholipid pool size and composition were unaltered in the SP-B(+/-) mice. Despite normal survival, pulmonary function studies demonstrated a consistent decrease in lung compliance in SP-B(+/-) mice. Abnormalities of inflation/deflation curves demonstrated airway collapse at low deflation pressures. Residual volumes were increased in the SP-B(+/-) mice. In summary, SP-B mRNA and SP-B protein were reduced by 50% in SP-B(+/-) mice, resulting in abnormalities of lung compliance and air trapping, suggesting a potential susceptibility to pulmonary dysfunction associated with SP-B deficiency.

  16. Altered social cognition in male BDNF heterozygous mice and following chronic methamphetamine exposure.

    PubMed

    Manning, Elizabeth E; van den Buuse, Maarten

    2016-05-15

    Growing clinical evidence suggests that persistent psychosis which occurs in methamphetamine users is closely related to schizophrenia. However, preclinical studies in animal models have focussed on psychosis-related behaviours following methamphetamine, and less work has been done to assess endophenotypes relevant to other deficits observed in schizophrenia. Altered social behaviour is a feature of both the negative symptoms and cognitive deficits in schizophrenia, and significantly impacts patient functioning. We recently found that brain-derived neurotrophic factor (BDNF) heterozygous mice show disrupted sensitization to methamphetamine, supporting other work suggesting an important role of this neurotrophin in the pathophysiology of psychosis and the neuronal response to stimulant drugs. In the current study, we assessed social and cognitive behaviours in methamphetamine-treated BDNF heterozygous mice and wildtype littermate controls. Following chronic methamphetamine exposure male wildtype mice showed a 50% reduction in social novelty preference. Vehicle-treated male BDNF heterozygous mice showed a similar impairment in social novelty preference, with a trend for no further disruption by methamphetamine exposure. Female mice were unaffected in this task, and no groups showed any changes in sociability or short-term spatial memory. These findings suggest that chronic methamphetamine alters behaviour relevant to disruption of social cognition in schizophrenia, supporting other studies which demonstrate a close resemblance between persistent methamphetamine psychosis and schizophrenia. Together these findings suggest that dynamic regulation of BDNF signalling is necessary to mediate the effects of methamphetamine on behaviours relevant to schizophrenia.

  17. Altered social cognition in male BDNF heterozygous mice and following chronic methamphetamine exposure.

    PubMed

    Manning, Elizabeth E; van den Buuse, Maarten

    2016-05-15

    Growing clinical evidence suggests that persistent psychosis which occurs in methamphetamine users is closely related to schizophrenia. However, preclinical studies in animal models have focussed on psychosis-related behaviours following methamphetamine, and less work has been done to assess endophenotypes relevant to other deficits observed in schizophrenia. Altered social behaviour is a feature of both the negative symptoms and cognitive deficits in schizophrenia, and significantly impacts patient functioning. We recently found that brain-derived neurotrophic factor (BDNF) heterozygous mice show disrupted sensitization to methamphetamine, supporting other work suggesting an important role of this neurotrophin in the pathophysiology of psychosis and the neuronal response to stimulant drugs. In the current study, we assessed social and cognitive behaviours in methamphetamine-treated BDNF heterozygous mice and wildtype littermate controls. Following chronic methamphetamine exposure male wildtype mice showed a 50% reduction in social novelty preference. Vehicle-treated male BDNF heterozygous mice showed a similar impairment in social novelty preference, with a trend for no further disruption by methamphetamine exposure. Female mice were unaffected in this task, and no groups showed any changes in sociability or short-term spatial memory. These findings suggest that chronic methamphetamine alters behaviour relevant to disruption of social cognition in schizophrenia, supporting other studies which demonstrate a close resemblance between persistent methamphetamine psychosis and schizophrenia. Together these findings suggest that dynamic regulation of BDNF signalling is necessary to mediate the effects of methamphetamine on behaviours relevant to schizophrenia. PMID:26965573

  18. Behavioral Analysis of Ste20 Kinase SPAK Knockout Mice

    PubMed Central

    Geng, Yang; Byun, Nellie; Delpire, Eric

    2009-01-01

    SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase [16]. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission. PMID:20006650

  19. Locomotory patterns, spatiotemporal organization of exploration and spatial memory in serotonin transporter knockout mice.

    PubMed

    Kalueff, Allan V; Jensen, Catherine L; Murphy, Dennis L

    2007-09-12

    Serotonin transporter knockout (SERT-/-) mice are extensively used as a genetic model of several neuropsychiatric disorders, and consistently display anxiety-like behaviors and inactivity in different tests. To better understand how these mice organize their behavior, we assessed the open field and elevated plus maze spatiotemporal patterning of activity in adult male SERT wild type (+/+), heterozygous (+/-) and -/- mice on C57BL/6J genetic background using new videotracking and analytic procedures. In addition, we analyzed their spatial memory, assessing within- and between-trial habituation, and examined specific motor characteristics of their movement in these two tests. In the open field test, SERT-/- mice showed reduced vertical exploration throughout the arena, reduced central (but not peripheral) horizontal exploration, unaltered within-trial habituation, and slightly poorer between-trial habituation for horizontal activity. In the elevated plus maze, SERT-/- mice demonstrated anxiety-like avoidance of open arms, hypoactivity, as well as unaltered within-trial and between-trial habituation (except for poorer between-trial habituation of total horizontal activity). In both tests, SERT-/- mice showed greater prevalence of horizontal over vertical dimension of their exploration in the areas protected by the walls (open field periphery, plus maze closed arms), but not in open aversive areas, such as the center of the open field or center or open arms of the maze. In both arenas, SERT-/- mice consistently displayed increased turning behavior, potentially representing a perseverance-like phenotype or aberrant spatial strategies in novel environments. Overall, using a fine-graded behavioral analysis in two different novelty tests, this study revealed alterations in motor and spatiotemporal patterning of activity in SERT-/- mice. Given the relevance of exploratory strategies to human personality traits and brain disorders, our data may be useful for developing

  20. Mice with a heterozygous Lrp6 deletion have impaired fracture healing

    PubMed Central

    Burgers, Travis A; Vivanco, Juan F; Zahatnansky, Juraj; Moren, Andrew J Vander; Mason, James J; Williams, Bart O

    2016-01-01

    Bone fracture non-unions, the failure of a fracture to heal, occur in 10%–20% of fractures and are a costly and debilitating clinical problem. The Wnt/β-catenin pathway is critical in bone development and fracture healing. Polymorphisms of linking low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt-binding receptor, have been associated with decreased bone mineral density and fragility fractures, although this remains controversial. Mice with a homozygous deletion of Lrp6 have severe skeletal abnormalities and are not viable, whereas mice with a heterozygous deletion have a combinatory effect with Lrp5 to decrease bone mineral density. As fracture healing closely models embryonic skeletal development, we investigated the process of fracture healing in mice heterozygous for Lrp6 (Lrp6 +/−) and hypothesized that the heterozygous deletion of Lrp6 would impair fracture healing. Mid-diaphyseal femur fractures were induced in Lrp6 +/− mice and wild-type controls (Lrp6 +/+). Fractures were analyzed using micro-computed tomography (μCT) scans, biomechanical testing, and histological analysis. Lrp6 +/− mice had significantly decreased stiffness and strength at 28 days post fracture (PF) and significantly decreased BV/TV, total density, immature bone density, and mature area within the callus on day-14 and -21 PF; they had significantly increased empty callus area at days 14 and 21 PF. Our results demonstrate that the heterozygous deletion of Lrp6 impairs fracture healing, which suggests that Lrp6 has a role in fracture healing. PMID:27635281

  1. Mice with a heterozygous Lrp6 deletion have impaired fracture healing

    PubMed Central

    Burgers, Travis A; Vivanco, Juan F; Zahatnansky, Juraj; Moren, Andrew J Vander; Mason, James J; Williams, Bart O

    2016-01-01

    Bone fracture non-unions, the failure of a fracture to heal, occur in 10%–20% of fractures and are a costly and debilitating clinical problem. The Wnt/β-catenin pathway is critical in bone development and fracture healing. Polymorphisms of linking low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt-binding receptor, have been associated with decreased bone mineral density and fragility fractures, although this remains controversial. Mice with a homozygous deletion of Lrp6 have severe skeletal abnormalities and are not viable, whereas mice with a heterozygous deletion have a combinatory effect with Lrp5 to decrease bone mineral density. As fracture healing closely models embryonic skeletal development, we investigated the process of fracture healing in mice heterozygous for Lrp6 (Lrp6 +/−) and hypothesized that the heterozygous deletion of Lrp6 would impair fracture healing. Mid-diaphyseal femur fractures were induced in Lrp6 +/− mice and wild-type controls (Lrp6 +/+). Fractures were analyzed using micro-computed tomography (μCT) scans, biomechanical testing, and histological analysis. Lrp6 +/− mice had significantly decreased stiffness and strength at 28 days post fracture (PF) and significantly decreased BV/TV, total density, immature bone density, and mature area within the callus on day-14 and -21 PF; they had significantly increased empty callus area at days 14 and 21 PF. Our results demonstrate that the heterozygous deletion of Lrp6 impairs fracture healing, which suggests that Lrp6 has a role in fracture healing.

  2. Mice with a heterozygous Lrp6 deletion have impaired fracture healing.

    PubMed

    Burgers, Travis A; Vivanco, Juan F; Zahatnansky, Juraj; Moren, Andrew J Vander; Mason, James J; Williams, Bart O

    2016-01-01

    Bone fracture non-unions, the failure of a fracture to heal, occur in 10%-20% of fractures and are a costly and debilitating clinical problem. The Wnt/β-catenin pathway is critical in bone development and fracture healing. Polymorphisms of linking low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt-binding receptor, have been associated with decreased bone mineral density and fragility fractures, although this remains controversial. Mice with a homozygous deletion of Lrp6 have severe skeletal abnormalities and are not viable, whereas mice with a heterozygous deletion have a combinatory effect with Lrp5 to decrease bone mineral density. As fracture healing closely models embryonic skeletal development, we investigated the process of fracture healing in mice heterozygous for Lrp6 (Lrp6 (+/-)) and hypothesized that the heterozygous deletion of Lrp6 would impair fracture healing. Mid-diaphyseal femur fractures were induced in Lrp6 (+/-) mice and wild-type controls (Lrp6 (+/+)). Fractures were analyzed using micro-computed tomography (μCT) scans, biomechanical testing, and histological analysis. Lrp6 (+/-) mice had significantly decreased stiffness and strength at 28 days post fracture (PF) and significantly decreased BV/TV, total density, immature bone density, and mature area within the callus on day-14 and -21 PF; they had significantly increased empty callus area at days 14 and 21 PF. Our results demonstrate that the heterozygous deletion of Lrp6 impairs fracture healing, which suggests that Lrp6 has a role in fracture healing. PMID:27635281

  3. Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

  4. Bone Growth and Turnover in Progesterone Receptor Knockout Mice

    PubMed Central

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jamie C.; Waters, Katrina M.; Lydon, John P.; O’Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-01-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and microcomputed tomography analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 wk of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain, and tibia longitudinal bone growth were normal in PRKO mice. In contrast, total, cancellous, and cortical bone mass were increased in the humerus of 12-wk-old PRKO mice, whereas cortical and cancellous bone mass in the tibia was normal. At 26 wk of age, cancellous bone area in the proximal tibia metaphysis of PRKO mice was 153% greater than age matched wild-type mice. The improved cancellous bone balance in 6-month-old PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice is not essential for bone growth and turnover. However, at some skeletal sites, PR signaling attenuates the accumulation of cortical and cancellous bone mass during adolescence. PMID:18276762

  5. Bone growth and turnover in progesterone receptor knockout mice.

    SciTech Connect

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O'Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  6. Knock-out of nexilin in mice leads to dilated cardiomyopathy and endomyocardial fibroelastosis.

    PubMed

    Aherrahrou, Zouhair; Schlossarek, Saskia; Stoelting, Stephanie; Klinger, Matthias; Geertz, Birgit; Weinberger, Florian; Kessler, Thorsten; Aherrahrou, Redouane; Moreth, Kristin; Bekeredjian, Raffi; Hrabě de Angelis, Martin; Just, Steffen; Rottbauer, Wolfgang; Eschenhagen, Thomas; Schunkert, Heribert; Carrier, Lucie; Erdmann, Jeanette

    2016-01-01

    Cardiomyopathy is one of the most common causes of chronic heart failure worldwide. Mutations in the gene encoding nexilin (NEXN) occur in patients with both hypertrophic and dilated cardiomyopathy (DCM); however, little is known about the pathophysiological mechanisms and relevance of NEXN to these disorders. Here, we evaluated the functional role of NEXN using a constitutive Nexn knock-out (KO) mouse model. Heterozygous (Het) mice were inter-crossed to produce wild-type (WT), Het, and homozygous KO mice. At birth, 32, 46, and 22 % of the mice were WT, Het, and KO, respectively, which is close to the expected Mendelian ratio. After postnatal day 6, the survival of the Nexn KO mice decreased dramatically and all of the animals died by day 8. Phenotypic characterizations of the WT and KO mice were performed at postnatal days 1, 2, 4, and 6. At birth, the relative heart weights of the WT and KO mice were similar; however, at day 4, the relative heart weight of the KO group was 2.3-fold higher than of the WT group. In addition, the KO mice developed rapidly progressive cardiomyopathy with left ventricular dilation and wall thinning and decreased cardiac function. At day 6, the KO mice developed a fulminant DCM phenotype characterized by dilated ventricular chambers and systolic dysfunction. At this stage, collagen deposits and some elastin deposits were observed within the left ventricle cavity, which resembles the features of endomyocardial fibroelastosis (EFE). Overall, these results further emphasize the role of NEXN in DCM and suggest a novel role in EFE.

  7. Heterozygous Che-1 KO mice show deficiencies in object recognition memory persistence.

    PubMed

    Zalcman, Gisela; Corbi, Nicoletta; Di Certo, Maria Grazia; Mattei, Elisabetta; Federman, Noel; Romano, Arturo

    2016-10-01

    Transcriptional regulation is a key process in the formation of long-term memories. Che-1 is a protein involved in the regulation of gene transcription that has recently been proved to bind the transcription factor NF-κB, which is known to be involved in many memory-related molecular events. This evidence prompted us to investigate the putative role of Che-1 in memory processes. For this study we newly generated a line of Che-1(+/-) heterozygous mice. Che-1 homozygous KO mouse is lethal during development, but Che-1(+/-) heterozygous mouse is normal in its general anatomical and physiological characteristics. We analyzed the behavioral characteristic and memory performance of Che-1(+/-) mice in two NF-κB dependent types of memory. We found that Che-1(+/-) mice show similar locomotor activity and thigmotactic behavior than wild type (WT) mice in an open field. In a similar way, no differences were found in anxiety-like behavior between Che-1(+/-) and WT mice in an elevated plus maze as well as in fear response in a contextual fear conditioning (CFC) and object exploration in a novel object recognition (NOR) task. No differences were found between WT and Che-1(+/-) mice performance in CFC training and when tested at 24h or 7days after training. Similar performance was found between groups in NOR task, both in training and 24h testing performance. However, we found that object recognition memory persistence at 7days was impaired in Che-1(+/-) heterozygous mice. This is the first evidence showing that Che-1 is involved in memory processes. PMID:27589891

  8. Heterozygous Che-1 KO mice show deficiencies in object recognition memory persistence.

    PubMed

    Zalcman, Gisela; Corbi, Nicoletta; Di Certo, Maria Grazia; Mattei, Elisabetta; Federman, Noel; Romano, Arturo

    2016-10-01

    Transcriptional regulation is a key process in the formation of long-term memories. Che-1 is a protein involved in the regulation of gene transcription that has recently been proved to bind the transcription factor NF-κB, which is known to be involved in many memory-related molecular events. This evidence prompted us to investigate the putative role of Che-1 in memory processes. For this study we newly generated a line of Che-1(+/-) heterozygous mice. Che-1 homozygous KO mouse is lethal during development, but Che-1(+/-) heterozygous mouse is normal in its general anatomical and physiological characteristics. We analyzed the behavioral characteristic and memory performance of Che-1(+/-) mice in two NF-κB dependent types of memory. We found that Che-1(+/-) mice show similar locomotor activity and thigmotactic behavior than wild type (WT) mice in an open field. In a similar way, no differences were found in anxiety-like behavior between Che-1(+/-) and WT mice in an elevated plus maze as well as in fear response in a contextual fear conditioning (CFC) and object exploration in a novel object recognition (NOR) task. No differences were found between WT and Che-1(+/-) mice performance in CFC training and when tested at 24h or 7days after training. Similar performance was found between groups in NOR task, both in training and 24h testing performance. However, we found that object recognition memory persistence at 7days was impaired in Che-1(+/-) heterozygous mice. This is the first evidence showing that Che-1 is involved in memory processes.

  9. Neuregulin 3 Knockout Mice Exhibit Behaviors Consistent with Psychotic Disorders.

    PubMed

    Hayes, Lindsay N; Shevelkin, Alexey; Zeledon, Mariela; Steel, Gary; Chen, Pei-Lung; Obie, Cassandra; Pulver, Ann; Avramopoulos, Dimitrios; Valle, David; Sawa, Akira; Pletnikov, Mikhail V

    2016-07-01

    Neuregulin 3 (NRG3) is a paralog of NRG1. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, and several intronic single nucleotide polymorphisms in NRG3 are associated with delusions in patients with schizophrenia. In order to gain insights into the biological function of the gene, we generated a novel Nrg3 knockout (KO) mouse model and tested for neurobehavioral phenotypes relevant to psychotic disorders. KO mice displayed novelty-induced hyperactivity, impaired prepulse inhibition of the acoustic startle response, and deficient fear conditioning. No gross cytoarchitectonic or layer abnormalities were noted in the brain of KO mice. Our findings suggest that deletion of the Nrg3 gene leads to alterations consistent with aspects of schizophrenia. We propose that KO mice will provide a valuable animal model to determine the role of the NRG3 in the molecular pathogenesis of schizophrenia and other psychotic disorders. PMID:27606322

  10. Phenotypic Characterization of Mice Heterozygous for a Null Mutation of Glutamate Carboxypeptidase II

    PubMed Central

    Han, Liqun; Picker, Jonathan D.; Schaevitz, Laura R.; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C.; Basu, Alo C.; Berger-Sweeney, Joanne; Coyle, Joseph T.

    2009-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of NMDA receptors has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-alpha L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate (NAA). NAAG is a neuropeptide that is an NMDA receptor antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDA receptor activation. To manipulate the expression of GCP II, loxP sites were inserted flanking exon 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N >200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory. PMID:19347959

  11. Screening methods to identify TALEN-mediated knockout mice.

    PubMed

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms.

  12. Salty taste deficits in CALHM1 knockout mice.

    PubMed

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

  13. Salty taste deficits in CALHM1 knockout mice.

    PubMed

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt. PMID:24846212

  14. Normal Taste Acceptance and Preference of PANX1 Knockout Mice.

    PubMed

    Tordoff, Michael G; Aleman, Tiffany R; Ellis, Hillary T; Ohmoto, Makoto; Matsumoto, Ichiro; Shestopalov, Val I; Mitchell, Claire H; Foskett, J Kevin; Poole, Rachel L

    2015-09-01

    Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1.

  15. Induction of mammary gland development in estrogen receptor-alpha knockout mice.

    PubMed

    Bocchinfuso, W P; Lindzey, J K; Hewitt, S C; Clark, J A; Myers, P H; Cooper, R; Korach, K S

    2000-08-01

    Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse. Implantation of an age-matched, heterozygous ERalpha pituitary isograft under the renal capsule of 25-day-old or 12-week-old alphaERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Grafted alphaERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the alphaERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in alphaERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce alphaERKO mammary growth. Estradiol and progesterone are required for the structural development of the alphaERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the alphaERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.

  16. Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice

    PubMed Central

    Reichmann, Florian; Wegerer, Vanessa; Jain, Piyush; Mayerhofer, Raphaela; Hassan, Ahmed M.; Fröhlich, Esther E.; Bock, Elisabeth; Pritz, Elisabeth; Herzog, Herbert; Holzer, Peter; Leitinger, Gerd

    2016-01-01

    Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for “enviromimetics”, therapeutics which reproduce the beneficial effects of enhanced environmental stimulation. PMID:27305846

  17. The evolution of thymic lymphomas in p53 knockout mice

    PubMed Central

    Dudgeon, Crissy; Chan, Chang; Kang, Wenfeng; Sun, Yvonne; Emerson, Ryan; Robins, Harlan

    2014-01-01

    Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors’ driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas. PMID:25452272

  18. Feeding-elicited cataplexy in orexin knockout mice

    PubMed Central

    Clark, Erika L.; Baumann, Christian R.; Cano, Georgina; Scammell, Thomas E.; Mochizuki, Takatoshi

    2009-01-01

    Mice lacking orexin/hypocretin signaling have sudden episodes of atonia and paralysis during active wakefulness. These events strongly resemble cataplexy, episodes of sudden muscle weakness triggered by strong positive emotions in people with narcolepsy, but it remains unknown whether murine cataplexy is triggered by positive emotions. To determine whether positive emotions elicit murine cataplexy, we placed orexin knockout (KO) mice on a scheduled feeding protocol with regular or highly palatable food. Baseline sleep/wake behavior was recorded with ad lib regular chow. Mice were then placed on a scheduled feeding protocol in which they received 60% of their normal amount of chow 3 hr after dark onset for the next 10 days. Wild-type and KO mice rapidly entrained to scheduled feeding with regular chow, with more wake and locomotor activity prior to the feeding time. On day 10 of scheduled feeding, orexin KO mice had slightly more cataplexy during the food-anticipation period and more cataplexy in the second half of the dark period, when they may have been foraging for residual food. To test whether more palatable food increases cataplexy, mice were then switched to scheduled feeding with an isocaloric amount of Froot Loops, a food often used as a reward in behavioral studies. With this highly palatable food, orexin KO mice had much more cataplexy during the food-anticipation period and throughout the dark period. The increase in cataplexy with scheduled feeding, especially with highly palatable food, suggests that positive emotions may trigger cataplexy in mice, just as in people with narcolepsy. Establishing this connection helps validate orexin KO mice as an excellent model of human narcolepsy and provides an opportunity to better understand the mechanisms that trigger cataplexy. PMID:19362119

  19. Spontaneous sleep and homeostatic sleep regulation in ghrelin knockout mice.

    PubMed

    Szentirmai, Eva; Kapás, Levente; Sun, Yuxiang; Smith, Roy G; Krueger, James M

    2007-07-01

    Ghrelin is well known for its feeding and growth hormone-releasing actions. It may also be involved in sleep regulation; intracerebroventricular administration and hypothalamic microinjections of ghrelin stimulate wakefulness in rats. Hypothalamic ghrelin, together with neuropeptide Y and orexin form a food intake-regulatory circuit. We hypothesized that this circuit also promotes arousal. To further investigate the role of ghrelin in the regulation of sleep-wakefulness, we characterized spontaneous and homeostatic sleep regulation in ghrelin knockout (KO) and wild-type (WT) mice. Both groups of mice exhibited similar diurnal rhythms with more sleep and less wakefulness during the light period. In ghrelin KO mice, spontaneous wakefulness and rapid-eye-movement sleep (REMS) were slightly elevated, and non-rapid-eye-movement sleep (NREMS) was reduced. KO mice had more fragmented NREMS than WT mice, as indicated by the shorter and greater number of NREMS episodes. Six hours of sleep deprivation induced rebound increases in NREMS and REMS and biphasic changes in electroencephalographic slow-wave activity (EEG SWA) in both genotypes. Ghrelin KO mice recovered from NREMS and REMS loss faster, and the delayed reduction in EEG SWA, occurring after sleep loss-enhanced increases in EEG SWA, was shorter-lasting compared with WT mice. These findings suggest that the basic sleep-wake regulatory mechanisms in ghrelin KO mice are not impaired and they are able to mount adequate rebound sleep in response to a homeostatic challenge. It is possible that redundancy in the arousal systems of the brain or activation of compensatory mechanisms during development allow for normal sleep-wake regulation in ghrelin KO mice. PMID:17409264

  20. The peripheral cannabinoid receptor knockout mice: an update

    PubMed Central

    Buckley, N E

    2007-01-01

    This review gives an overview of the CB2 receptor (CB2R) knockout (CB2R−/−) mice phenotype and the work that has been carried out using this mutant mouse. Using the CB2R−/− mice, investigators have discovered the involvement of CB2R on immune cell function and development, infection, embryonic development, bone loss, liver disorders, pain, autoimmune inflammation, allergic dermatitis, atherosclerosis, apoptosis and chemotaxis. Using the CB2R−/− mice, investigators have also found that this receptor is not involved in cannabinoid-induced hypotension. In addition, the CB2R−/− mice have been used to determine specific tissue CB2R expression. The specificity of synthetic cannabinoid agonists, antagonists and anti-CB2R antibodies has been screened using tissues from CB2R−/− mice. Thus, the use of this mouse model has greatly helped reveal the diverse events involving the CB2R, and has aided in drug and antibody screening. PMID:17965741

  1. Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice.

    PubMed

    Stirling, L Caroline; Forlani, Greta; Baker, Mark D; Wood, John N; Matthews, Elizabeth A; Dickenson, Anthony H; Nassar, Mohammed A

    2005-01-01

    NaV1.8 is a voltage-gated sodium channel expressed only in a subset of sensory neurons of which more than 85% are nociceptors. In order to delete genes in nociceptive neurons, we generated heterozygous transgenic mice expressing Cre recombinase under the control of the NaV1.8 promoter. Functional Cre recombinase expression replicated precisely the expression pattern of NaV1.8. Cre expression began at embryonic day 14 in small diameter neurons in dorsal root, trigeminal and nodose ganglia, but was absent in non-neuronal or CNS tissues into adulthood. Sodium channel subtypes were normal in isolated DRG neurons. Pain behaviour in response to mechanical or thermal stimuli, and in acute, inflammatory and neuropathic pain was also normal. These data demonstrate that the heterozygous NaV1.8-Cre mouse line is a useful tool to analyse the effects of deleting floxed genes on pain behaviour. PMID:15621361

  2. Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice.

    PubMed Central

    Nicoletti, A; Kaveri, S; Caligiuri, G; Bariéty, J; Hansson, G K

    1998-01-01

    Atherosclerosis is associated with immune activation. T cells and macrophages infiltrate atherosclerotic plaques and disease progression is associated with formation of autoantibodies to oxidized lipoproteins. In the apo E knockout mouse, a genetic model of cholesterol-induced atherosclerosis, congenital deficiency of macrophages, lymphocytes, or interferon-gamma receptors result in reduced lesion formation. We have now evaluated whether immune modulation in the adult animal affects disease development. Injections of 7-wk-old male apo E knockout mice with polyclonal immunoglobulin preparations (ivIg) during a 5-d period reduced fatty streak formation over a 2-mo period on cholesterol diet by 35%. Fibrofatty lesions induced by diet treatment for 4 mo were reduced by 50% in mice receiving ivIg after 2 mo on the diet. ivIg treatment also reduced IgM antibodies to oxidized LDL and led to inactivation of spleen and lymph node T cells. These data indicate that ivIg inhibits atherosclerosis, that it is effective both during the fatty streak and plaque phases, and that it may act by modulating T cell activity and/or antibody production. Therefore, immunomodulation may be an effective way to prevent and/or treat atherosclerosis. PMID:9727059

  3. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice.

    PubMed

    Wu, Xudong; Indzhykulian, Artur A; Niksch, Paul D; Webber, Roxanna M; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A; Corey, David P

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.

  4. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

    PubMed Central

    Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  5. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice.

    PubMed

    Wu, Xudong; Indzhykulian, Artur A; Niksch, Paul D; Webber, Roxanna M; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A; Corey, David P

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  6. BDNF restricted knockout mice as an animal model for aggression

    PubMed Central

    Ito, Wataru; Chehab, Mahmoud; Thakur, Siddarth; Li, Jiayang; Morozov, Alexei

    2011-01-01

    Mice with global deletion of one 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, 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 of BDNF-dependent aggression and object recognition deficiency. PMID:21255268

  7. Generation of mice carrying a knockout-first and conditional-ready allele of transforming growth factor beta2 gene.

    PubMed

    Ishtiaq Ahmed, A S; Bose, Gracelyn C; Huang, Li; Azhar, Mohamad

    2014-09-01

    Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell-matrix remodeling, epithelial-mesenchymal transition, and wound healing in a highly context-dependent and tissue-specific manner. Tgfb2(-/-) mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2(βgeo) (knockout-first lacZ-tagged insertion) gene-trap allele and Tgfb2(flox) conditional allele. Tgfb2(βgeo/βgeo) or Tgfb2(βgeo/-) mice died at perinatal stage from the same congenital heart defects as Tgfb2(-/-) mice. β-galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2(βgeo) fetal tissue sections. Tgfb2(flox) mice were produced by crossing the Tgfb2(+/βgeo) mice with the FLPeR mice. Tgfb2(flox/-) mice were viable. Tgfb2 conditional knockout (Tgfb2(cko/-) ) fetuses were generated by crossing of Tgfb2(flox/-) mice with Tgfb2(+/-) ; EIIaCre mice. Systemic Tgfb2(cko/-) embryos developed cardiac defects which resembled the Tgfb2(βgeo/βgeo) , Tgfb2(βgeo/-) , and Tgfb2(-/-) fetuses. In conclusion, Tgfb2(βgeo) and Tgfb2(flox) mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis

  8. Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

    PubMed Central

    Boini, Krishna M.; Xia, Min; Abais, Justine M.; Xu, Ming; Li, Cai-xia; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs+/− and Asm+/− mice. Given that the homozygotes of Cbs−/−/Asm−/− mice could not survive for 3 weeks. Cbs+/−/Asm+/+, Cbs+/−/Asm+/− and Cbs+/−/Asm−/− as well as their Cbs wild type littermates were used to study the role of Asm−/− under a background of Cbs+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs+/−) mice with different copies of Asm gene compared to Cbs+/+ mice with different Asm gene copies. Cbs+/−/Asm+/+ mice had significantly increased renal Asm activity, ceramide production and O2.− level compared to Cbs+/+/Asm+/+, while Cbs+/−/Asm−/− mice showed significantly reduced renal Asm activity, ceramide production and O2.− level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs+/−/Asm−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O2.− production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or

  9. Effects of chronic deoxynivalenol exposure on p53 heterozygous and p53 homozygous mice.

    PubMed

    Bondy, G S; Coady, L; Curran, I; Caldwell, D; Armstrong, C; Aziz, S A; Nunnikhoven, A; Gannon, A M; Liston, V; Shenton, J; Mehta, R

    2016-10-01

    Deoxynivalenol (DON) is a secondary metabolite associated with Fusarium species pathogenic to important food crops. A two-year feeding study reported that DON was non-carcinogenic in B6C3F1 mice. The present study was conducted to further characterize the chronic effects of DON by exposing cancer-prone transgenic p53 heterozygous (p53+/-) male mice and p53 homozygous (p53+/+) male mice to 0, 1, 5, or 10 mg DON/kg in diet for 26 weeks. Gross and microscopic organ-specific neoplastic and non-neoplastic changes and expression profiles of key hepatic and renal genes were assessed. Few toxicologic differences between p53+/+ and p53+/- mice were observed, and no tumours were observed due to DON. The results indicated that DON was non-carcinogenic and that reduced expression of the p53 gene did not play a key role in responses to DON toxicity. The lack of inflammatory and proliferative lesions in mice may be attributed to the anorectic effects of DON, which resulted in dose-dependent reductions in body weight in p53+/+ and p53+/- mice. Hepatic and renal gene expression analyses confirmed that chronic exposure to DON was noninflammatory. The effects of 26-week DON exposure on p53+/+ and p53+/-mice were consistent with those previously seen in B6C3F1 mice exposed to DON for two years.

  10. Reduced ultrasonic vocalizations in vasopressin 1b knockout mice.

    PubMed

    Scattoni, M L; McFarlane, H G; Zhodzishsky, V; Caldwell, H K; Young, W S; Ricceri, L; Crawley, J N

    2008-03-01

    The neuropeptides oxytocin and vasopressin have been implicated in rodent social and affiliative behaviors, including social bonding, parental care, social recognition, social memory, vocalizations, territoriality, and aggression, as well as components of human social behaviors and the etiology of autism. Previous investigations of mice with various manipulations of the oxytocin and vasopressin systems reported unusual levels of ultrasonic vocalizations in social settings. We employed a vasopressin 1b receptor (Avpr1b) knockout mouse to evaluate the role of the vasopressin 1b receptor subtype in the emission of ultrasonic vocalizations in adult and infant mice. Avpr1b null mutant female mice emitted fewer ultrasonic vocalizations, and their vocalizations were generally at lower frequencies, during a resident-intruder test. Avpr1b null mutant pups emitted ultrasonic vocalizations similar to heterozygote and wildtype littermates when separated from the nest on postnatal days 3, 6, 9, and 12. However, maternal potentiation of ultrasonic vocalizations in Avpr1b null and heterozygote mutants was absent, when tested at postnatal day 9. These results indicate that Avpr1b null mutant mice are impaired in the modulation of ultrasonic vocalizations within different social contexts at infant and adult ages.

  11. Decreased vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function in knockout mice affects aging of dopaminergic systems

    PubMed Central

    Hall, F. S.; Itokawa, K.; Schmitt, A.; Moessner, R.; Sora, I.; Lesch, K. P.; Uhl, G. R.

    2013-01-01

    Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2). These transporters work at the plasma and vesicular membranes of dopaminergic neurons, respectively, and thus regulate levels of DA in neuronal compartments that include the extravesicular cytoplasmic compartment. DA in this compartment has been hypothesized to contribute to oxidative damage that can reduce the function of dopaminergic neurons in aging brains and may contribute to reductions in dopaminergic neurochemical markers, locomotor behavior and responses to dopaminergic drugs that are found in aged animals. The studies reported here examined aged mice with heterozygous deletions of VMAT2 or of DAT, which each reduce transporter expression to about 50% of levels found in wild-type (WT) mice. Aged mice displayed reduced locomotor responses under a variety of circumstances, including in response to locomotor stimulants, as well as changes in monoamine levels and metabolites in a regionally dependent manner. Several effects of aging were more pronounced in heterozygous VMAT2 knockout (KO) mice, including aging induced reductions in locomotion and reduced locomotor responses to cocaine. By contrast, some effects of aging were reduced or not observed in heterozygous DAT KO mice. These findings support the idea that altered DAT and VMAT2 expression affect age-related changes in dopaminergic function. These effects are most likely mediated by alterations in DA compartmentalization, and might be hypothesized to be more exacerbated by other factors that affect the metabolism of cytosolic DA. PMID:23978383

  12. P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

    PubMed Central

    Viering, Daan H. H. M.; Bos, Caro; Bindels, René J. M.; Hoenderop, Joost G. J.

    2016-01-01

    ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg2+ and Na+ reabsorption, but its role in ion transport remains unknown. In this study, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na+, K+, Mg2+ and Ca2+ were not detected between P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions. PMID:27254077

  13. Comprehensive Behavioral Analysis of Cluster of Differentiation 47 Knockout Mice

    PubMed Central

    Koshimizu, Hisatsugu; Takao, Keizo; Matozaki, Takashi; Ohnishi, Hiroshi; Miyakawa, Tsuyoshi

    2014-01-01

    Cluster of differentiation 47 (CD47) is a member of the immunoglobulin superfamily which functions as a ligand for the extracellular region of signal regulatory protein α (SIRPα), a protein which is abundantly expressed in the brain. Previous studies, including ours, have demonstrated that both CD47 and SIRPα fulfill various functions in the central nervous system (CNS), such as the modulation of synaptic transmission and neuronal cell survival. We previously reported that CD47 is involved in the regulation of depression-like behavior of mice in the forced swim test through its modulation of tyrosine phosphorylation of SIRPα. However, other potential behavioral functions of CD47 remain largely unknown. In this study, in an effort to further investigate functional roles of CD47 in the CNS, CD47 knockout (KO) mice and their wild-type littermates were subjected to a battery of behavioral tests. CD47 KO mice displayed decreased prepulse inhibition, while the startle response did not differ between genotypes. The mutants exhibited slightly but significantly decreased sociability and social novelty preference in Crawley’s three-chamber social approach test, whereas in social interaction tests in which experimental and stimulus mice have direct contact with each other in a freely moving setting in a novel environment or home cage, there were no significant differences between the genotypes. While previous studies suggested that CD47 regulates fear memory in the inhibitory avoidance test in rodents, our CD47 KO mice exhibited normal fear and spatial memory in the fear conditioning and the Barnes maze tests, respectively. These findings suggest that CD47 is potentially involved in the regulation of sensorimotor gating and social behavior in mice. PMID:24586890

  14. Altered motor activity, exploration and anxiety in heterozygous neuregulin 1 mutant mice: implications for understanding schizophrenia.

    PubMed

    Karl, T; Duffy, L; Scimone, A; Harvey, R P; Schofield, P R

    2007-10-01

    Human genetic studies have shown that neuregulin 1 (NRG1) is a potential susceptibility gene for schizophrenia. Nrg1 influences various neurodevelopmental processes, which are potentially related to schizophrenia. The neurodevelopmental theory of schizophrenia suggests that interactions between genetic and environmental factors are responsible for biochemical alterations leading to schizophrenia. To investigate these interactions and to match experimental design with the pathophysiology of schizophrenia, we applied a comprehensive behavioural phenotyping strategy for motor activity, exploration and anxiety in a heterozygous Nrg1 transmembrane domain mutant mouse model (Nrg1 HET) using different housing conditions and age groups. We observed a locomotion- and exploration-related hyperactive phenotype in Nrg1 HETs. Increased age had a locomotion- and exploration-inhibiting effect, which was significantly attenuated in mutant mice. Environmental enrichment (EE) had a stimulating influence on locomotion and exploration. The impact of EE was more pronounced in Nrg1 hypomorphs. Our study also showed a moderate task-specific anxiolytic-like phenotype for Nrg1 HETs, which was influenced by external factors. The behavioural phenotype detected in heterozygous Nrg1 mutant mice is not specific to schizophrenia per se, but the increased sensitivity of mutant mice to exogenous factors is consistent with the pathophysiology of schizophrenia and the neurodevelopmental theory. Our findings reinforce the importance of carefully controlling experimental designs for external factors and of comprehensive, integrative phenotyping strategies. Thus, Nrg1 HETs may, in combination with other genetic and drug models, help to clarify pathophysiological mechanisms behind schizophrenia.

  15. Subtype-specific reduction of olfactory bulb interneurons in Pax6 heterozygous mutant mice.

    PubMed

    Haba, Hasumi; Nomura, Tadashi; Suto, Fumikazu; Osumi, Noriko

    2009-09-01

    Interneurons in the olfactory bulb (OB) play essential roles in the processing of olfactory information. They are classified into several subpopulations by the expression of different neurochemical markers. Here we focused on a transcription factor Pax6, and examined its expression and function in distinct subtypes of OB interneurons. We identified Pax6 expression in specific subtypes of interneurons in the external plexiform layer (EPL). The number of these interneuron subtypes was dramatically decreased in Pax6 heterozygous mutant mice. These results indicate that Pax6 is required for differentiation and/or maintenance of EPL interneurons in the adult mouse OB.

  16. Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

    ERIC Educational Resources Information Center

    Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

    2006-01-01

    CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

  17. Chronic Toxoplasma gondii in Nurr1-null heterozygous mice exacerbates elevated open field activity.

    PubMed

    Eells, Jeffrey B; Varela-Stokes, Andrea; Guo-Ross, Shirley X; Kummari, Evangel; Smith, Holly M; Cox, Erin; Lindsay, David S

    2015-01-01

    Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.

  18. Chronic Toxoplasma gondii in Nurr1-null heterozygous mice exacerbates elevated open field activity.

    PubMed

    Eells, Jeffrey B; Varela-Stokes, Andrea; Guo-Ross, Shirley X; Kummari, Evangel; Smith, Holly M; Cox, Erin; Lindsay, David S

    2015-01-01

    Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice. PMID:25855987

  19. SNARE function analyzed in synaptobrevin/VAMP knockout mice.

    PubMed

    Schoch, S; Deák, F; Königstorfer, A; Mozhayeva, M; Sara, Y; Südhof, T C; Kavalali, E T

    2001-11-01

    SNAREs (soluble NSF-attachment protein receptors) are generally acknowledged as central components of membrane fusion reactions, but their precise function has remained enigmatic. Competing hypotheses suggest roles for SNAREs in mediating the specificity of fusion, catalyzing fusion, or actually executing fusion. We generated knockout mice lacking synaptobrevin/VAMP 2, the vesicular SNARE protein responsible for synaptic vesicle fusion in forebrain synapses, to make use of the exquisite temporal resolution of electrophysiology in measuring fusion. In the absence of synaptobrevin 2, spontaneous synaptic vesicle fusion and fusion induced by hypertonic sucrose were decreased approximately 10-fold, but fast Ca2+-triggered fusion was decreased more than 100-fold. Thus, synaptobrevin 2 may function in catalyzing fusion reactions and stabilizing fusion intermediates but is not absolutely required for synaptic fusion.

  20. Altered Sleep Homeostasis in Rev-erbα Knockout Mice

    PubMed Central

    Mang, Géraldine M.; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A.; Albrecht, Urs; Franken, Paul

    2016-01-01

    Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1–4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. Citation: Mang GM, La Spada F, Emmenegger Y, Chappuis S, Ripperger JA, Albrecht U, Franken P. Altered sleep homeostasis in Rev

  1. Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice.

    PubMed

    Al-Samir, Samer; Goossens, Dominique; Cartron, Jean-Pierre; Nielsen, Søren; Scherbarth, Frank; Steinlechner, Stephan; Gros, Gerolf; Endeward, Volker

    2016-01-01

    We have measured maximal oxygen consumption ([Formula: see text]O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that [Formula: see text]O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of [Formula: see text]O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of [Formula: see text]O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of [Formula: see text]O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced [Formula: see text]O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced [Formula: see text]O2,max, which constitutes a new phenotype of these mice. PMID:27559317

  2. Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice

    PubMed Central

    Al-Samir, Samer; Goossens, Dominique; Cartron, Jean-Pierre; Nielsen, Søren; Scherbarth, Frank; Steinlechner, Stephan; Gros, Gerolf; Endeward, Volker

    2016-01-01

    We have measured maximal oxygen consumption (V˙O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V˙O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V˙O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V˙O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V˙O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V˙O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V˙O2,max, which constitutes a new phenotype of these mice. PMID:27559317

  3. TNF-α knockout mice have increased corpora cavernosa relaxation

    PubMed Central

    2010-01-01

    Introduction Erectile dysfunction (ED) is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression. Aim Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-α actions would increase cavernosal smooth muscle relaxation through an increase in NOS expression. Methods In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-α knockout (TNF-α KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 min.). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively. Main Outcome Measures Corpora cavernosa from TNF-α KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression. Results Cavernosal strips from TNF-α KO mice displayed increased endothelium-dependent [97.4±5.3 vs Control: 76.3±6.3, %] and nonadrenergic-noncholinergic (NANC) [93.3±3.0 vs Control: 67.5±16.0; 16 Hz] relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (p<0.05). Sympathetic-mediated [0.69±0.16 vs Control: 1.22±0.22; 16 Hz] as well as phenylephrine-induced contractile responses [1.6±0.1 vs Control: 2.5±0.1, mN] were attenuated in cavernosal strips from TNF-α KO mice. Additionally, corpora cavernosa from TNF-α KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-α KO mice display increased number of spontaneous erections. Conclusion Corpora cavernosa from

  4. Mice heterozygous for cathepsin D deficiency exhibit mania-related behavior and stress-induced depression.

    PubMed

    Zhou, Rui; Lu, Yi; Han, Yong; Li, Xia; Lou, Huifang; Zhu, Liya; Zhen, Xuechu; Duan, Shumin

    2015-12-01

    Mutations in cathepsin D (CTSD), an aspartic protease in the endosomal-lysosomal system, underlie congenital neuronal ceroid-lipofuscinosis (cNCL, also known as CLN10), a devastating neurodegenerative disease. CLN10 patients die within the first few days of life, and in the few patients who live into adulthood psychopathological symptoms have not been reported. Extensive neuropathology and altered neurotransmission have been reported in CTSD-deficient mice; however signs of neuropsychiatric behavior in these mice are not well characterized due to the severe movement disorder and premature death of the animal. In the present study, we show that heterozygous CTSD-deficient (CTSD HET) mice display an overall behavioral profile that is similar to human mania, including hyperlocomotion, d-amphetamine-induced hyperactivity, sleep-disturbance, and reduced anxiety-like behavior. However, under stressful conditions CTSD HET mice manifest depressive-like behavior, including anhedonia, behavioral despair, and enhanced learned helplessness. Chronic administration of lithium chloride or valproic acid, two clinically effective mood stabilizers, reverses the majority of these behavioral abnormalities. In addition, CTSD HET mice display stress-induced hypersecretion of corticosterone. These findings suggest an important role for CTSD in the regulation of mood stabilization.

  5. Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

    PubMed Central

    Choi, Jin-Hwa; Nguyen, Minh-Phuong; Lee, Dongjin; Oh, Goo-Taeg; Lee, You-Mie

    2014-01-01

    Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT+/− EPCs. In AGT+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis. PMID:24938229

  6. Abolished thermal and mechanical antinociception but retained visceral chemical antinociception induced by butorphanol in mu-opioid receptor knockout mice.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Ishihara, Kumatoshi; Uhl, George R; Satoh, Masamichi; Sora, Ichiro; Ikeda, Kazutaka

    2008-06-01

    Butorphanol is hypothesized to induce analgesia via opioid pathways, although the precise mechanisms for its effects remain unknown. In this study, we investigated the role of the mu-opioid receptor (MOP) in thermal, mechanical, and visceral chemical antinociception induced by butorphanol using MOP knockout (KO) mice. Butorphanol-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from our butorphanol-induced mechanical antinociception experiments, assessed by the Randall-Selitto test, were similar to the results obtained from the thermal antinociception experiments in these mice. Interestingly, however, butorphanol retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice. The butorphanol-induced visceral chemical antinociception that was retained in homozygous MOP-KO mice was completely blocked by pretreatment with nor-binaltorphimine, a kappa-opioid receptor (KOP) antagonist. In vitro binding and cyclic adenosine monophosphate assays also showed that butorphanol possessed higher affinity for KOPs and MOPs than for delta-opioid receptors. These results molecular pharmacologically confirmed previous studies implicating MOPs, and partially KOPs, in mediating butorphanol-induced analgesia. PMID:18417173

  7. The antidepressant-like effects of glutamatergic drugs ketamine and AMPA receptor potentiator LY 451646 are preserved in bdnf⁺/⁻ heterozygous null mice.

    PubMed

    Lindholm, Jesse S O; Autio, Henri; Vesa, Liisa; Antila, Hanna; Lindemann, Lothar; Hoener, Marius C; Skolnick, Phil; Rantamäki, Tomi; Castrén, Eero

    2012-01-01

    Accumulating evidence suggests that biogenic amine-based antidepressants act, at least in part, via regulation of brain-derived neurotrophic factor (BDNF) signaling. Biogenic amine-based antidepressants increase BDNF synthesis and activate its signaling pathway through TrkB receptors. Moreover, the antidepressant-like effects of these molecules are abolished in BDNF deficient mice. Glutamate-based drugs, including the NMDA antagonist ketamine, and the AMPA receptor potentiator LY 451646, mimic the effects of antidepressants in preclinical tests with high predictive validity. In humans, a single intravenous dose of ketamine produces an antidepressant effect that is rapid, robust and persistent. In this study, we examined the role of BDNF in expression of the antidepressant-like effects of ketamine and an AMPA receptor potentiator (LY 451646) in the forced swim test (FST). Ketamine and LY 451646 produced antidepressant-like effects in the FST in mice at 45 min after a single injection, but no effects were observed one week after a single ketamine injection. As previously reported, the effects of imipramine in the forced swim test were blunted in heterozygous BDNF knockout (bdnf(+/-)) mice. However ketamine and LY 451646 produced similar antidepressant-like responses in wildtype and bdnf(+/-) mice. Neither ketamine nor LY 451646 significantly influenced the levels BDNF or TrkB phosphorylation in the hippocampus when assessed at 45 min or 7 days after the drug administration. These data demonstrate that under the conditions tested, neither ketamine nor the AMPA-potentiator LY 451656 activate BDNF signaling, but produce a characteristic antidepressant-like response in heterozygous bdnf(+/-) mice. These data indicate that unlike biogenic amine-based agents, BDNF signaling does not play a pivotal role in the antidepressant effects of glutamate-based compounds. This article is part of a Special Issue entitled 'Anxiety and Depression'.

  8. Disabled-2 heterozygous mice are predisposed to endometrial and ovarian tumorigenesis and exhibit sex-biased embryonic lethality in a p53-null background.

    PubMed

    Yang, Dong-Hua; Fazili, Zia; Smith, Elizabeth R; Cai, Kathy Qi; Klein-Szanto, Andres; Cohen, Cynthia; Horowitz, Ira R; Xu, Xiang-Xi

    2006-07-01

    Disabled-2 (Dab2) is a phosphoprotein involved in cellular signal transduction and endocytic trafficking. The expression of Dab2 is frequently lost or suppressed in several epithelial tumors, and studies of its cellular function and growth suppressive activity when re-expressed in cancer cells led to the suggestion that Dab2 is a tumor suppressor. A role for Dab2 in epithelial cell positioning organization was derived from study of knockout mice: homozygous deletion of dab2 results in early embryonic lethality due to the disorganization of the primitive endoderm, the first epithelium in early embryos. We now report that dab2 heterozygous mice develop uterine hyperplasia and ovarian preneoplastic morphological changes at a high frequency. Crossing into a p53(-/-) background unexpectedly produced few female dab2(+/-):p53(-/-) mice, while the male dab2(+/-):p53(-/-) were born at the expected Mendelian frequency. The tumor-prone phenotype of dab2(+/-) mice provides additional support for a role of human Dab2 as a tumor suppressor, and the sex-biased embryonic lethality suggests a genetic interaction between p53 and dab2 genes in female mice. PMID:16816378

  9. Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for the GDNF mutation.

    PubMed

    Gerlai, R; McNamara, A; Choi-Lundberg, D L; Armanini, M; Ross, J; Powell-Braxton, L; Phillips, H S

    2001-10-01

    Exogenous glial cell line-derived neurotrophic factor (GDNF) exhibits potent survival-promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinson's disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities. PMID:11683907

  10. Effects of LSD on grooming behavior in serotonin transporter heterozygous (Sert⁺/⁻) mice.

    PubMed

    Kyzar, Evan J; Stewart, Adam Michael; Kalueff, Allan V

    2016-01-01

    Serotonin (5-HT) plays a crucial role in the brain, modulating mood, cognition and reward. The serotonin transporter (SERT) is responsible for the reuptake of 5-HT from the synaptic cleft and regulates serotonin signaling in the brain. In humans, SERT genetic variance is linked to the pathogenesis of various psychiatric disorders, including anxiety, autism spectrum disorders (ASD) and obsessive-compulsive disorder (OCD). Rodent self-grooming is a complex, evolutionarily conserved patterned behavior relevant to stress, ASD and OCD. Genetic ablation of mouse Sert causes various behavioral deficits, including increased anxiety and grooming behavior. The hallucinogenic drug lysergic acid diethylamide (LSD) is a potent serotonergic agonist known to modulate human and animal behavior. Here, we examined heterozygous Sert(+/-) mouse behavior following acute administration of LSD (0.32 mg/kg). Overall, Sert(+/-) mice displayed a longer duration of self-grooming behavior regardless of LSD treatment. In contrast, LSD increased serotonin-sensitive behaviors, such as head twitching, tremors and backwards gait behaviors in both Sert(+/+) and Sert(+/-) mice. There were no significant interactions between LSD treatment and Sert gene dosage in any of the behavioral domains measured. These results suggest that Sert(+/-) mice may respond to the behavioral effects of LSD in a similar manner to wild-type mice. PMID:26340513

  11. Effects of LSD on grooming behavior in serotonin transporter heterozygous (Sert⁺/⁻) mice.

    PubMed

    Kyzar, Evan J; Stewart, Adam Michael; Kalueff, Allan V

    2016-01-01

    Serotonin (5-HT) plays a crucial role in the brain, modulating mood, cognition and reward. The serotonin transporter (SERT) is responsible for the reuptake of 5-HT from the synaptic cleft and regulates serotonin signaling in the brain. In humans, SERT genetic variance is linked to the pathogenesis of various psychiatric disorders, including anxiety, autism spectrum disorders (ASD) and obsessive-compulsive disorder (OCD). Rodent self-grooming is a complex, evolutionarily conserved patterned behavior relevant to stress, ASD and OCD. Genetic ablation of mouse Sert causes various behavioral deficits, including increased anxiety and grooming behavior. The hallucinogenic drug lysergic acid diethylamide (LSD) is a potent serotonergic agonist known to modulate human and animal behavior. Here, we examined heterozygous Sert(+/-) mouse behavior following acute administration of LSD (0.32 mg/kg). Overall, Sert(+/-) mice displayed a longer duration of self-grooming behavior regardless of LSD treatment. In contrast, LSD increased serotonin-sensitive behaviors, such as head twitching, tremors and backwards gait behaviors in both Sert(+/+) and Sert(+/-) mice. There were no significant interactions between LSD treatment and Sert gene dosage in any of the behavioral domains measured. These results suggest that Sert(+/-) mice may respond to the behavioral effects of LSD in a similar manner to wild-type mice.

  12. Cannabinoid 1 receptor knockout mice display cold allodynia, but enhanced recovery from spared-nerve injury-induced mechanical hypersensitivity

    PubMed Central

    Piskoun, Boris; Russo, Lori; Norcini, Monica; Blanck, Thomas; Recio-Pinto, Esperanza

    2016-01-01

    Background The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R−/−) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. Results At baseline, CB1R−/− mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R−/− mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R−/− mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/−) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R−/− mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different

  13. Knockout of the TauT Gene Predisposes C57BL/6 Mice to Streptozotocin-Induced Diabetic Nephropathy

    PubMed Central

    Han, Xiaobin; Patters, Andrea B.; Ito, Takashi; Schaffer, Stephen W.; Chesney, Russell W.

    2015-01-01

    Diabetic nephropathy is the leading cause of end stage renal disease in the world. Although tremendous efforts have been made, scientists have yet to identify an ideal animal model that can reproduce the characteristics of human diabetic nephropathy. In this study, we hypothesize that taurine insufficiency is a critical risk factor for development of diabetic nephropathy associated with diabetes mellitus. This hypothesis was tested in vivo in TauT heterozygous (TauT+/-) and homozygous (TauT-/-) knockout in C57BL/6 background mice. We have shown that alteration of the TauT gene (also known as SLC6A6) has a substantial effect on the susceptibility to development of extensive diabetic kidney disease in both TauT+/- and TauT-/-mouse models of diabetes. These animals developed histological changes characteristic of human diabetic nephropathy that included glomerulosclerosis, nodular lesions, arteriosclerosis, arteriolar dilation, and tubulointerstitial fibrosis. Immunohistochemical staining of molecular markers of smooth muscle actin, CD34, Ki67 and collagen IV further confirmed these observations. Our results demonstrated that both homozygous and heterozygous TauT gene deletion predispose C57BL/6 mice to develop end-stage diabetic kidney disease, which closely replicates the pathological features of diabetic nephropathy in human diabetic patients. PMID:25629817

  14. Alterations of Retinal Vasculature in Cystathionine–β-Synthase Heterozygous Mice

    PubMed Central

    Tawfik, Amany; Markand, Shanu; Al-Shabrawey, Mohamed; Mayo, Jamie N.; Reynolds, Jason; Bearden, Shawn E.; Ganapathy, Vadivel; Smith, Sylvia B.

    2015-01-01

    Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine–β-synthase (Cbs+/−) to evaluate retinal vascular integrity. The Cbs+/+ (wild type) and Cbs+/− (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti–zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti–pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs+/− mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs+/− retinas. Retinal vein occlusion was observed in some Cbs+/− mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs+/− mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion. PMID:25016930

  15. Generation and Behavior Characterization of CaMKIIβ Knockout Mice

    PubMed Central

    Tu, Tao; Goulding, Danielle S.; Haiech, Jacques; Watterson, D. Martin; Van Eldik, Linda J.

    2014-01-01

    The calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the brain, where it makes important contributions to synaptic organization and homeostasis, including playing an essential role in synaptic plasticity and memory. Four genes encode isoforms of CaMKII (α, β, δ, γ), with CaMKIIα and CaMKIIβ highly expressed in the brain. Decades of molecular and cellular research, as well as the use of a large number of CaMKIIα mutant mouse lines, have provided insight into the pivotal roles of CaMKIIα in brain plasticity and cognition. However, less is known about the CaMKIIβ isoform. We report the development and extensive behavioral and phenotypic characterization of a CaMKIIβ knockout (KO) mouse. The CaMKIIβ KO mouse was found to be smaller at weaning, with an altered body mass composition. The CaMKIIβ KO mouse showed ataxia, impaired forelimb grip strength, and deficits in the rotorod, balance beam and running wheel tasks. Interestingly, the CaMKIIβ KO mouse exhibited reduced anxiety in the elevated plus maze and open field tests. The CaMKIIβ KO mouse also showed cognitive impairment in the novel object recognition task. Our results provide a comprehensive behavioral characterization of mice deficient in the β isoform of CaMKII. The neurologic phenotypes and the construction of the genotype suggest the utility of this KO mouse strain for future studies of CaMKIIβ in brain structure, function and development. PMID:25127391

  16. Nature vs. nurture: can enrichment rescue the behavioural phenotype of BDNF heterozygous mice?

    PubMed

    Chourbaji, Sabine; Brandwein, Christiane; Vogt, Miriam A; Dormann, Christof; Hellweg, Rainer; Gass, Peter

    2008-10-10

    In earlier experiments we have demonstrated that group-housing in a rather impoverished "standard" environment can be a crucial stress factor in male C57Bl/6 mice. The present study aimed at investigating the effect of combining a probable genetic vulnerability--postulated by the "Neurotrophin Hypothesis of Depression"--with the potentially modulating influence of a stressful environment such as "impoverished" standard housing conditions. For that purpose mice with a partial deletion of brain-derived neurotrophic factor (BDNF) were group-housed under standard and enriched housing conditions and analysed in a well-established test battery for emotional behaviours. Standard group-housing affected emotional behaviour in male and female BDNF heterozygous mice, causing an increase in anxiety, changes in exploration as well as nociception. Providing the animals' cages with supplementary enrichment, however, led to a rescue of emotional alterations, which emphasises the significance of external factors and their relevance for a valid investigation of genetic aspects in these mutants as well as others, which may be examined in terms of stress-responsiveness or emotionality.

  17. Nature vs. nurture: can enrichment rescue the behavioural phenotype of BDNF heterozygous mice?

    PubMed

    Chourbaji, Sabine; Brandwein, Christiane; Vogt, Miriam A; Dormann, Christof; Hellweg, Rainer; Gass, Peter

    2008-10-10

    In earlier experiments we have demonstrated that group-housing in a rather impoverished "standard" environment can be a crucial stress factor in male C57Bl/6 mice. The present study aimed at investigating the effect of combining a probable genetic vulnerability--postulated by the "Neurotrophin Hypothesis of Depression"--with the potentially modulating influence of a stressful environment such as "impoverished" standard housing conditions. For that purpose mice with a partial deletion of brain-derived neurotrophic factor (BDNF) were group-housed under standard and enriched housing conditions and analysed in a well-established test battery for emotional behaviours. Standard group-housing affected emotional behaviour in male and female BDNF heterozygous mice, causing an increase in anxiety, changes in exploration as well as nociception. Providing the animals' cages with supplementary enrichment, however, led to a rescue of emotional alterations, which emphasises the significance of external factors and their relevance for a valid investigation of genetic aspects in these mutants as well as others, which may be examined in terms of stress-responsiveness or emotionality. PMID:18538870

  18. Mildly impaired water maze performance in male Fmr1 knockout mice.

    PubMed

    D'Hooge, R; Nagels, G; Franck, F; Bakker, C E; Reyniers, E; Storm, K; Kooy, R F; Oostra, B A; Willems, P J; De Deyn, P P

    1997-01-01

    Fmr1 knockout mice constitute a putative model of fragile X syndrome, the most common form of heritable mental disability in humans. We have compared the performance of transgenic mice with an Fmr1 knockout with that of normal littermates in hidden- and visible-platform water maze learning, and showed that knockouts exhibit subnormal spatial learning abilities and marginal motor performance deficits. During 12 training trials of the hidden-platform task, escape latency and path length decreased significantly in knockouts and control littermates, and no effect of genotype was found. During four ensuing reversal trials, however, significant differences were found between knockouts and control littermates both in escape latency and path length. During the visible-platform condition, the reversal trials also revealed a difference between knockouts and normal littermates in escape latency, but not in path length. Possibly due to marginal motor incapacity, knockouts swam significantly slower than controls during these latter trials. During both probe trials of the hidden-platform task, knockouts as well as normal littermates spent more time in the target quadrant than in the other quadrants, and percent of time spent in the target quadrant was the same in both groups; swimming velocity was not significantly different between knockouts and normal littermates during these trials. Entries in the target area during the probe trials did show a significant effect of genotype on number of entries. The present results largely confirm and extend our previous findings. Impaired spatial abilities in Fmr1 knockouts might have been due to relatively low response flexibility or high memory interference in Fmr1 knockouts. It remains unclear, however, which brain region or neurochemical system might be involved in these disabilities. We conclude that Fmr1 knockout mice might be a valid model of fragile X mental retardation.

  19. Impact of partial dopamine depletion on cognitive flexibility in BDNF heterozygous mice

    PubMed Central

    Parikh, Vinay; Naughton, Sean X.; Yegla, Brittney; Guzman, Dawn M.

    2016-01-01

    Rationale Cognitive flexibility is a key component of executive function and is disrupted in major psychiatric disorders. Brain-derived neurotrophic factor (BDNF) exerts neuromodulatory effects on synaptic transmission and cognitive/affective behaviors. However the causal mechanisms linking BDNF hypofunction with executive deficits are not well understood. Objectives Here, we assessed the consequences of BDNF hemizygosity on cognitive flexibility in mice performing an operant conditioning task. As dopaminergic-glutamatergic interaction in the striatum is important for cognitive processing, and BDNF heterozygous (BDNF+/−) mice display a higher dopamine tone in the dorsal striatum, we also assessed the effects of partial striatal dopamine depletion on task performance and glutamate release. Results BDNF+/− mice acquired discrimination learning as well as new rule learning during set-shifting as efficiently as wild-type mice. However, partial removal of striatal dopaminergic inputs with 6-hydroxydopamine (6-OHDA) impaired these cognitive processes by impeding the maintenance of a new learning strategy in both genotypes. BDNF mutants exhibited performance impairments during reversal learning and these deficits were associated with increased perseveration to the previously acquired strategy. Partial dopamine depletion of the striatum reversed these cognitive impairments. Additionally, reduction in depolarization-evoked glutamate release noted in the dorsal striatum of BDNF+/− mice was not observed in 6-OHDA-infused BDNF mutants indicating normalization of glutamatergic transmission in these animals. Conclusions Our data illustrate that BDNF signaling regulates cognitive control processes presumably by maintaining striatal dopamine-glutamate balance. Moreover, aberrations in BDNF signaling may act as a common neurobiological substrate that accounts for executive dysfunction observed in multiple psychiatric conditions. PMID:26861892

  20. Chronic hyperglycemia induced via the heterozygous knockout of Pdx1 worsens neuropathological lesion in an Alzheimer mouse model

    PubMed Central

    Guo, Chuang; Zhang, Shuai; Li, Jia-Yi; Ding, Chen; Yang, Zhao-Hui; Chai, Rui; Wang, Xu; Wang, Zhan-You

    2016-01-01

    Compelling evidence has indicated that dysregulated glucose metabolism links Alzheimer’s disease (AD) and diabetes mellitus (DM) via glucose metabolic products. Nevertheless, because of the lack of appropriate animal models, whether chronic hyperglycemia worsens AD pathologies in vivo remains to be confirmed. Here, we crossed diabetic mice (Pdx1+/− mice) with Alzheimer mice (APP/PS1 transgenic mice) to generate Pdx1+/−/APP/PS1. We identified robust increases in tau phosphorylation, the loss of the synaptic spine protein, amyloid-β (Aβ) deposition and plaque formation associated with increased microglial and astrocyte activation proliferation, which lead to exacerbated memory and cognition deficits. More importantly, we also observed increased glucose intolerance accompanied by Pdx1 reduction, the formation of advanced glycation end-products (AGEs), and the activation of the receptor for AGEs (RAGE) signaling pathways during AD progression; these changes are thought to contribute to the processing of Aβ precursor proteins and result in increased Aβ generation and decreased Aβ degradation. Protein glycation, increased oxidative stress and inflammation via hyperglycemia are the primary mechanisms involved in the pathophysiology of AD. These results indicate the pathological relationship between these diseases and provide novel insights suggesting that glycemic control may be beneficial for decreasing the incidence of AD in diabetic patients and delaying AD progression. PMID:27406855

  1. Chronic hyperglycemia induced via the heterozygous knockout of Pdx1 worsens neuropathological lesion in an Alzheimer mouse model.

    PubMed

    Guo, Chuang; Zhang, Shuai; Li, Jia-Yi; Ding, Chen; Yang, Zhao-Hui; Chai, Rui; Wang, Xu; Wang, Zhan-You

    2016-01-01

    Compelling evidence has indicated that dysregulated glucose metabolism links Alzheimer's disease (AD) and diabetes mellitus (DM) via glucose metabolic products. Nevertheless, because of the lack of appropriate animal models, whether chronic hyperglycemia worsens AD pathologies in vivo remains to be confirmed. Here, we crossed diabetic mice (Pdx1(+/-) mice) with Alzheimer mice (APP/PS1 transgenic mice) to generate Pdx1(+/-)/APP/PS1. We identified robust increases in tau phosphorylation, the loss of the synaptic spine protein, amyloid-β (Aβ) deposition and plaque formation associated with increased microglial and astrocyte activation proliferation, which lead to exacerbated memory and cognition deficits. More importantly, we also observed increased glucose intolerance accompanied by Pdx1 reduction, the formation of advanced glycation end-products (AGEs), and the activation of the receptor for AGEs (RAGE) signaling pathways during AD progression; these changes are thought to contribute to the processing of Aβ precursor proteins and result in increased Aβ generation and decreased Aβ degradation. Protein glycation, increased oxidative stress and inflammation via hyperglycemia are the primary mechanisms involved in the pathophysiology of AD. These results indicate the pathological relationship between these diseases and provide novel insights suggesting that glycemic control may be beneficial for decreasing the incidence of AD in diabetic patients and delaying AD progression. PMID:27406855

  2. Vmat2 Heterozygous Mutant Mice Display a Depressive-Like Phenotype

    PubMed Central

    Fukui, Masato; Rodriguiz, Ramona M.; Zhou, Jiechun; Jiang, Sara X.; Phillips, Lindsey E.; Caron, Marc G.; Wetsel, William C.

    2010-01-01

    The vesicular monoamine transporter 2 (VMAT2) is localized primarily within the CNS and is responsible for transporting monoamines from the cytoplasm into secretory vesicles. Because reserpine (a VMAT inhibitor) can precipitate depressive-like symptoms in humans, we investigated whether Vmat2 heterozygous (HET) mice present with depressive-like behaviors. The mutants showed locomotor and rearing retardation in the open field and appeared anhedonic to 1 and 1.5% sucrose solutions. Immobility times for Vmat2 heterozygotes were prolonged in forced swim and imipramine normalized this behavior. HET animals also showed enhanced immobility in tail suspension and this response was alleviated by fluoxetine, reboxetine, and bupropion. Stimulated GTPγS binding indicated that α2-adrenergic receptors in HET hippocampus were more sensitive to UK 14,304 (5-bromo-N-(4,5-dihydro-1-H-imidazol-2-yl)-6-quinoxalinamine) stimulation than in wild type (WT) mice. In learned helplessness, mice were exposed to a shuttle box for 4 d or were given inescapable foot-shocks for the same time period. On day 5, all animals were tested in shock escape. Failure rates and the latency to escape were similar for WT and HET mice that were only pre-exposed to the test apparatus. In foot-shock groups, learned helplessness was more robust in heterozygotes than in WT controls. Basal secretion of serum corticosterone was not distinguished by genotype; however, corticosterone levels in mutants were more responsive to stress. Anxiety-like responses of WT and HET animals in the open field, light-dark exploration, zero maze, and novelty-suppressed feeding tests were indistinguishable. Collectively, these findings suggest that Vmat2 heterozygotes display a depressive-like phenotype that is devoid of anxiety-like behavior. PMID:17898223

  3. Altered expression of neuropeptides in FoxG1-null heterozygous mutant mice.

    PubMed

    Frullanti, Elisa; Amabile, Sonia; Lolli, Maria Grazia; Bartolini, Anna; Livide, Gabriella; Landucci, Elisa; Mari, Francesca; Vaccarino, Flora M; Ariani, Francesca; Massimino, Luca; Renieri, Alessandra; Meloni, Ilaria

    2016-02-01

    Foxg1 gene encodes for a transcription factor essential for telencephalon development in the embryonic mammalian forebrain. Its complete absence is embryonic lethal while Foxg1 heterozygous mice are viable but display microcephaly, altered hippocampal neurogenesis and behavioral and cognitive deficiencies. In order to evaluate the effects of Foxg1 alteration in adult brain, we performed expression profiling in total brains from Foxg1+/- heterozygous mutants and wild-type littermates. We identified statistically significant differences in expression levels for 466 transcripts (P<0.001), 29 of which showed a fold change ≥ 1.5. Among the differentially expressed genes was found a group of genes expressed in the basal ganglia and involved in the control of movements. A relevant (three to sevenfold changes) and statistically significant increase of expression, confirmed by qRT-PCR, was found in two highly correlated genes with expression restricted to the hypothalamus: Oxytocin (Oxt) and Arginine vasopressin (Avp). These neuropeptides have an important role in maternal and social behavior, and their alteration is associated with impaired social interaction and autistic behavior. In addition, Neuronatin (Nnat) levels appear significantly higher both in Foxg1+/- whole brain and in hippocampal neurons after silencing Foxg1, strongly suggesting that it is directly or indirectly repressed by Foxg1. During fetal and neonatal brain development, Nnat may regulate neuronal excitability, receptor trafficking and calcium-dependent signaling and, in the adult brain, it is predominantly expressed in parvalbumin-positive GABAergic interneurons. Overall, these results implicate the overexpression of a group of neuropeptides in the basal ganglia, hypothalamus, cortex and hippocampus in the pathogenesis FOXG1 behavioral impairments.

  4. Effects of Chronic Ochratoxin A Exposure on p53 Heterozygous and p53 Homozygous Mice.

    PubMed

    Bondy, Genevieve S; Caldwell, Donald S; Aziz, Syed A; Coady, Laurie C; Armstrong, Cheryl L; Curran, Ivan H A; Koffman, Robyn L; Kapal, Kamla; Lefebvre, David E; Mehta, Rekha

    2015-07-01

    Exposure to the mycotoxin ochratoxin A (OTA) causes nephropathy in domestic animals and rodents and renal tumors in rodents and poultry. Humans are exposed to OTA by consuming foods made with contaminated cereal grains and other commodities. Management of human health risks due to OTA exposure depends, in part, on establishing a mode of action (MOA) for OTA carcinogenesis. To further investigate OTA's MOA, p53 heterozygous (p53+/-) and p53 homozygous (p53+/+) mice were exposed to OTA in diet for 26 weeks. The former are susceptible to tumorigenesis upon chronic exposure to genotoxic carcinogens. OTA-induced renal damage but no tumors were observed in either strain, indicating that p53 heterozygosity conferred little additional sensitivity to OTA. Renal changes included dose-dependent increases in cellular proliferation, apoptosis, karyomegaly, and tubular degeneration in proximal tubules, which were consistent with ochratoxicosis. The lowest observed effect level for renal changes in p53+/- and p53+/+ mice was 200 μg OTA/kg bw/day. Based on the lack of tumors and the severity of renal and body weight changes at a maximum tolerated dose, the results were interpreted as suggestive of a primarily nongenotoxic (epigenetic) MOA for OTA carcinogenesis in this mouse model.

  5. Long-Term Effects of Prenatal Hypoxia on Schizophrenia-Like Phenotype in Heterozygous Reeler Mice.

    PubMed

    Howell, Kristy R; Pillai, Anilkumar

    2016-07-01

    Prenatal hypoxia (PHX) is a well-known environmental factor implicated in the pathophysiology of schizophrenia. However, the long-term effects of PHX on schizophrenia-related neuroplasticity are poorly understood. Using behavioral tasks, MRI imaging, and biochemical studies, we examined the long-term effects of PHX in heterozygous reeler mice (HRM; mice deficient for reelin, a candidate gene for schizophrenia). PHX at E17 failed to induce any significant deficits in prepulse inhibition, spatial memory, anxiety-like behavior, or blood flow in wild type (WT) and HRM at 6 months of age. However, PHX induced a significant increase in frontal cortex volume in WT whereas the higher frontal cortical volume found in HRM was significantly reduced by PHX. A significant decrease in reelin levels was observed in frontal cortex of WT and HRM and hippocampus of HRM following PHX. In addition, PHX induced significant reductions in hypoxia inducible factor-1α (HIF-1α) levels in frontal cortex and hippocampus of HRM. Although no significant effect of PHX was observed in vascular endothelial growth factor (VEGF) protein levels in frontal cortex and hippocampus of WT and HRM, serum VEGF levels were found higher in HRM following PHX. Moreover, glucocorticoid receptor (GR) protein levels were significantly lower in frontal cortex of WT and HRM and hippocampus of HRM following PHX. We found a significant reduction in serum corticosterone levels of PHX-treated WT mice. These findings suggest that future experiments addressing gene-environment interaction in schizophrenia should consider age-dependent effects of the environmental factor, in addition to the specificity of the gene of interest. PMID:26059812

  6. Abnormal corneal epithelial maintenance in mice heterozygous for the micropinna microphthalmia mutation Mp.

    PubMed

    Douvaras, Panagiotis; Dorà, Natalie J; Mort, Richard L; Lodge, Emily J; Hill, Robert E; West, John D

    2016-08-01

    We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2(MP) protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2(MP) protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice. PMID:27235794

  7. Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

    PubMed

    Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

    2001-10-01

    Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These

  8. Developmental Divergence of Sleep-Wake Patterns in Orexin Knockout and Wild-Type Mice

    PubMed Central

    Coleman, Cassandra M.; Johnson, Eric D.; Shaw, Cynthia

    2008-01-01

    Narcolepsy, a disorder characterized by fragmented bouts of sleep and wakefulness during the day and night as well as cataplexy, has been linked in humans and non-human animals to the functional integrity of the orexinergic system. Adult orexin knockout mice and dogs with a mutation of the orexin receptor exhibit symptoms that mirror those seen in narcoleptic humans. As with narcolepsy, infant sleep-wake cycles in humans and rats are highly fragmented, with consolidated bouts of sleep and wakefulness developing gradually. Based on these common features of narcoleptics and infants, we hypothesized that the development of sleep-wake fragmentation in orexin knockout mice would be expressed as a developmental divergence between knockouts and wild-types, with the knockouts lagging behind the wild-types. We tested this hypothesis by recording the sleep-wake patterns of infant orexin knockout and wild-type mice across the first three postnatal weeks. Both knockouts and wild-types exhibited age-dependent, and therefore orexin-independent, quantitative and qualitative changes in sleep-wake patterning. At 3 weeks of age, however, by which time the sleep and wake bouts of the wild-types had consolidated further, the knockouts lagged behind the wild-types and exhibited significantly more bout fragmentation. These findings suggest the possibility that the fragmentation of behavioral states that characterizes narcolepsy in adults reflects reversion back toward the more fragmented sleep-wake patterns that characterize infancy. PMID:17284193

  9. PROXIMAL GUT MUCOSAL EPITHELIAL HOMEOSTASIS IN AGED IL-1 TYPE I RECEPTOR KNOCKOUT MICE AFTER STARVATION

    PubMed Central

    Song, Juquan; Wolf, Steven E.; Wu, Xiao-Wu; Finnerty, Celeste C.; Herndon, David N.; Jeschke, Marc G.

    2010-01-01

    Background Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. Materials and Methods Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. Results Aged IL-1R knockout mice were larger than aged-matched wild-type mice (p<0.05). Proximal gut mucosal height and mucosal cell number were not different between aged IL-1R knockout and wild-type groups. The apoptosis index in gut epithelial cells was higher in fed IL-1R knockout versus wild-type mice (p<0.05), while no significant difference in cell proliferation between both groups. Mucosal atrophy was induced in both aged IL-1R knockout and wild-type groups by starvation (p<0.05), however, aged IL-1R knockout mice experienced greater losses in proximal gut weight, mucosal length, and corresponding cell number than did wild-type mice at the 12-hour time point (p<0.05). The apoptosis index in gut epithelial cells significantly increased in both groups after starvation (p<0.05). Starvation decreased cell proliferation in IL-1R knockout mice (p<0.05), but not in wild-type mice. Conclusions The response in aged IL-1R knockout mice differs from wild-type mice in that starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis. PMID:20605606

  10. Alpha-Ca2+/calmodulin-dependent protein kinase II contributes to the developmental programming of anxiety in serotonin receptor 1A knock-out mice.

    PubMed

    Lo Iacono, Luisa; Gross, Cornelius

    2008-06-11

    Mice lacking the serotonin receptor 1A [Htr1aknock-out (Htr1a(KO))] display increased innate and conditioned anxiety-related behavior. Expression of the receptor in the mouse forebrain during development is sufficient to restore normal anxiety-related behavior to knock-out mice, demonstrating a role for serotonin in the developmental programming of anxiety circuits. However, the precise developmental period as well as the signaling pathways and neural substrates involved in this phenomenon are unknown. Here, we show that pharmacological blockade of the receptor from postnatal day 13 (P13)-P34 is sufficient to reproduce the knock-out phenotype in adulthood, thus defining a role for serotonin in the maturation and refinement of anxiety circuits during a limited postnatal period. Furthermore, we identify increases in the phosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) at threonine 286 in the hippocampus of young Htr1a(KO) mice under anxiety-provoking conditions. Increases in alphaCaMKII phosphorylation were most pronounced in the CA1 region of the hippocampus and were localized to the extrasynaptic compartment, consistent with a tissue-specific effect of the receptor. No changes in alphaCaMKII phosphorylation were found in adult knock-out mice, suggesting a transient role of alphaCaMKII as a downstream target of the receptor. Finally, the anxiety phenotype was abolished when knock-out mice were crossed to mice in which alphaCaMKII phosphorylation was compromised by the heterozygous mutation of threonine 286 into alanine. These findings suggest that modulation of alphaCaMKII function by serotonin during a restricted postnatal period contributes to the developmental programming of anxiety-related behavior. PMID:18550767

  11. MR histology of advanced atherosclerotic lesions of ApoE- knockout mice

    NASA Astrophysics Data System (ADS)

    Naumova, A.; Yarnykh, V.; Ferguson, M.; Rosenfeld, M.; Yuan, C.

    2016-02-01

    The purposes of this study were to examine the feasibility of determining the composition of advanced atherosclerotic plaques in fixed ApoE-knockout mice and to develop a time-efficient microimaging protocol for MR histological imaging on mice. Five formalin-fixed transgenic ApoE-knockout mice were imaged at the 9.4T Bruker BioSpec MR scanner using 3D spoiled gradient-echo sequence with an isotropic field of view of 24 mm3; TR 20.8 ms; TE 2.6 ms; flip angle 20°, resulted voxel size 47 × 63 × 94 pm3. MRI examination has shown that advanced atherosclerotic lesions of aorta, innominate and carotid arteries in ApoE-knockout mice are characterized by high calcification and presence of the large fibrofatty nodules. MRI quantification of atherosclerotic lesion components corresponded to histological assessment of plaque composition with a correlation coefficient of 0.98.

  12. A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice.

    PubMed

    Ren, S; Li, M; Cai, H; Hudgins, S; Furth, P A

    2001-03-01

    Polymerase chain reaction (PCR) amplification of DNA is the most widely used technique for screening of large numbers of genetically engineered transgenic or knockout mice (Mus musculus). In this report, we present a new DNA preparation procedure for running diagnostic PCR. In this procedure, mouse ear tissue was used directly for PCR after the tissue underwent brief digestion in a solution containing only proteinase K. Using this method, we have successfully screened several lines of single, double, and triple transgenic and knockout mice. The results are reliable and reproducible. The advantage of this new method is that DNA purification by organic extraction or isolation kit was omitted. DNA purification is the limiting factor in terms of time and money when screening transgenic and knockout mice by PCR. In addition, using ear instead of tail tissue can reduce distress of animals because the samples can be obtained when the mice are labeled by ear punch.

  13. Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice

    PubMed Central

    Bosse, Kelly E.; Mathews, Tiffany A.

    2010-01-01

    Drugs of abuse like ethanol have the ability to stimulate forebrain dopaminergic pathways. Although the positive reinforcing properties of abused substances are largely attributed to their effects on dopamine transmission, alcohol addiction involves complex interactions between numerous molecular mediators. Brain-derived neurotrophic factor (BDNF) is suggested to have a protective role in regulating the reinforcing effects of ethanol. In the present study, we evaluated the effects of an acute, systemic injection of ethanol (2 g/kg) on BDNF protein levels and extracellular dopamine concentrations, measured by in vivo microdialysis, in the caudate-putamen of wildtype and heterozygous BDNF mice. In both genotypes, the peak increase in extracellular dopamine following ethanol coincided temporally with a decrease in BDNF protein levels following a similar ethanol treatment. Moreover, the effect of ethanol to increase extracellular dopamine was blunted in heterozygous BDNF mice compared to wildtype mice. While the magnitude of decrease in BDNF protein induced by ethanol was similar between genotypes (two-fold), ethanol treatment induced significantly lower BDNF protein levels in heterozygous BDNF mice overall. These findings suggest the effects of ethanol are influenced by an interaction between BDNF and dopamine transmission, which may relate to the pathway through which BDNF regulates ethanol intake. PMID:21163332

  14. Cysteamine treatment ameliorates alterations in GAD67 expression and spatial memory in heterozygous reeler mice.

    PubMed

    Kutiyanawalla, Ammar; Promsote, Wanwisa; Terry, Alvin; Pillai, Anilkumar

    2012-09-01

    Brain-derived neurotrophic factor (BDNF) signalling through its receptor, TrkB is known to regulate GABAergic function and glutamic acid decarboxylase (GAD) 67 expression in neurons. Alterations in BDNF signalling have been implicated in the pathophysiology of schizophrenia and as a result, they are a potential therapeutic target. Interestingly, heterozygous reeler mice (HRM) have decreased GAD67 expression in the frontal cortex and hippocampus and they exhibit many behavioural and neurochemical abnormalities similar to schizophrenia. In this study, we evaluated the potential of cysteamine, a neuroprotective compound to improve the deficits in GAD67 expression and cognitive function in HRM. We found that cysteamine administration (150 mg/kg.d, through drinking water) for 30 d significantly ameliorated the decreases in GAD67, mature BDNF and full-length TrkB protein levels found in frontal cortex and hippocampus of HRM. A significant attenuation of the increased levels of truncated BDNF in frontal cortex and hippocampus, as well as truncated TrkB in frontal cortex of HRM was also observed following cysteamine treatment. In behavioural studies, HRM were impaired in a Y-maze spatial recognition memory task, but not in a spontaneous alternation task or a sensorimotor, prepulse inhibition (PPI) procedure. Cysteamine improved Y-maze spatial recognition in HRM to the level of wide-type controls and it improved PPI in both wild-type and HRM. Finally, mice deficient in TrkB, showed a reduced response to cysteamine in GAD67 expression suggesting that TrkB signalling plays an important role in GAD67 regulation by cysteamine.

  15. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

    PubMed

    Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

    2015-06-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

  16. Narp knockout mice show normal reactivity to novelty but attenuated recovery from neophobia.

    PubMed

    Blouin, Ashley M; Lee, Jongah J; Tao, Bo; Smith, Dani R; Johnson, Alexander W; Baraban, Jay M; Reti, Irving M

    2013-11-15

    Narp knockout (KO) mice demonstrate cognitive inflexibility and addictive behavior, which are associated with abnormal reactivity to a novel stimulus. To assess reactivity to novelty, we tested Narp KO and wild-type (WT) mice on a neophobia procedure. Both Narp KO and WT mice showed a similar decrease in consumption upon initial exposure to a novel flavor, but Narp KO mice did not increase consumption with subsequent exposures to the novel flavor like the WT mice. Therefore, Narp KO mice do not have abnormal reactivity to novelty but show deficits in adapting behavior to reflect the updated value of a stimulus.

  17. Maltodextrin and fat preference deficits in "taste-blind" P2X2/P2X3 knockout mice.

    PubMed

    Sclafani, Anthony; Ackroff, Karen

    2014-07-01

    Adenosine triphosphate is a critical neurotransmitter in the gustatory response to the 5 primary tastes in mice. Genetic deletion of the purinergic P2X2/P2X3 receptor greatly reduces the neural and behavioral response to prototypical primary taste stimuli. In this study, we examined the behavioral response of P2X double knockout mice to maltodextrin and fat stimuli, which appear to activate additional taste channels. P2X double knockout and wild-type mice were given 24-h choice tests (vs. water) with ascending concentrations of Polycose and Intralipid. In Experiment 1, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.5-4%) Polycose solutions but preferred concentrated (8-32%) Polycose to water. In a retest, the Polycose-experienced double knockout mice, like wild-type mice, preferred all Polycose concentrations. In Experiment 2, naive double knockout mice, unlike wild-type mice, were indifferent to dilute (0.313-2.5%) Intralipid emulsions but preferred concentrated (5-20%) Intralipid to water. In a retest, the fat-experienced double knockout mice, like wild-type mice, strongly preferred 0.313-5% Intralipid to water. These results indicate that the inherent preferences of mice for maltodextrin and fat are dependent upon adenosine triphosphate taste cell signaling. With experience, however, P2X double knockout mice develop strong preferences for the nontaste flavor qualities of maltodextrin and fat conditioned by the postoral actions of these nutrients.

  18. Transgenic knockout mice with exclusively human sickle hemoglobinand sickle cell disease

    SciTech Connect

    Paszty, C.; Brion, C.; Manci, E.; Witkowska, E.; Stevens, M.; Narla, M.; Rubin, E.

    1997-06-13

    To create mice expressing exclusively human sicklehemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, andbeta[S]-globin were generated and bred with knockout mice that haddeletions of the murine alpha- and beta-globin genes. These sickle cellmice have the major features (irreversibly sickled red cells, anemia,multiorgan pathology) found in humans with sickle cell disease and, assuch, represent a useful in vivo system to accelerate the development ofimproved therapies for this common genetic disease.

  19. Interleukin-10 Deficiency Increases Atherosclerosis, Thrombosis, and Low-density Lipoproteins in Apolipoprotein E Knockout Mice

    PubMed Central

    Caligiuri, Giuseppina; Rudling, Mats; Ollivier, Véronique; Jacob, Marie-Paule; Michel, Jean-Baptiste; Hansson, Göran K; Nicoletti, Antonino

    2003-01-01

    Interleukin (IL)-10 is an anti-inflammatory cytokine that may play a protective role in atherosclerosis. The aim of this study was to assess the effect of IL-10 deficiency in the apolipoprotein E knockout mouse. Apolipoprotein E deficient (E−/−) and IL-10 deficient (−/−) mice were crossed to generate E−/− × IL-10−/− double knockout mice. By 16 wk, cholesterol and triglycerides were similar in double and single knockouts but the lack of IL-10 led to increased low-density lipoprotein cholesterol whereas very-low-density lipoprotein was reduced. In parallel, T-helper 1 responses and lesion size were dramatically increased in double knockout compared with E−/− controls. At 48 wk, matrix metalloproteinases and tissue factor activities were increased in lesions of double-knockout mice. Furthermore, markers of systemic coagulation were increased, and vascular thrombosis in response to i.v. thrombin occurred more frequently in E−/− × IL-10−/− than in E−/− mice. Our findings suggest that IL-10 deficiency plays a deleterious role in atherosclerosis. The early phase of lesion development was increased, and the proteolytic and procoagulant activity was elevated in advanced lesions. These data show that IL-10 may reduce atherogenesis and improve the stability of plaques. PMID:12765335

  20. Reproductive toxicity of ethylene glycol monoethyl ether in Aldh2 knockout mice.

    PubMed

    Wang, Rui-Sheng; Ohtani, Katsumi; Suda, Megumi; Kitagawa, Kyoko; Nakayama, Keiichi; Kawamoto, Toshihiro; Nakajima, Tamie

    2007-08-01

    Ethylene glycol monoethyl ether (EGEE) can cause damage to testes and sperm, and its metabolites are believed to play an important role in its toxicity. Aldehyde dehydrogenase 2 (ALDH2) is involved in the metabolism of this chemical. To investigate whether and how the enzyme affects the toxicity of EGEE, we conducted experiments comparing Aldh2 knockout mice with wild-type mice. Administration of EGEE at 100 and 600 mg/kg/day for one week did not induce any significant change in the weight and body weight ratios of testes, prostate and epididymides in either Aldh2 knockout or wild-type mice. However, motion of sperm from the spermaduct, as analyzed with a Hamilton-Thorne Sperm analyzer, was slightly decreased in the low dose group, and significantly lower in the high dose group; and the percentage of progressive sperm was also reduced in the two EGEE groups. This effect of EGEE treatment was observed in the wild-type, but not in the Aldh2 knockout mice. Sperm motion from the cauda epididymides was not affected. On the other hand, the concentration of ethoxyacetic acid, a metabolite of EGEE, in 24 h pooled urine of EGEE-treated Aldh2 knockout mice was not significantly lower than that of the wild-type mice on most days of urine sampling. These results suggest that inactivation of the ALDH2 enzyme due to gene mutation may be linked to differences in the susceptibility to EGEE-induced sperm toxicity. PMID:17878629

  1. The spatial learning phenotype of heterozygous leaner mice is robust to systematic variation of the housing environment.

    PubMed

    Marques, Joana M; Alonso, Isabel; Santos, Cristina; Silveira, Isabel; Olsson, I Anna S

    2009-04-01

    Providing stimulation and allowing the performance of motivated behaviors through environmental enrichment improves learning and memory in rodents and delays cognitive impairment in neurodegenerative disease models. The leaner mutation affects the Ca(v)2.1 voltage-gated calcium channel alpha(1A)-subunit gene, and homozygous mice show severe phenotypic alterations. Although several authors have described heterozygous mice as normal, recent studies in our laboratory indicate motor and cognitive impairment in tg(la)/+ mice. In the present study, we evaluated whether this impairment is robust to systematic variation of the housing environment from barren to standard and furnished (enriched) cages. Wildtype (n = 55) and tg(la)/+ (n = 79) C57Bl/6J mice were assigned randomly to 1 of the 3 housing systems and tested on the Morris water maze at 6, 12, and 20 mo of age. The results confirmed impaired performance in tg(la)/+ mice, particularly in older mice. At 12 and 20 mo, only wildtype (and not tg(la)/+) mice showed evidence of learning (spending increased time in the target quadrant) during the probe trial. Housing also affected performance: at 12 mo, only mice from furnished cages showed evidence of learning, and in aged mice (20 mo), only those housed in more complex environments showed long-term memory (8 mo after previous testing) of the platform position. In conclusion, a heterozygous mutation in a Ca(2+) channel gene causes cognitive deficits in leaner mice that are robust to environmental variation but attenuated by physical and behavioral stimulation.

  2. Heterozygous deletion of ventral anterior homeobox (vax1) causes subfertility in mice.

    PubMed

    Hoffmann, Hanne M; Tamrazian, Anika; Xie, Huimin; Pérez-Millán, María Inés; Kauffman, Alexander S; Mellon, Pamela L

    2014-10-01

    The known genetic causes of idiopathic hypogonadotropic hypogonadism (IHH) are often associated with the loss of GnRH neurons, leading to the disruption of the hypothalamic pituitary gonadal axis and subfertility. The majority of IHH cases have unknown origins and likely arise from compound mutations in more than one gene. Here we identify the homeodomain transcription factor ventral anterior homeobox1 (Vax1) as a potential genetic contributor to polygenic IHH. Although otherwise healthy, male and female Vax1 heterozygous (HET) mice are subfertile, indicating dosage sensitivity for the Vax1 allele. Although Vax1 mRNA is expressed in the pituitary, hypothalamus, and testis, we did not detect Vax1 mRNA in the sperm, ovary, or isolated pituitary gonadotropes. Whereas Vax1 HET females produced normal numbers of superovulated oocytes, corpora lutea numbers were reduced along with a slight increase in circulating basal LH and estrogen. The subfertility originated in the hypothalamus in which kisspeptin and GnRH transcripts were altered along with a substantial reduction of GnRH neuron number. Although the pituitary responded normally to a GnRH challenge, diestrus females had reduced LHβ and FSHβ in diestrus. Furthermore, Vax1 HET males had reduced GnRH mRNA and neuron numbers, whereas the pituitary had normal transcript levels and response to GnRH. Interestingly, the Vax1 HET males had an 88% reduction of motile sperm. Taken together, our data suggest that Vax1 HET subfertility originates in the hypothalamus by disrupting the hypothalamic-pituitary-gonadal axis. In addition, male subfertility may also be due to an unknown effect of Vax1 in the testis.

  3. Lack of self-administration of cocaine in dopamine D1 receptor knock-out mice.

    PubMed

    Caine, S Barak; Thomsen, Morgane; Gabriel, Kara I; Berkowitz, Jill S; Gold, Lisa H; Koob, George F; Tonegawa, Susumu; Zhang, Jianhua; Xu, Ming

    2007-11-28

    Evidence suggests a critical role for dopamine in the reinforcing effects of cocaine in rats and primates. However, self-administration has been less often studied in the mouse species, and, to date, "knock-out" of individual dopamine-related genes in mice has not been reported to reduce the reinforcing effects of cocaine. We studied the dopamine D1 receptor and cocaine self-administration in mice using a combination of gene-targeted mutation and pharmacological tools. Two cohorts with varied breeding and experimental histories were tested, and, in both cohorts, there was a significant decrease in the number of D1 receptor knock-out mice that met criteria for acquisition of cocaine self-administration (2 of 23) relative to wild-type mice (27 of 32). After extinction of responding with saline self-administration, dose-response studies showed that cocaine reliably and dose dependently maintained responding greater than saline in all wild-type mice but in none of the D1 receptor knock-out mice. The D1-like agonist SKF 82958 (2,3,4,5,-tetrahydro-6-chloro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine hydrobromide) and the D2-like agonist quinelorane both functioned as positive reinforcers in wild-type mice but not in D1 receptor mutant mice, whereas food and intravenous injections of the opioid agonist remifentanil functioned as positive reinforcers in both genotypes. Finally, pretreatment with the D1-like antagonist SCH 23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-01] produced surmountable antagonism of the reinforcing effects of cocaine in the commonly used strain C57BL/6J. We conclude that D1 receptor knock-out mice do not reliably self-administer cocaine and that the D1 receptor is critical for the reinforcing effects of cocaine and other dopamine agonists, but not food or opioids, in mice.

  4. Simultaneous generation of fra-2 conditional and fra-2 knock-out mice.

    PubMed

    Eferl, Robert; Zenz, Rainer; Theussl, Hans-Christian; Wagner, Erwin F

    2007-07-01

    Loss of function mouse models comprise knock-out mice, where a gene is deleted in the germline, and conditional knock-out mice with somatic deletion of a floxed allele in defined tissues. Both types of mice are used for comprehensive studies of gene functions in vivo. Here, we describe a simple method for simultaneous generation of mice with conditional or knock-out alleles for the transcription factor fra-2 (Fos-related antigen 2) using a single embryonic stem (ES) cell clone. ES cells with a floxed fra-2 allele were transiently transfected with a Cre-recombinase expression plasmid and plated at low density. Most of the resulting ES cell colonies consisted of a mixture of cells that have either retained or lost the conditional allele. We demonstrate that these mixed ES cell clones can be directly used for generation of chimeras that give rise to offspring with conditional or knock-out alleles simultaneously. This strategy shortens the time and reduces the number of germline transmission events to generate genetically modified mice.

  5. True Niacin Deficiency in Quinolinic Acid Phosphoribosyltransferase (QPRT) Knockout Mice.

    PubMed

    Shibata, Katsumi

    2015-01-01

    Pyridine nucleotide coenzymes (PNCs) are involved in over 500 enzyme reactions. PNCs are biosynthesized from the amino acid L-tryptophan (L-Trp), as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-) or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and the qprt(+/-) mice. On the other hand, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (p<0.01) and 70% (p<0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at day 23, respectively. The nutritional levels of niacin such as blood and liver NAD concentrations were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of quinolinic acid was greater in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice (p<0.01). These data suggest that we generated truly niacin-deficient mice. PMID:26598832

  6. True Niacin Deficiency in Quinolinic Acid Phosphoribosyltransferase (QPRT) Knockout Mice.

    PubMed

    Shibata, Katsumi

    2015-01-01

    Pyridine nucleotide coenzymes (PNCs) are involved in over 500 enzyme reactions. PNCs are biosynthesized from the amino acid L-tryptophan (L-Trp), as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-) or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and the qprt(+/-) mice. On the other hand, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (p<0.01) and 70% (p<0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at day 23, respectively. The nutritional levels of niacin such as blood and liver NAD concentrations were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of quinolinic acid was greater in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice (p<0.01). These data suggest that we generated truly niacin-deficient mice.

  7. Generation and behavioral characterization of beta-catenin forebrain-specific conditional knock-out mice.

    PubMed

    Gould, Todd D; O'Donnell, Kelley C; Picchini, Alyssa M; Dow, Eliot R; Chen, Guang; Manji, Husseini K

    2008-05-16

    The canonical Wnt pathway and beta-catenin have been implicated in the pathophysiology of mood disorders. We generated forebrain-specific CRE-mediated conditional beta-catenin knock-out mice to begin exploring the behavioral implications of decreased Wnt pathway signaling in the central nervous system. In situ hybridization revealed a progressive knock-out of beta-catenin that began between 2 and 4 weeks of age, and by 12 weeks resulted in considerably decreased beta-catenin expression in regions of the forebrain, including the frontal cortex, hippocampus, and striatum. A significant decrease in protein levels of beta-catenin in these brain regions was observed by Western blot. Behavioral characterization of these mice in several tests (including the forced swim test, tail suspension test (TST), learned helplessness, response and sensitization to stimulants, and light/dark box among other tests) revealed relatively circumscribed alterations. In the TST, knock-out mice spent significantly less time struggling (a depression-like phenotype). However, knock-out mice did not differ from their wild-type littermates in the other behavioral tests of mood-related or anxiety-related behaviors. These results suggest that a 60-70% beta-catenin reduction in circumscribed brain regions is only capable of inducing subtle behavioral changes. Alternatively, regulating beta-catenin may modulate drug effects rather than being a model of mood disorder pathophysiology per se.

  8. Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice

    PubMed Central

    Jeoung, Nam Ho; Rahimi, Yasmeen; Wu, Pengfei; Lee, W. N. Paul; Harris, Robert A.

    2015-01-01

    The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4. PMID:22360721

  9. SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS

    EPA Science Inventory

    SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS. L.F. Strader*, S.D. Perreault, J.C. Luft*, and D.J. Dix*. US EPA/ORD, Reproductive Toxicology Div., Research Triangle Park, NC
    Heat shock proteins (HSPs) protect cells from environm...

  10. Kv4.2 Knockout Mice Have Hippocampal-Dependent Learning and Memory Deficits

    ERIC Educational Resources Information Center

    Lugo, Joaquin N.; Brewster, Amy L.; Spencer, Corinne M.; Anderson, Anne E.

    2012-01-01

    Kv4.2 channels contribute to the transient, outward K[superscript +] current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit…

  11. Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice.

    PubMed

    Slätis, Katharina; Gåfvels, Mats; Kannisto, Kristina; Ovchinnikova, Olga; Paulsson-Berne, Gabrielle; Parini, Paolo; Jiang, Zhao-Yan; Eggertsen, Gösta

    2010-11-01

    To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

  12. SAMHD1 knockout mice: modeling retrovirus restriction in vivo.

    PubMed

    Wu, Li

    2013-11-20

    The host dNTP hydrolase SAMHD1 acts as a viral restriction factor to inhibit the replication of several retroviruses and DNA viruses in non-cycling human immune cells. However, understanding the physiological role of mammalian SAMHD1 has been elusive due to the lack of an animal model. Two recent studies reported the generation of samhd1 knockout mouse models for investigating the restriction of HIV-1 vectors and endogenous retroviruses in vivo. Both studies suggest that SAMHD1 is important for regulating the intracellular dNTP pool and the intrinsic immunity against retroviral infection, despite different outcomes of HIV-1 vector transduction in these mouse models. Here I discuss the significance of these new findings and the future directions in studying SAMHD1-mediated retroviral restriction.

  13. Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

    SciTech Connect

    Antonson, P.; Omoto, Y.; Humire, P.; Gustafsson, J.-A.

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established a new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

  14. Somatostatin Receptor 1 and 5 Double Knockout Mice Mimic Neurochemical Changes of Huntington's Disease Transgenic Mice

    PubMed Central

    Rajput, Padmesh S.; Kharmate, Geetanjali; Norman, Michael; Liu, Shi-He; Sastry, Bhagavatula R.; Brunicardi, Charles F.; Kumar, Ujendra

    2011-01-01

    Background Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD). However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST) positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5). Methods and Findings To delineate subtype selective biological responses we have here investigated changes in SSTR1 and 5 double knockout mice brain and compared with HD transgenic mouse model (R6/2). Our study revealed significant loss of dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32) and comparable changes in SST, N-methyl-D-aspartic acid receptors subtypes, calbindin and brain nitric oxide synthase expression as well as in key signaling proteins including calpain, phospho-extracellular-signal-regulated kinases1/2, synapsin-IIa, protein kinase C-α and calcineurin in SSTR1/5−/− and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice. Conclusions This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of

  15. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

    PubMed

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-05-19

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders.

  16. Behavioral characterization of striatal-enriched protein tyrosine phosphatase (STEP) knockout mice.

    PubMed

    Sukoff Rizzo, S J; Lotarski, S M; Stolyar, P; McNally, T; Arturi, C; Roos, M; Finley, J E; Reinhart, V; Lanz, T A

    2014-09-01

    Striatal-enriched protein tyrosine phosphatase (STEP) has been described as a regulator of multiple kinases and glutamate receptor subunits critical for synaptic plasticity. Published behavioral and biochemical characterization from the founder line of STEP knockout (KO) mice revealed superior cognitive performance, with enhanced phosphorylation of substrates such as ERK, Fyn and GluN2B; suggesting that inhibitors of STEP may have potential as therapeutic agents for the treatment of neuropsychiatric disorders. The objectives of this work aimed to replicate and extend the previously reported behavioral consequences of STEP knockout. Consistent with previous reported data, STEP KO mice demonstrated exploratory activity levels and similar motor coordination relative to WT littermate controls as well as intact memory in a Y-maze spatial novelty test. Interestingly, KO mice demonstrated deficits in pre-pulse inhibition as well as reduced seizure threshold relative to WT controls. Immunohistochemical staining of brains revealed the expected gene-dependent reduction in STEP protein confirming knockout in the mice. The present data confirm expression and localization of STEP and the absence in KO mice, and describe functional downstream implications of reducing STEP levels in vivo.

  17. Memory formation and retention are affected in adult miR-132/212 knockout mice.

    PubMed

    Hernandez-Rapp, Julia; Smith, Pascal Y; Filali, Mohammed; Goupil, Claudia; Planel, Emmanuel; Magill, Stephen T; Goodman, Richard H; Hébert, Sébastien S

    2015-01-01

    The miR-132/212 family is thought to play an important role in neural function and plasticity, while its misregulation has been observed in various neurodegenerative disorders. In this study, we analyzed 6-month-old miR-132/212 knockout mice in a battery of cognitive and non-cognitive behavioral tests. No significant changes were observed in reflexes and basic sensorimotor functions as determined by the SHIRPA primary screen. Accordingly, miR-132/212 knockout mice did not differ from wild-type controls in general locomotor activity in an open-field test. Furthermore, no significant changes of anxiety were measured in an elevated plus maze task. However, the mutant mice showed retention phase defects in a novel object recognition test and in the T-water maze. Moreover, the learning and probe phases in the Barnes maze were clearly altered in knockout mice when compared to controls. Finally, changes in BDNF, CREB, and MeCP2 were identified in the miR-132/212-deficient mice, providing a potential mechanism for promoting memory loss. Taken together, these results further strengthen the role of miR-132/212 in memory formation and retention, and shed light on the potential consequences of its deregulation in neurodegenerative diseases.

  18. Axonal and Periaxonal Swelling Precede Peripheral Neurodegeneration in KCC3 Knockout Mice

    PubMed Central

    Byun, Nellie; Delpire, Eric

    2007-01-01

    We have previously reported CNS and locomotor deficits in KCC3 knockout mice, an animal model of agenesis of the corpus callosum associated with peripheral neuropathy (ACCPN) (Howard, et al., 2002)). To assess the role of KCC3 in peripheral axon and/or myelin development and maintenance, we determined its expression and performed a detailed morphometric analysis of sciatic nerves. Sciatic nerves of juvenile wild-type mice, but not in adult, express KCC3. In the knockout, Schwann cell/myelin development appears normal at P3, but axons are swollen. At P8 and into P30, some fibers accumulate fluid periaxonally. These initial swelling pathologies are followed by myelin degeneration in adult nerves, leading to reduction in nerve conduction velocity. Mutant mice also exhibit decreased sensitivity to noxious pain. This evidence for swollen axons and fluid-related axonopathy, which ultimately result in neurodegeneration, implicates cell volume regulation as a critical component of peripheral nerve maintenance. PMID:17659877

  19. Defective excitation-contraction coupling is partially responsible for impaired contractility in hindlimb muscles of Stac3 knockout mice

    PubMed Central

    Cong, Xiaofei; Doering, Jonathan; Grange, Robert W.; Jiang, Honglin

    2016-01-01

    The Stac3 gene is exclusively expressed in skeletal muscle, and Stac3 knockout is perinatal lethal in mice. Previous data from Stac3-deleted diaphragms indicated that Stac3-deleted skeletal muscle could not contract because of defective excitation-contraction (EC) coupling. In this study, we determined the contractility of Stac3-deleted hindlimb muscle. In response to frequent electrostimulation, Stac3-deleted hindlimb muscle contracted but the maximal tension generated was only 20% of that in control (wild type or heterozygous) muscle (P < 0.05). In response to high [K+], caffeine, and 4-chloro-m-cresol (4-CMC), the maximal tensions generated in Stac3-deleted muscle were 29% (P < 0.05), 58% (P = 0.08), and 55% (P < 0.05) of those in control muscle, respectively. In response to 4-CMC or caffeine, over 90% of myotubes formed from control myoblasts contracted, but only 60% of myotubes formed from Stac3-deleted myoblasts contracted (P = 0.05). However, in response to 4-CMC or caffeine, similar increases in intracellular calcium concentration were observed in Stac3-deleted and control myotubes. Gene expression and histological analyses revealed that Stac3-deleted hindlimb muscle contained more slow type-like fibers than control muscle. These data together confirm a critical role of STAC3 in EC coupling but also suggest that STAC3 may have additional functions in skeletal muscle, at least in the hindlimb muscle. PMID:27184118

  20. Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

    PubMed

    Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

    2015-05-01

    Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications.

  1. Deficits of learning and memory in Hemojuvelin knockout mice.

    PubMed

    Li, Jinglong; Zhang, Peng; Liu, Hongju; Ren, Wei; Song, Jinjing; Rao, Elizabeth; Takahashi, Eiki; Zhou, Ying; Li, Weidong; Chen, Xiaoping

    2015-10-01

    Iron is involved in various physiological processes of the human body to maintain normal functions. Abnormal iron accumulation in brain has been reported as a pathogenesis of several neurodegenerative disorders and cognitive impairments. Hemojuvelin (HVJ) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis. Although iron accumulation in brain has been related to neurodegenerative diseases, it remains unknown the effect of mutation of HVJ gene on cognitive performance. In our studies, HJV(-/-) mice showed deficits in novel object recognition and Morris water maze tests. Furthermore, the expression ration of apoptotic marker Bax and anti-apoptotic marker Bcl-2 in the hippocampus and prefrontal cortex showed higher levels in HJV(-/-) mice. Our results suggested that deletion of HJV gene could increase apoptosis in brain which might contribute to learning and memory deficits in mutant mice. These results indicated that HJV(-/-) mice would be a useful model to study cognitive impairment induced by iron overload in brain.

  2. Tumor suppressor gene co-operativity in compound Patched1 and Suppressor of fused heterozygous mutant mice

    PubMed Central

    Svärd, Jessica; Rozell, Björn; Toftgård, Rune; Teglund, Stephan

    2008-01-01

    Dysregulation of the Hedgehog signaling pathway is central to the development of certain tumor types, including medulloblastoma and basal cell carcinoma (BCC). Patched1 (Ptch1) and Suppressor of fused (Sufu) are two essential negative regulators of the pathway with tumor suppressor activity. Ptch1+/− mice are predisposed to developing medulloblastoma and rhabdomyosarcoma, while Sufu+/− mice develop a skin phenotype characterized by basaloid epidermal proliferations. Here, we have studied tumor development in Sufu+/−Ptch1+/− mice to determine the effect of compound heterozygosity on the onset, incidence, and spectrum of tumors. We found significantly more (2.3-fold) basaloid proliferations in Sufu+/−Ptch1+/− compared to Sufu+/− female, but not male, mice. For medulloblastoma, the cumulative one-year incidence was 1.5-fold higher in Sufu+/−Ptch1+/− compared to Ptch1+/− female mice but this strong trend was not statistically significant. Together this suggests a weak genetic interaction of the two tumor suppressor genes. We noted a few rhabdomyosarcomas and pancreatic cysts in the Sufu+/−Ptch1+/− mice, but the numbers were not significantly different from the single heterozygous mice. Hydrocephalus developed in ∼20% of the Ptch1+/− and Sufu+/−Ptch1+/− but not in Sufu+/− mice. Interestingly, most of the medulloblastomas from the Sufu+/−Ptch1+/− mice had lost expression of the remaining Ptch1 wild-type allele but not the Sufu wild-type allele. On the contrary, Sufu as well as Gli1 and Gli2 expression was upregulated in the medulloblastomas compared to adult cerebellum in Ptch1+/− and Sufu+/−Ptch1+/− mice. This suggests that Sufu expression may be regulated by Hedgehog pathway activity and could constitute another negative feedback loop in the pathway. PMID:18781608

  3. GnRH-Deficient Phenotypes in Humans and Mice with Heterozygous Variants in KISS1/Kiss1

    PubMed Central

    Chan, Yee-Ming; Broder-Fingert, Sarabeth; Paraschos, Sophia; Lapatto, Risto; Au, Margaret; Hughes, Virginia; Bianco, Suzy D. C.; Min, Le; Plummer, Lacey; Cerrato, Felecia; De Guillebon, Adelaide; Wu, I-Hsuan; Wahab, Fazal; Dwyer, Andrew; Kirsch, Susan; Quinton, Richard; Cheetham, Timothy; Ozata, Metin; Ten, Svetlana; Chanoine, Jean-Pierre; Pitteloud, Nelly; Martin, Kathryn A.; Schiffmann, Raphael; Van der Kamp, Hetty J.; Nader, Shahla; Hall, Janet E.; Kaiser, Ursula B.

    2011-01-01

    Context: KISS1 is a candidate gene for GnRH deficiency. Objective: Our objective was to identify deleterious mutations in KISS1. Patients and Methods: DNA sequencing and assessment of the effects of rare sequence variants (RSV) were conducted in 1025 probands with GnRH-deficient conditions. Results: Fifteen probands harbored 10 heterozygous RSV in KISS1 seen in less than 1% of control subjects. Of the variants that reside within the mature kisspeptin peptide, p.F117L (but not p.S77I, p.Q82K, p.H90D, or p.P110T) reduces inositol phosphate generation. Of the variants that lie within the coding region but outside the mature peptide, p.G35S and p.C53R (but not p.A129V) are predicted in silico to be deleterious. Of the variants that lie outside the coding region, one (g.1-3659C→T) impairs transcription in vitro, and another (c.1-7C→T) lies within the consensus Kozak sequence. Of five probands tested, four had abnormal baseline LH pulse patterns. In mice, testosterone decreases with heterozygous loss of Kiss1 and Kiss1r alleles (wild-type, 274 ± 99, to double heterozygotes, 69 ± 16 ng/dl; r2 = 0.13; P = 0.03). Kiss1/Kiss1r double-heterozygote males have shorter anogenital distances (13.0 ± 0.2 vs. 15.6 ± 0.2 mm at P34, P < 0.001), females have longer estrous cycles (7.4 ± 0.2 vs. 5.6 ± 0.2 d, P < 0.01), and mating pairs have decreased litter frequency (0.59 ± 0.09 vs. 0.71 ± 0.06 litters/month, P < 0.04) and size (3.5 ± 0.2 vs. 5.4 ± 0.3 pups/litter, P < 0.001) compared with wild-type mice. Conclusions: Deleterious, heterozygous RSV in KISS1 exist at a low frequency in GnRH-deficient patients as well as in the general population in presumably normal individuals. As in Kiss1+/−/Kiss1r+/− mice, heterozygous KISS1 variants in humans may work with other genetic and/or environmental factors to cause abnormal reproductive function. PMID:21880801

  4. Impaired Sperm Maturation in Rnase9 Knockout Mice1

    PubMed Central

    Westmuckett, Andrew D.; Nguyen, Edward B.; Herlea-Pana, Oana M.; Alvau, Antonio; Salicioni, Ana M.; Moore, Kevin L.

    2014-01-01

    ABSTRACT Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9−/− mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9−/− mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9−/− males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10–90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation. PMID:24719258

  5. Characterization of sensory deficits in TrkB knockout mice.

    PubMed

    Perez-Pinera, P; García-Suarez, O; Germanà, A; Díaz-Esnal, B; de Carlos, F; Silos-Santiago, I; del Valle, M E; Cobo, J; Vega, J A

    2008-03-01

    The sensory deficit in TrkB deficient mice was evaluated by counting the neuronal loss in lumbar dorsal root ganglia (DRG), the absence of sensory receptors (cutaneous--associated to the hairy and glabrous skin - muscular and articular), and the percentage and size of the neurocalcin-positive DRG neurons (a calcium-binding protein which labels proprioceptive and mechanoceptive neurons). Mice lacking TrkB lost 32% of neurons, corresponding to the intermediate-sized and neurocalcin-positive ones. This neuronal lost was accomplished by the absence of Meissner corpuscles, and reduction of hair follicle-associated sensory nerve endings and Merkel cells. The mutation was without effect on Pacinian corpuscles, Golgi's organs and muscle spindles. Present results further characterize the sensory deficit of the TrkB-/- mice demonstrating that the intermediate-sized neurons in lumbar DRG, as well as the cutaneous rapidly and slowly adapting sensory receptors connected to them, are under the control of TrkB for survival and differentiation. This study might serve as a baseline for future studies in experimentally induced neuropathies affecting TrkB positive DRG neurons and their peripheral targets, and to use TrkB ligands in the treatment of neuropathies in which cutaneous mechanoreceptors are primarily involved.

  6. Role of connexin 32 in acetaminophen toxicity in a knockout mice model.

    PubMed

    Igarashi, Isao; Maejima, Takanori; Kai, Kiyonori; Arakawa, Shingo; Teranishi, Munehiro; Sanbuissho, Atsushi

    2014-03-01

    Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

  7. Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

    PubMed

    Cao, Qiang; Cui, Xin; Wu, Rui; Zha, Lin; Wang, Xianfeng; Parks, John S; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-06-01

    Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6C(high) monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. PMID:26822081

  8. Bioinformatic analysis of miRNA expression patterns in TFF2 knock-out mice.

    PubMed

    Yin, Y; Shan, H Q; Huang, W; Wu, Y M; Lu, H; Jin, Y

    2014-10-20

    Trefoil factors, which bear a unique 3-loop trefoil domain, are a family of small secretory protease-resistant peptides (7-12 kDa) discovered in the 1980s. Trefoil factor 2 (TFF2) is a unique member of trefoil factors family that plays important roles in gastrointestinal mucosal defense and repair. However, few studies have characterized the miRNA expression patterns in TFF2 knock-out mice. In this study, we investigated the regulatory role of miRNAs in TFF2 knock-out mice. Whole miRNome profiling for TFF2 knock-out mice and wild-type mice were downloaded from the Gene Expression Omnibus database. A total of 14 differentially expressed miRNAs were identified using the limma package. Target genes for 2 differentially expressed miRNAs were retrieved from 2 databases. After mapping these target genes into STRING, an interaction network was constructed. Gene Ontology analysis suggested that the differentially expressed miRNAs are involved in cyclic AMP metabolism and the growth process. Additionally, dysregulated miRNAs target pathways of transforming growth factor-beta signaling pathway and cytokine-cytokine receptor interaction. Our results suggest that miRNAs may play important regulatory roles in processes involving TFF2, particularly in the regulation of signal transduction pathways. However, further validation of our results is needed.

  9. Otoancorin Knockout Mice Reveal Inertia is the Force for Hearing

    NASA Astrophysics Data System (ADS)

    Weddell, Thomas; Legan, P. Kevin; Lukashkina, Victoria A.; Goodyear, Richard J.; Welstead, Lindsy; Petit, Chistine; Russell, Ian J.; Lukashkin, Andrei N.; Richardson, Guy P.

    2011-11-01

    We demonstrate that in Otoa-/- mice, in which the inner-ear-specific protein otoancorin is absent, excitation of the outer hair cells and cochlear amplification is normal. This finding is remarkable because the tectorial membrane (TM), although remaining functionally attached to the outer hair cell bundles, is completely detached from the spiral limbus. Therefore, as in ancestral vertebrate auditory organs, where inertia provides the excitatory force to the hair cells, it is the inertia of the TM that must be important for exciting the outer hair cells, setting the sensitivity of their transducer conductance, and determining the precise timing of cochlear amplification.

  10. Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.

    PubMed

    Mehta, Jawahar L; Sanada, Nobuhito; Hu, Chang Ping; Chen, Jiawei; Dandapat, Abhijit; Sugawara, Fumiaki; Satoh, Hiroo; Inoue, Kazuhiko; Kawase, Yosuke; Jishage, Kou-ichi; Suzuki, Hiroshi; Takeya, Motohiro; Schnackenberg, Laura; Beger, Richard; Hermonat, Paul L; Thomas, Maria; Sawamura, Tatsuya

    2007-06-01

    Atherosclerosis is associated with oxidative stress and inflammation, and upregulation of LOX-1, an endothelial receptor for oxidized LDL (oxLDL). Here, we describe generation of LOX-1 knockout (KO) mice in which binding of oxLDL to aortic endothelium was reduced and endothelium-dependent vasorelaxation preserved after treatment with oxLDL (P<0.01 versus wild-type mice). To address whether endothelial functional preservation might lead to reduction in atherogenesis, we crossed LOX-1 KO mice with LDLR KO mice and fed these mice 4% cholesterol/10% cocoa butter diet for 18 weeks. Atherosclerosis was found to cover 61+/-2% of aorta in the LDLR KO mice, but only 36+/-3% of aorta in the double KO mice. Luminal obstruction and intima thickness were significantly reduced in the double KO mice (versus LDLR KO mice). Expression of redox-sensitive NF-kappaB and the inflammatory marker CD68 in LDLR KO mice was increased (P<0.01 versus wild-type mice), but not in the double KO mice. On the other hand, antiinflammatory cytokine IL-10 expression and superoxide dismutase activity were low in the LDLR KO mice (P<0.01 versus wild-type mice), but not in the double KO mice. Endothelial nitric oxide synthase expression was also preserved in the double KO mice. The proinflammatory signal MAPK P38 was activated in the LDLR KO mice, and LOX-1 deletion reduced this signal. In conclusion, LOX-1 deletion sustains endothelial function leading to a reduction in atherogenesis in association with reduction in proinflammatory and prooxidant signals. PMID:17478727

  11. Rapid phenotyping of knockout mice to identify genetic determinants of bone strength

    PubMed Central

    Freudenthal, Bernard; Logan, John; Croucher, Peter I

    2016-01-01

    The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease. PMID:27535945

  12. Rapid phenotyping of knockout mice to identify genetic determinants of bone strength.

    PubMed

    Freudenthal, Bernard; Logan, John; Croucher, Peter I; Williams, Graham R; Bassett, J H Duncan

    2016-10-01

    The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease. PMID:27535945

  13. Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

    PubMed

    Knutson, M D; Levy, J E; Andrews, N C; Wessling-Resnick, M

    2001-05-01

    Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

  14. Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

    PubMed

    Manzini, S; Pinna, C; Busnelli, M; Cinquanta, P; Rigamonti, E; Ganzetti, G S; Dellera, F; Sala, A; Calabresi, L; Franceschini, G; Parolini, C; Chiesa, G

    2015-11-01

    Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

  15. Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance.

    PubMed

    Nguyen, Xuan V; Masse, James; Kumar, Ashok; Vijitruth, Rattanavijit; Kulik, Cynthia; Liu, Mei; Choi, Dong-Young; Foster, Thomas C; Usynin, Ivan; Bakalkin, Georgy; Bing, Guoying

    2005-06-20

    Dynorphins, endogenous kappa-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin (Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13-17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3-6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

  16. CAF1-knockout mice are more susceptive to lipopolysaccharide-induced acute lung injury

    PubMed Central

    Shi, Jia-Xin; Li, Jia-Shu; Hu, Rong; Li, Xiao-Min; Wang, Hong

    2016-01-01

    The carbon catabolite repressor protein 4 (CCR4)–negative on TATA (NOT) complex includes multiple subunits and is conserved in the eukaryotic cells. The CCR4–NOT complex can regulate gene expression at different levels. Two subunits of the CCR4–NOT complex, CCR4 and CCR4-associated factor 1 (CAF1), possess deadenylase activity. In yeast, the deadenylase activity is mainly provided by the CCR4 subunit; however, the deadenylase activity is provided by both CCR4 and CAF1 in other eukaryotes. A previous study reported that CAF1 but not CCR4 is required for the decay of a reporter mRNA with AU-rich elements. Our previous study showed that CAF1 is involved in the regulation of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) expression. Both ICAM-1 and IL-8 play crucial roles in acute lung injury. In the present study, we examined the effects of CAF1 deficiency on IL-8 and ICAM-1 expression and acute lung injury in mice. Here we showed that there were no differences between the wild-type and CAF1-knockout mice on phenotypes. The lung histology and protein and mRNA levels of IL-8 and ICAM-1 in unstimulated wild-type mice were comparable to those in unstimulated CAF1-knockout mice. However, lipopolysaccharide stimulation led to more severe lung histological injury and greatly higher IL-8 and ICAM-1 expression in CAF1-knockout mice compared to the wild-type mice. These results, together with our previous study, suggest that CAF1 is involved in the regulation of lipopolysaccharide-stimulated IL-8 and ICAM-1 expression in vivo and affects the progression of acute lung injury. PMID:27358572

  17. Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

    PubMed

    Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

    2014-01-01

    Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy. PMID:25064116

  18. Peptidylarginine deiminase expression and activity in PAD2 knock-out and PAD4-low mice.

    PubMed

    van Beers, Joyce J B C; Zendman, Albert J W; Raijmakers, Reinout; Stammen-Vogelzangs, Judith; Pruijn, Ger J M

    2013-02-01

    Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2-/- mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2-/- mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.

  19. Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

    PubMed

    Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

    2014-01-01

    Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

  20. Long-term potentiation in the hippocampus of fragile X knockout mice

    SciTech Connect

    Godfraind, J.M.; Reyniers, E.; De Boulle, K.

    1996-08-09

    To gain more insight in the physiological function of the fragile X gene (FMR1) and the mechanisms leading to fragile X syndrome, the Fmr1 gene has been inactivated in mice by gene targeting techniques. In the Morris water maze test, the Fmr1 knockout mice learn to find the hidden platform nearly as well as the control animals, but show impaired performance after the position of the platform has been modified. As malperformance in the Morris water maze test has been associated with impaired long-term potentiation (LTP), electrophysiological studies were performed in hippocampal slices of Fmr1 knockout mice to check for the presence of LTP. Judged by field extracellular excitatory postsynaptic potential recordings in the CA1 hippocampal area, Fmr1 knockout mice express LTP to a similar extent as their wild type littermates during the first 1-2 hr after high frequency stimulation. Also, short-term potentiation (STP) was similar in both types of mice. To investigate whether Fmr1 is involved in the latter stages of LTP as an immediate early gene, we compared Fmr1 mRNA quantities on northern blots after chemical induction of seizures. A transient increase in the transcription of immediate early genes is thought to be essential for the maintenance of LTP. As no increase in Fmr1 mRNA could be detected, neither in cortex nor in total brain, during the first 2{1/2} hr after pentylenetetrazol-induced seizures, it is unlikely that Fmr1 is an immediate early gene in mice. In conclusion, we found no evidence for a function of FMR1 in STP or LTP. 37 refs., 4 figs.

  1. Dose-dependent mesothelioma induction by intraperitoneal administration of multi-wall carbon nanotubes in p53 heterozygous mice.

    PubMed

    Takagi, Atsuya; Hirose, Akihiko; Futakuchi, Mitsuru; Tsuda, Hiroyuki; Kanno, Jun

    2012-08-01

    Among various types of multi-wall carbon nanotubes (MWCNT) are those containing fibrous particles longer than 5 μm with an aspect ratio of more than three (i.e. dimensions similar to mesotheliomagenic asbestos). A previous study showed that micrometer-sized MWCNT (μm-MWCNT) administered intraperitoneally at a dose of 3000 μg/mouse corresponding to 1 × 10(9) fibers per mouse induced mesotheliomas in p53 heterozygous mice. Here, we report a dose-response study; three groups of p53 heterozygous mice (n = 20) were given a single intraperitoneal injection of 300 μg/mouse of μm-MWCNT (corresponding to 1 × 10(8) fibers), 30 μg/mouse (1 × 10(7)) or 3 μg/mouse (1 × 10(6)), respectively, and observed for up to 1 year. The cumulative incidence of mesotheliomas was 19/20, 17/20 and 5/20, respectively. The severity of peritoneal adhesion and granuloma formation were dose-dependent and minimal in the lowest dose group. However, the time of tumor onset was apparently independent of the dose. All mice in the lowest dose group that survived until the terminal kill had microscopic atypical mesothelial hyperplasia considered as a precursor lesion of mesothelioma. Right beneath was a mononuclear cell accumulation consisting of CD45- or CD3-positive lymphocytes and CD45/CD3-negative F4/80 faintly positive macrophages; some of the macrophages contained singular MWCNT in their cytoplasm. The lesions were devoid of epithelioid cell granuloma and fibrosis. These findings were in favor of the widely proposed mode of action of fiber carcinogenesis, that is, frustrated phagocytosis where the mesotheliomagenic microenvironment on the peritoneal surface is neither qualitatively altered by the density of the fibers per area nor by the formation of granulomas against agglomerates.

  2. Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

    PubMed Central

    Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

    2014-01-01

    Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

  3. Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

    PubMed

    Flodby, Per; Kim, Yong Ho; Beard, LaMonta L; Gao, Danping; Ji, Yanbin; Kage, Hidenori; Liebler, Janice M; Minoo, Parviz; Kim, Kwang-Jin; Borok, Zea; Crandall, Edward D

    2016-09-01

    Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung. PMID:27064541

  4. Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice.

    PubMed

    Lyon, Louisa; Kew, James N C; Corti, Corrado; Harrison, Paul J; Burnet, Philip W J

    2008-11-01

    Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice. PMID:18720515

  5. Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice.

    PubMed

    van der Merwe, Elizabeth L; Kidson, Susan H

    2016-05-01

    Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma.

  6. Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice.

    PubMed

    van der Merwe, Elizabeth L; Kidson, Susan H

    2016-05-01

    Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma. PMID:27068508

  7. Perineuronal net formation and structure in aggrecan knockout mice.

    PubMed

    Giamanco, K A; Morawski, M; Matthews, R T

    2010-11-10

    Perineuronal nets (PNNs) are specialized substructures of the neural extracellular matrix (ECM) which envelop the cell soma and proximal neurites of particular sets of neurons with apertures at sites of synaptic contact. Previous studies have shown that PNNs are enriched with chondroitin sulfate proteoglycans (CSPGs) and hyaluronan, however, a complete understanding of their precise molecular composition has been elusive. In addition, identifying which specific PNN components are critical to the formation of this structure has not been demonstrated. Previous work in our laboratory has demonstrated that the CSPG, aggrecan, is a key activity-dependent component of PNNs in vivo. In order to assess the contribution of aggrecan to PNN formation, we utilized cartilage matrix deficiency (cmd) mice, which lack aggrecan. Herein, we utilized an in vitro model, dissociated cortical culture, and an ex vivo model, organotypic slice culture, to specifically investigate the role aggrecan plays in PNN formation. Our work demonstrates that staining with the lectin, Wisteria floribunda agglutinin (WFA), considered a broad PNN marker, is eliminated in the absence of aggrecan, suggesting the loss of PNNs. However, in contrast, we found that the expression patterns of other PNN markers, including hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R, brevican, and hyaluronan are unaffected by the absence of aggrecan. Lastly, we determined that while all PNN components are bound to the surface in a hyaluronan-dependent manner, only HAPLN1 remains attached to the cell surface when neurons are treated with chondroitinase. These results suggest a different model for the molecular association of PNNs to the cell surface. Together our work has served to assess the contribution of aggrecan to PNN formation while providing key evidence concerning the molecular composition of PNNs in addition to determining how these components ultimately form PNNs.

  8. Perineuronal net formation and structure in aggrecan knockout mice.

    PubMed

    Giamanco, K A; Morawski, M; Matthews, R T

    2010-11-10

    Perineuronal nets (PNNs) are specialized substructures of the neural extracellular matrix (ECM) which envelop the cell soma and proximal neurites of particular sets of neurons with apertures at sites of synaptic contact. Previous studies have shown that PNNs are enriched with chondroitin sulfate proteoglycans (CSPGs) and hyaluronan, however, a complete understanding of their precise molecular composition has been elusive. In addition, identifying which specific PNN components are critical to the formation of this structure has not been demonstrated. Previous work in our laboratory has demonstrated that the CSPG, aggrecan, is a key activity-dependent component of PNNs in vivo. In order to assess the contribution of aggrecan to PNN formation, we utilized cartilage matrix deficiency (cmd) mice, which lack aggrecan. Herein, we utilized an in vitro model, dissociated cortical culture, and an ex vivo model, organotypic slice culture, to specifically investigate the role aggrecan plays in PNN formation. Our work demonstrates that staining with the lectin, Wisteria floribunda agglutinin (WFA), considered a broad PNN marker, is eliminated in the absence of aggrecan, suggesting the loss of PNNs. However, in contrast, we found that the expression patterns of other PNN markers, including hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R, brevican, and hyaluronan are unaffected by the absence of aggrecan. Lastly, we determined that while all PNN components are bound to the surface in a hyaluronan-dependent manner, only HAPLN1 remains attached to the cell surface when neurons are treated with chondroitinase. These results suggest a different model for the molecular association of PNNs to the cell surface. Together our work has served to assess the contribution of aggrecan to PNN formation while providing key evidence concerning the molecular composition of PNNs in addition to determining how these components ultimately form PNNs. PMID:20732394

  9. Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion.

    PubMed

    Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C; Wang, Tong

    2016-03-01

    Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule. PMID:26728465

  10. Voluntary Exercise Decreases Atherosclerosis in Nephrectomised ApoE Knockout Mice

    PubMed Central

    Shing, Cecilia M.; Fassett, Robert G.; Peake, Jonathan M.; Coombes, Jeff S.

    2015-01-01

    Cardiovascular disease is the main cause of morbidity and mortality in patients with kidney disease. The effectiveness of exercise for cardiovascular disease that is accelerated by the presence of chronic kidney disease remains unknown. The present study utilized apolipoprotein E knockout mice with 5/6 nephrectomy as a model of combined kidney disease and cardiovascular disease to investigate the effect of exercise on aortic plaque formation, vascular function and systemic inflammation. Animals were randomly assigned to nephrectomy or control and then to either voluntary wheel running exercise or sedentary. Following 12-weeks, aortic plaque area was significantly (p<0.05, d=1.2) lower in exercising nephrectomised mice compared to sedentary nephrectomised mice. There was a strong, negative correlation between average distance run each week and plaque area in nephrectomised and control mice (r=–0.76, p=0.048 and r=–0.73, p=0.062; respectively). In vitro aortic contraction and endothelial-independent and endothelial-dependent relaxation were not influenced by exercise (p>0.05). Nephrectomy increased IL-6 and TNF-α concentrations compared with control mice (p<0.001 and p<0.05, respectively), while levels of IL-10, MCP-1 and MIP-1α were not significantly influenced by nephrectomy or voluntary exercise (p>0.05). Exercise was an effective non-pharmacologic approach to slow cardiovascular disease in the presence of kidney disease in the apolipoprotein E knockout mouse. PMID:25799529

  11. ABSENCE OF THE SP/SP RECEPTOR CIRCUITRY IN THE SP PRECURSOR KNOCKOUT MICE OR SP-RECEPTOR, NEUROKININ (NK1) KNOCKOUT MICE LEADS TO AN INHIBITED CYTOKINE RESPONSE IN GRANULOMAS ASSOCIATED WITH MURINE TAENIA CRASSICEPS INFECTION

    PubMed Central

    Garza, Armandina; Weinstock, Joel; Robinson, Prema

    2008-01-01

    Neurocysticercosis, caused by the cestode Taenia solium, is the most common parasitic infection of the human central nervous system that leads to seizures. Taenia crassiceps cysticercosis in mice is an experimental model for Taenia solium cysticercosis. Similar to the human infection, live parasites cause little or no granulomatous inflammation. Dying parasites initiate a granulomatous reaction. The neuropeptide, Substance P (SP), stimulates Th1 cytokine production. In the current studies, we determined if absence of SP/SP receptor circuitry in the SP precursor, preprotachykinin knockout or SP-receptor, neurokinin (NK1) knockout mice, affected granuloma cytokine production. We hence compared the levels of Th1 cytokines, IL-2 and IFN-γ, and levels of Th2/immunoregulatory cytokines, IL-4 and IL-10, by ELISA in T. crassiceps-induced granulomas derived from infected C57BL/6 wild type (WT) versus SP-Precursor knockout and NK1 knockout mice. We found that mean levels of IL-2, IFN-γ, IL-4, and IL-10 in infected, WT-derived granulomas were significantly higher than those of granulomas derived from infected SP-Precursor knockout or the NK1 receptor knockout mice. Levels of Th2/immunoregulatory cytokines, IL-4 and IL-10, were higher in early stage granulomas (histologically-staged on basis of evidence of parasite remnants) versus late stage granulomas (no parasite-remnants) of both knockouts, whereas the reverse was noted in WT-derived granulomas. These studies established that the absence of an SP/SP receptor circuitry in the SP precursor knockout mice or NK1 receptor knockout led to an inhibited cytokine response. PMID:18576810

  12. Mapping ecologically relevant social behaviours by gene knockout in wild mice.

    PubMed

    Chalfin, Lea; Dayan, Molly; Levy, Dana Rubi; Austad, Steven N; Miller, Richard A; Iraqi, Fuad A; Dulac, Catherine; Kimchi, Tali

    2014-08-05

    The laboratory mouse serves as an important model system for studying gene, brain and behavioural interactions. Powerful methods of gene targeting have helped to decipher gene-function associations in human diseases. Yet, the laboratory mouse, obtained after decades of human-driven artificial selection, inbreeding, and adaptation to captivity, is of limited use for the study of fitness-driven behavioural responses that characterize the ancestral wild house mouse. Here, we demonstrate that the backcrossing of wild mice with knockout mutant laboratory mice retrieves behavioural traits exhibited exclusively by the wild house mouse, thereby unmasking gene functions inaccessible in the domesticated mutant model. Furthermore, we show that domestication had a much greater impact on females than on males, erasing many behavioural traits of the ancestral wild female. Hence, compared with laboratory mice, wild-derived mutant mice constitute an improved model system to gain insights into neuronal mechanisms underlying normal and pathological sexually dimorphic social behaviours.

  13. Interferon α/β receptor knockout mice as a model to study bluetongue virus infection.

    PubMed

    Ortego, Javier; de la Poza, Francisco; Marín-López, Alejandro

    2014-03-01

    Bluetongue is an arthropod-borne disease caused by a virus of the genus Orbivirus, the bluetongue virus (BTV), which affects ruminant livestock such as cattle, sheep, and goats and wild ruminants such as deer, and camelids. Recently, adult mice with gene knockouts of the interferon α/β receptor (IFNAR-/-) have been described as a model of lethal BTV infection. IFNAR(-/-) mice are highly susceptible to BTV-1, BTV-4 and BTV-8 infection when the virus is administered intravenously or subcutaneosuly. Disease progression and pathogenesis closely mimics signs of bluetongue disease in ruminants. In the present paper we review the studies where IFNAR(-/-) mice have been used as an animal model to study BTV transmission, pathogenesis, virulence, and protective efficacy of inactivated and new recombinant marker BTV vaccines. Furthermore, we report new data on protective efficacy of different strategies of BTV vaccination and also on induction of interferon α/β and proinflammatory immune responses in IFNAR(-/-) mice infected with BTV.

  14. Alleviation of high-fat diet-induced fatty liver damage in group IVA phospholipase A2-knockout mice.

    PubMed

    Ii, Hiromi; Yokoyama, Naoki; Yoshida, Shintaro; Tsutsumi, Kae; Hatakeyama, Shinji; Sato, Takashi; Ishihara, Keiichi; Akiba, Satoshi

    2009-12-01

    Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A(2) (IVA-PLA(2)), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA(2)-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA(2)-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA(2)-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA(2)-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA(2)-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E(2), which has a fat storage effect, was lower in IVA-PLA(2)-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA(2) alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA(2) metabolites, such as prostaglandin E(2). IVA-PLA(2) could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.

  15. Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high-calorie diet.

    PubMed

    Britton, Laurence; Jaskowski, Lesley; Bridle, Kim; Santrampurwala, Nishreen; Reiling, Janske; Musgrave, Nick; Subramaniam, V Nathan; Crawford, Darrell

    2016-06-01

    Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high-calorie diet (HCD). Eight-week-old wild-type and Hfe(+/-) mice received 8 weeks of a control diet or HCD Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe(+/-) mice of both dietary groups. HCD resulted in a hepcidin-independent reduction in HIC Hfe(+/-) mice demonstrated raised fasting serum glucose concentrations and HOMA-IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP-1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model.

  16. Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high-calorie diet.

    PubMed

    Britton, Laurence; Jaskowski, Lesley; Bridle, Kim; Santrampurwala, Nishreen; Reiling, Janske; Musgrave, Nick; Subramaniam, V Nathan; Crawford, Darrell

    2016-06-01

    Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high-calorie diet (HCD). Eight-week-old wild-type and Hfe(+/-) mice received 8 weeks of a control diet or HCD Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe(+/-) mice of both dietary groups. HCD resulted in a hepcidin-independent reduction in HIC Hfe(+/-) mice demonstrated raised fasting serum glucose concentrations and HOMA-IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP-1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model. PMID:27354540

  17. Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

    SciTech Connect

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J. Last, Jerold A.

    2009-02-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, N{omega}-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the

  18. Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice.

    PubMed

    Wood, Tyler T; Winden, Duane R; Marlor, Derek R; Wright, Alex J; Jones, Cameron M; Chavarria, Michael; Rogers, Geraldine D; Reynolds, Paul R

    2014-11-15

    The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in proinflammatory signaling, and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short-term secondhand smoke (SHS) exposure in RAGE knockout and control mice compared with identical animals exposed to room air only. Quantitative PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after 4 wk of SHS exposure and an anticipated absence of RAGE expression in RAGE knockout mice regardless of smoke exposure. Ras activation, NF-κB activity, and cytokine elaboration were assessed to characterize the molecular basis of SHS-induced inflammation in the mouse lung. Furthermore, bronchoalveolar lavage fluid was procured from RAGE knockout and control animals for the assessment of inflammatory cells and molecules. As a general theme, inflammation coincident with leukocyte recruitment was induced by SHS exposure and significantly influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to SHS. However, ongoing research is still warranted to fully explain roles for RAGE and other receptors in cells coping with involuntary smoke exposure for prolonged periods of time.

  19. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear

    PubMed Central

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-01-01

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant D-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifest by freezing during the presentation of a tone 48 hours after it had been paired with a shock. During the 30 minutes following tone presentation they showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders. PMID:25841792

  20. Systemic inflammation and insulin sensitivity in obese IFN-γ knockout mice

    PubMed Central

    O’Rourke, Robert W.; White, Ashley E.; Metcalf, Monja D.; Winters, Brian R.; Diggs, Brian S.; Zhu, Xinxia; Marks, Daniel L.

    2012-01-01

    Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype. PMID:22386937

  1. Myo5b knockout mice as a model of microvillus inclusion disease

    PubMed Central

    Cartón-García, Fernando; Overeem, Arend W.; Nieto, Rocio; Bazzocco, Sarah; Dopeso, Higinio; Macaya, Irati; Bilic, Josipa; Landolfi, Stefania; Hernandez-Losa, Javier; Schwartz, Simo; Ramon y Cajal, Santiago; van Ijzendoorn, Sven C. D.; Arango, Diego

    2015-01-01

    Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches. PMID:26201991

  2. Behavioral characterization of CD36 knockout mice with SHIRPA primary screen.

    PubMed

    Zhang, Shuxiao; Wang, Wei; Li, Juan; Cheng, Ke; Zhou, Jingjing; Zhu, Dan; Yang, Deyu; Liang, Zihong; Fang, Liang; Liao, Li; Xie, Peng

    2016-02-15

    CD36 is a member of the class B scavenger receptor family of cell surface proteins, which plays a major role in fatty acid, glucose and lipid metabolism. Besides, CD36 functions as a microglial surface receptor for amyloid beta peptide. Regarding this, we suggest CD36 might also contribute to neuropsychiatric disease. The aim of this study was to achieve a behavioral phenotype of CD36 knockout (CD36(-/-)) mice. We characterized the behavior of CD36(-/-) mice and C57BL/6J mice by subjecting them to a series of tests, which include SHIRPA primary behavioral screen test, 1% sucrose preference test, elevated plus-maze test, open-field test and forced swimming test. The results showed that CD36(-/-) mice traversed more squares, emitted more defecation, exhibited higher tail elevation and had more aggressive behaviors than C57BL/6J mice. The CD36(-/-) mice spent more time and traveled longer distance in periphery zone in the open-field test. Meanwhile, the numbers that CD36(-/-) mice entered in the open arms of elevated plus-maze were reduced. These findings suggest that CD36(-/-) mice present an anxious phenotype and might be involved in neuropsychiatric disorders.

  3. Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

    PubMed

    Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-10-01

    Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth. PMID:27423518

  4. Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

    PubMed

    Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-10-01

    Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth.

  5. Ethanol-induced neurodegeneration in NRSF/REST neuronal conditional knockout mice.

    PubMed

    Cai, L; Bian, M; Liu, M; Sheng, Z; Suo, H; Wang, Z; Huang, F; Fei, J

    2011-05-01

    The transcription regulator, neuron-restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor (REST), plays an important role in neurogenesis and various neuronal diseases such as ischaemia, epilepsy, and Huntington's disease. In these disease processes, neuronal loss is associated with abnormal expression and/or localization of NRSF. Previous studies have demonstrated that NRSF regulates the effect of ethanol on neuronal cells in vitro, however, the role of NRSF in ethanol-induced neuronal cell death remains unclear. We generated nrsf conditional knockout mice using the Cre-loxP system to disrupt neuronal expression of nrsf and its truncated forms. At postnatal day 6, ethanol significantly increased the expression of REST4, a neuron-specific truncated form of NRSF, in the brains of wild type mice, and this effect was diminished in nrsf conditional knockout mice. The apoptotic effect of ethanol was pronounced in multiple brain regions of nrsf conditional mutant mice. These results indicate that NRSF, specifically REST4, may protect the developing brain from ethanol, and provide new evidence that NRSF can be a therapeutic target in foetal alcohol syndrome (FAS). PMID:21396985

  6. Changes in liver gene expression of Azin1 knock-out mice.

    PubMed

    Wan, Tao; Hu, Yuan; Zhang, Wenlu; Huang, Ailong; Yamamura, Ken-ichi; Tang, Hua

    2010-01-01

    The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change > or = 2) and 1466 downregulated genes (fold change < or = 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

  7. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis.

  8. Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice

    PubMed Central

    Paixão-Cavalcante, D; Botto, M; Cook, H T; Pickering, M C

    2009-01-01

    Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin-producing enteropathic bacteria (Stx-associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx-associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx-2. Stx-2 was administered together with lipopolysaccharide to wild-type and Cfh+/− C57BL/6 animals. Forty-eight hours after administration of the first Stx-2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild-type or Cfh+/− Stx-2-treated mice. Uraemia and renal tubular apoptosis did not develop in wild-type or Cfh+/− animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx-2 treated animals. In summary, Stx-2 administration did not result in TMA in either Cfh+/− or wild-type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx-2-induced renal tubular injury. PMID:19040606

  9. Acquisition and extinction of a conditioned passive avoidance reflex in mice with genetic knockout of monoamine oxidase A.

    PubMed

    Dubrovina, N I; Popova, N K; Gilinskii, M A; Tomilenko, R A; Seif, I

    2006-05-01

    We report here the results obtained from comparative analysis of learning and the dynamics of extinction of a conditioned passive avoidance response in mice with genetic knockout of monoamine oxidase A (MAO A) and the progenitor line C3H. Mice of both lines acquired the conditioned passive avoidance reaction efficiently. Mice with genetic knockout of MAO A were characterized by prolonged retention of reproduction of the memory trace, as compared with rapid extinction in C3H mice. Smaller numbers of transfers, and vertical rearings on days 7-13 and the numbers of glances into and rom the dark sector on days 11-13 of extinction in MAO A-knockout mice appear to reflect their more marked fear reactions when confronted with the "dangerous" sector, along with increased anxiety, these facilitating longer-lasting retention of the memory trace. PMID:16583159

  10. [Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

    PubMed

    Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei

    2014-08-25

    The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

  11. Effect of peripheral administration of cholecystokinin on food intake in apolipoprotein AIV knockout mice.

    PubMed

    Yoshimichi, Go; Lo, Chunmin C; Tamashiro, Kellie L K; Ma, Liyun; Lee, Dana M; Begg, Denovan P; Liu, Min; Sakai, Randall R; Woods, Stephen C; Yoshimatsu, Hironobu; Tso, Patrick

    2012-06-01

    Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are satiation factors secreted by the small intestine in response to lipid meals. Apo AIV and CCK-8 has an additive effect to suppress food intake relative to apo AIV or CCK-8 alone. In this study, we determined whether CCK-8 (1, 3, or 5 μg/kg ip) reduces food intake in fasted apo AIV knockout (KO) mice as effectively as in fasted wild-type (WT) mice. Food intake was monitored by the DietMax food system. Apo AIV KO mice had significantly reduced 30-min food intake following all doses of CCK-8, whereas WT mice had reduced food intake only at doses of 3 μg/kg and above. Post hoc analysis revealed that the reduction of 10-min and 30-min food intake elicited by each dose of CCK-8 was significantly larger in the apo AIV KO mice than in the WT mice. Peripheral CCK 1 receptor (CCK1R) gene expression (mRNA) in the duodenum and gallbladder of the fasted apo AIV KO mice was comparable to that in WT mice. In contrast, CCK1R mRNA in nodose ganglia of the apo AIV KO mice was upregulated relative to WT animals. Similarly, upregulated CCK1R gene expression was found in the brain stem of apo AIV KO mice by in situ hybridization. Although it is possible that the increased satiating potency of CCK in apo AIV KO mice is mediated by upregulation of CCK 1R in the nodose ganglia and nucleus tractus solitarius, additional experiments are required to confirm such a mechanism.

  12. Host resistance of CD18 knockout mice against systemic infection with Listeria monocytogenes

    NASA Technical Reports Server (NTRS)

    Wu, Huaizhu; Prince, Joseph E.; Brayton, Cory F.; Shah, Chirayu; Zeve, Daniel; Gregory, Stephen H.; Smith, C. Wayne; Ballantyne, Christie M.

    2003-01-01

    Mice with targeted mutations of CD18, the common beta2 subunit of CD11/CD18 integrins, have leukocytosis, impaired transendothelial neutrophil emigration, and reduced host defense to Streptococcus pneumoniae, a gram-positive extracellular bacterium. Previous studies using blocking monoclonal antibodies suggested roles for CD18 and CD11b in hepatic neutrophil recruitment and host innate response to Listeria monocytogenes, a gram-positive intracellular bacterium. We induced systemic listeriosis in CD18 knockout (CD18-ko) and wild-type (WT) mice by tail vein injection with Listeria. By 14 days postinjection (dpi), 8 of 10 WT mice died, compared with 2 of 10 CD18-ko mice (P < 0.01). Quantitative organ culture showed that numbers of Listeria organisms in livers and spleens were similar in both groups at 20 min postinfection. By 3, 5, and 7 dpi, however, numbers of Listeria organisms were significantly lower in livers and spleens of CD18-ko mice than in WT mice. Histopathology showed that following Listeria infection, CD18-ko mice had milder inflammatory and necrotizing lesions in both spleens and livers than did WT mice. Cytokine assays indicated that baseline interleukin-1beta and granulocyte colony-stimulating factor (G-CSF) levels were higher in CD18-ko mice than in WT mice and that CD18-ko splenocytes produced higher levels of interleukin-1beta and G-CSF than WT splenocytes under the same amount of Listeria stimulation. These findings show that CD18 is not an absolute requirement for antilisterial innate immunity or hepatic neutrophil recruitment. We propose that the absence of CD18 in the mice results in the priming of innate immunity, as evidenced by elevated cytokine expression, and neutrophilic leukocytosis, which augments antilisterial defense.

  13. Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice.

    PubMed Central

    Su, H; Feilzer, K; Caldwell, H D; Morrison, R P

    1997-01-01

    The importance of antibody-mediated immunity in primary and secondary Chlamydia trachomatis genital tract infections was examined by using a definitive model of B-cell deficiency, the microMT/microMT gene knockout mouse. Vaginally infected B-cell-deficient microMT/microMT mice developed a self-limiting primary infection that was indistinguishable from infection of control C57BL/6 mice. Sera and vaginal secretions from infected mice were analyzed for anti-Chlamydia antibodies. C57BL/6 mice produced high-titered serum anti-Chlamydia immunoglobulin G2a (IgG2a), IgG2b, and IgA antibodies, and vaginal washes contained predominately anti-Chlamydia IgA. Serum and vaginal washes from infected B-cell-deficient mice were negative for anti-Chlamydia antibody. T-cell proliferation and delayed-type hypersensitivity assays were used as measures of Chlamydia-specific cell-mediated immunity and were found to be comparable for C57BL/6 and B-cell-deficient mice. Seventy days following primary infection, mice were rechallenged to assess acquired immunity. B-cell-deficient mice which lack anti-Chlamydia antibodies were more susceptible to reinfection than immunocompetent C57BL/6 mice. However, acquired immune resistance was evident in both strains of mice and characterized by decreased shedding of chlamydiae and an infection of shorter duration. Thus, this study demonstrates that cell-mediated immune responses alone were capable of resolving chlamydial infection; however, in the absence of specific antibody, mice were more susceptible to reinfection. Therefore, these data suggest that both humoral and cell-mediated immune responses were important mediators of immune protection in this model, though cell-mediated immune responses appear to play a more dominant role. PMID:9169723

  14. Gomafu lncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine.

    PubMed

    Ip, Joanna Y; Sone, Masamitsu; Nashiki, Chieko; Pan, Qun; Kitaichi, Kiyoyuki; Yanaka, Kaori; Abe, Takaya; Takao, Keizo; Miyakawa, Tsuyoshi; Blencowe, Benjamin J; Nakagawa, Shinichi

    2016-01-01

    The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes. PMID:27251103

  15. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.

    PubMed

    Sung, Young Hoon; Kim, Jong Min; Kim, Hyun-Taek; Lee, Jaehoon; Jeon, Jisun; Jin, Young; Choi, Jung-Hwa; Ban, Young Ho; Ha, Sang-Jun; Kim, Cheol-Hee; Lee, Han-Woong; Kim, Jin-Soo

    2014-01-01

    RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F₀ and F₁ mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

  16. Systemic autoimmunity in TAM triple knockout mice causes inflammatory brain damage and cell death.

    PubMed

    Li, Qiutang; Lu, Qingjun; Lu, Huayi; Tian, Shifu; Lu, Qingxian

    2013-01-01

    The Tyro3, Axl and Mertk (TAM) triply knockout (TKO) mice exhibit systemic autoimmune diseases, with characteristics of increased proinflammatory cytokine production, autoantibody deposition and autoreactive lymphocyte infiltration into a variety of tissues. Here we show that TKO mice produce high level of serum TNF-α and specific autoantibodies deposited onto brain blood vessels. The brain-blood barrier (BBB) in mutant brains exhibited increased permeability for Evans blue and fluorescent-dextran, suggesting a breakdown of the BBB in the mutant brains. Impaired BBB integrity facilitated autoreactive T cells infiltrating into all regions of the mutant brains. Brain autoimmune disorder caused accumulation of the ubiquitin-reactive aggregates in the mutant hippocampus, and early formation of autofluorescent lipofuscins in the neurons throughout the entire brains. Chronic neuroinflammation caused damage of the hippocampal mossy fibers and neuronal apoptotic death. This study shows that chronic systemic inflammation and autoimmune disorders in the TKO mice cause neuronal damage and death.

  17. Gomafu lncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine

    PubMed Central

    Ip, Joanna Y.; Sone, Masamitsu; Nashiki, Chieko; Pan, Qun; Kitaichi, Kiyoyuki; Yanaka, Kaori; Abe, Takaya; Takao, Keizo; Miyakawa, Tsuyoshi; Blencowe, Benjamin J.; Nakagawa, Shinichi

    2016-01-01

    The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes. PMID:27251103

  18. Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry.

    PubMed

    Weber, Allan; Ni, Jinsong; Ling, Kah-Hiing John; Acheampong, Andrew; Tang-Liu, Diane D-S; Burk, Robert; Cravatt, Benjamin F; Woodward, David

    2004-04-01

    We investigated the formation of PGF(2alpha) 1-ethanolamide, PGE(2) 1-ethanolamide, and PGD(2) 1-ethanolamide (prostamides F(2alpha), E(2), and D(2), respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH -/-) male mice. One group of three normal mice was not dosed (naïve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH -/- mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F(2alpha), prostamide E(2), prostamide D(2), and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F(2alpha) was detected in tissues in FAAH -/- mice after administration of anandamide. Concentrations of anandamide, prostamide E(2), and prostamide D(2) in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH -/- mice than those of the anandamide-treated control mice. This report demonstrates that prostamides, including prostamide F(2alpha), were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking. PMID:14729864

  19. Inhibition of fibrous adhesion formation in the temporomandibular joint of tenascin-C knockout mice.

    PubMed

    Shinohara, Y; Okamoto, K; Goh, Y; Kiga, N; Tojyo, I; Fujita, S

    2014-10-22

    Tenascin-C (TNC) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has been reported that TNC is expressed in inflamed synovial membranes and deformed discs of temporomandibular joint (TMJ) disorder. However, the role of TNC in TMJ is not fully known. In this study, the role of TNC in fibrous adhesion formation of TMJ was examined using TNC knockout (TNCKO) mice. Hypermobility was produced by excessive mouth opening method on the TMJ of both wild-type (WT) and TNCKO mice. TMJ wound healing was compared histologically, and the expression of TNC, fibronectin (FN) and α-smooth muscle actin (α-SMA) in the wounded TMJ was examined by immunohistochemical and immunoblot analyses. Based on histologic analysis, fibrous adhesions were observed in the TMJ of both TNCKO and wild-type (WT) mice after excessive mouth opening. However, fibrous adhesion formation in TNCKO mice occurred later than in WT mice. TNC was expressed in the wounded TMJ disc and mandibular fossa. Although FN and α-SMA expression in the TMJ of TNCKO and WT mice was up-regulated after excessive mouth opening, FN and α-SMA protein levels were higher in WT mice at the same time points. In the wounded TMJ, TNC appears to enhance the expression of FN and α-SMA, and a lack of TNC may reduce fibrous adhesion formation in the TMJ. TNC plays an important role in TMJ wound healing, especially for wounds generated by mechanical stress.

  20. Recombination and synaptic adjustment in oocytes of mice heterozygous for a large paracentric inversion.

    PubMed

    Torgasheva, Anna A; Rubtsov, Nikolai B; Borodin, Pavel M

    2013-03-01

    Homologous chromosome synapsis in inversion heterozygotes results in the formation of inversion loops. These loops might be transformed into straight, non-homologously paired bivalents via synaptic adjustment. Synaptic adjustment was discovered 30 years ago; however, its relationship with recombination has remained unclear. We analysed this relationship in female mouse embryos heterozygous for large paracentric inversion In(1)1Rk using immunolocalisation of the synaptonemal complex (SYCP3) and mature recombination nodules (MLH1) proteins. The frequency of cells containing bivalents with inversion loops decreased from 69 % to 28 % during pachytene. If an MLH1 focus was present in the non-homologously paired inverted region of the straight bivalent, it was always located in the middle of the inversion. Most of the small, incompletely adjusted loops contained MLH1 foci near the points at which pairing partners were switched. This observation indicates that the degree of synaptic adjustment depended on the crossover position. Complete synaptic adjustment was only possible if a crossover (CO) was located exactly in the middle of the inversion. If a CO was located at any other site, this interrupted synaptic adjustment and resulted in inversion loops of different sizes with an MLH1 focus at or near the edge of the remaining loop.

  1. Reduced beta-amyloid production and increased inflammatory responses in presenilin conditional knock-out mice.

    PubMed

    Beglopoulos, Vassilios; Sun, Xiaoyan; Saura, Carlos A; Lemere, Cynthia A; Kim, Richard D; Shen, Jie

    2004-11-01

    Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional double knock-out mice lacking both presenilins in the postnatal forebrain (PS cDKO mice) exhibit memory and synaptic plasticity impairments followed by progressive neurodegeneration in the cerebral cortex. Here we further investigate the molecular events that may underlie the observed phenotypes and identify additional neuropathological markers in the PS cDKO brain. Enzyme-linked immunosorbent assay analysis showed reduced levels of the toxic beta-amyloid (Abeta) peptides in the cerebral cortex of PS cDKO mice. Interestingly, the reduction in Abeta40 and Abeta42 peptides is similar in PS1 conditional knock-out and PS cDKO mice. We further examined the gene expression profile by oligonucleotide microarrays in the PS cDKO cerebral cortex and found that a high number of genes are differentially expressed, most notably a group of up-regulated inflammatory genes. Quantitative real-time reverse transcription PCR and Western analyses confirmed the elevated levels of glial fibrillary acidic protein, complement component C1q, and cathepsin S, up-regulation of which has been associated with inflammatory responses in various neurodegenerative processes. Immunohistochemical analysis revealed that the increase in complement component C1q is confined to the hippocampal formation, whereas glial fibrillary acidic protein and cathepsin S are up-regulated throughout the entire neocortex and hippocampus. In addition, strong microglial activation occurs in the hippocampus and the deeper cortical layers of PS cDKO mice. These results indicate that the memory impairment and neurodegeneration in PS cDKO mice are not caused by Abeta accumulation and that loss of PS function leads to differential up-regulation of inflammatory markers in the cerebral cortex.

  2. Emotional disorders in adult mice heterozygous for the transcription factor Phox2b.

    PubMed

    Bollen, Bieke; Ramanantsoa, Nelina; Naert, Arne; Matrot, Boris; Van den Bergh, Omer; D'Hooge, Rudi; Gallego, Jorge

    2015-03-15

    Phox2b is an essential transcription factor for the development of the autonomic nervous system. Mice carrying one invalidated Phox2b allele (Phox2b(+/-)) show mild autonomic disorders including sleep apneas, and impairments in chemosensitivity and thermoregulation that recover within 10days of postnatal age. Because Phox2b is not expressed above the pons nor in the cerebellum, this mutation is not expected to affect brain development and cognitive functioning directly. However, the transient physiological disorders in Phox2b(+/-) mice might impair neurodevelopment. To examine this possibility, we conducted a behavioral test battery of emotional, motor, and cognitive functioning in adult Phox2b(+/-) mice and their wildtype littermates (Phox2b(+/+)). Adult Phox2b(+/-) mice showed altered exploratory behavior in the open field and in the elevated plus maze, both indicative of anxiety. Phox2b(+/-) mice did not show cognitive or motor impairments. These results suggest that also mild autonomic control deficits may disturb long-term emotional development. PMID:25582512

  3. Emotional disorders in adult mice heterozygous for the transcription factor Phox2b.

    PubMed

    Bollen, Bieke; Ramanantsoa, Nelina; Naert, Arne; Matrot, Boris; Van den Bergh, Omer; D'Hooge, Rudi; Gallego, Jorge

    2015-03-15

    Phox2b is an essential transcription factor for the development of the autonomic nervous system. Mice carrying one invalidated Phox2b allele (Phox2b(+/-)) show mild autonomic disorders including sleep apneas, and impairments in chemosensitivity and thermoregulation that recover within 10days of postnatal age. Because Phox2b is not expressed above the pons nor in the cerebellum, this mutation is not expected to affect brain development and cognitive functioning directly. However, the transient physiological disorders in Phox2b(+/-) mice might impair neurodevelopment. To examine this possibility, we conducted a behavioral test battery of emotional, motor, and cognitive functioning in adult Phox2b(+/-) mice and their wildtype littermates (Phox2b(+/+)). Adult Phox2b(+/-) mice showed altered exploratory behavior in the open field and in the elevated plus maze, both indicative of anxiety. Phox2b(+/-) mice did not show cognitive or motor impairments. These results suggest that also mild autonomic control deficits may disturb long-term emotional development.

  4. Impaired lipid accumulation in the liver of Tsc2-heterozygous mice during liver regeneration

    SciTech Connect

    Obayashi, Yoko; Campbell, Jean S.; Fausto, Nelson; Yeung, Raymond S.

    2013-07-19

    Highlights: •Tuberin phosphorylation correlated with mTOR activation in early liver regeneration. •Liver regeneration in the Tsc2+/− mice was not enhanced. •The Tsc2+/− livers failed to accumulate lipid bodies during liver regeneration. •Mortality rate increased in Tsc2+/− mice after partial hepatectomy. •Tuberin plays a critical role in hepatic lipid accumulation to support regeneration. -- Abstract: Tuberin is a negative regulator of mTOR pathway. To investigate the function of tuberin during liver regeneration, we performed 70% hepatectomy on wild-type and Tsc2+/− mice. We found the tuberin phosphorylation correlated with mTOR activation during early liver regeneration in wild-type mice. However, liver regeneration in the Tsc2+/− mice was not enhanced. Instead, the Tsc2+/− livers failed to accumulate lipid bodies, and this was accompanied by increased mortality. These findings suggest that tuberin plays a critical role in liver energy balance by regulating hepatocellular lipid accumulation during early liver regeneration. These effects may influence the role of mTORC1 on cell growth and proliferation.

  5. Genetic or Pharmacologic Activation of Nrf2 Signaling Fails to Protect Against Aflatoxin Genotoxicity in Hypersensitive GSTA3 Knockout Mice

    PubMed Central

    Kensler, Kevin H.; Slocum, Stephen L.; Chartoumpekis, Dionysios V.; Dolan, Patrick M.; Johnson, Natalie M.; Ilic, Zoran; Crawford, Dana R.; Sell, Stewart; Groopman, John D.; Kensler, Thomas W.; Egner, Patricia A.

    2014-01-01

    Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N7-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N7-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was >200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice. PMID:24675090

  6. Uptake and catabolism of modified LDL in scavenger-receptor class A type I/II knock-out mice.

    PubMed Central

    Van Berkel, T J; Van Velzen, A; Kruijt, J K; Suzuki, H; Kodama, T

    1998-01-01

    The liver is the major organ responsible for the uptake of modified low-density lipoprotein (LDL) from the blood circulation, with endothelial and Kupffer cells as major cellular uptake sites. Scavenger-receptors, which include various classes, are held responsible for this uptake. Mice deficient in scavenger-receptor class A types I and II were created and the fate of acetylated LDL (Ac-LDL) in vivo and its interaction with liver endothelial, Kupffer and peritoneal macrophages was characterized. Surprisingly, the decay in vivo (t12 < 2 min), tissue distribution and liver uptake (at 5 min it was 77.4 +/- 4.6% of the injected dose) of Ac-LDL in the knock-out mice were not significantly different from control mice (t12 < 2 min and liver uptake 79.1 +/- 4.6% of the injected dose). A separation of mice liver cells into parenchymal, endothelial and Kupffer cells 10 min after injection of Ac-LDL indicated that in both control and knock-out mice the liver endothelial cells were responsible for more than 70% of the liver uptake. Both in control and knock-out mice, preinjection of polyinosinic acid (poly I, 200 microg) completely blocked the liver uptake, indicating that both in control and knock-out mice the scavenger-receptors are sensitive to poly I. Preinjection of suboptimal poly I concentrations (20 and 50 microg) provided evidence that the serum decay and liver uptake of Ac-LDL is more readily inhibited in the knock-out mice as compared with the control mice, indicating less efficient removal of Ac-LDL in vivo in the knock-out mice under these conditions. Studies in vitro with isolated liver endothelial and Kupffer cells from knock-out mice indicate that the cell association of Ac-LDL during 2 h at 37 degrees C is 50 and 53% of the control, respectively, whereas the degradation reaches values of 58 and 63%. For peritoneal macrophages from knock-out mice the cell association of Ac-LDL was identical to the control mice whereas the Ac-LDL degradation in cells from the

  7. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

    SciTech Connect

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Bohlooly-Y, Mohammad . E-mail: mohammad.bohlooly@astrazeneca.com

    2006-10-20

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys, gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

  8. Parkin expression profile in dopamine d3 receptor knock-out mice brains.

    PubMed

    D'Agata, Velia; Tiralongo, Adriana; Castorina, Alessandro; Leggio, Gian Marco; Micale, Vincenzo; Carnazza, Maria Luisa; Drago, Filippo

    2009-02-01

    Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms' distribution pattern and in exons' expression both in wild type controls and dopamine D3 receptor's knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.

  9. CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation.

    PubMed

    Ranganathan, Punithavathi; Jayakumar, Calpurnia; Manicassamy, Santhakumar; Ramesh, Ganesan

    2013-11-15

    Organ cross talk exists in many diseases of the human and animal models of human diseases. A recent study demonstrated that inflammatory mediators can cause acute kidney injury and neutrophil infiltration in a mouse model of dextran sodium sulfate (DSS)-colitis. However, the chemokines and their receptors that may mediate distant organ effects in colitis are unknown. We hypothesized that keratinocyte chemoattractant (KC)/IL-8 receptor chemokine (C-X-C motif) ligand 2 (CXCL2) mediates DSS-colitis-induced acute kidney injury. Consistent with our hypothesis, wild-type (WT) mice developed severe colitis with DSS treatment, which was associated with inflammatory cytokine and chemokine expression and neutrophil infiltration in the colon. DSS-colitis in WT was accompanied by acute kidney injury and enhanced expression of inflammatory cytokines in the kidney. However, CXCR2 knockout mice were protected against DSS-colitis as well as acute kidney injury. Moreover, the expression of cytokines and chemokines and neutrophil infiltration was blunted in CXCR2 knockout mice in the colon and kidney. Administration of recombinant KC exacerbated DSS-colitis-induced acute kidney injury. Our results suggest that KC/IL-8 and its receptor CXCR2 are critical and major mediators of organ cross talk in DSS colitis and neutralization of CXCR2 will help to reduce the incidence of acute kidney injury due to ulcerative colitis and Crohn's disease in humans.

  10. Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

    PubMed Central

    2010-01-01

    Background Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. Methods To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1+/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Results Six Men1+/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Conclusion Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland. PMID:20663219

  11. Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice.

    PubMed

    Huang, F L; Huang, K-P

    2012-10-01

    Neurogranin (Ng), a brain-specific calmodulin-binding protein, is expressed highly in hippocampus, and is important for cognitive function. Deletion of the Ng gene from mice caused attenuation of signal reaction cascade in hippocampus, impairments in learning and memory and high frequency stimulation-induced long-term potentiation (LTP). Environmental enrichment alone failed to improve cognitive function. In this study, behavioral testing revealed that Ng knockout (NgKO) mice were both hyperactive and socially withdrawn. Methylphenidate (MPH) was given to mice while they were also kept under an enrichment condition. MPH treatment reduced the hyperactivity of NgKO mice tested in both the open field and forced swim chamber. MPH improved their social abilities such that mice recognized and interacted better with novel subjects. The cognitive memories of MPH-treated mutants were improved in both water maze and contextual fear conditioning tests. High frequency stimulation-induced LTP of NgKO mice was also improved by MPH. The present treatment regimen, however, did not fully reverse the deficits of the mutant mice. In contrast, MPH exerted only a minimal effect on the wild type mice. At the cellular level, MPH increased the number of glial fibrillary acidic protein-positive cells in hippocampus, particularly within the dentate gyrus of NgKO mice. Therefore it will be of interest to determine the nature of MPH-mediated astrocyte activation and how it may modulate behavior in future studies. Taken together these NgKO mice may be useful for the development of better drug treatment to improve cognitive and behavioral impairments.

  12. The protective role of tacrine and donepezil in the retina of acetylcholinesterase knockout mice

    PubMed Central

    Yi, Yun-Min; Cai, Li; Shao, Yi; Xu, Man; Yi, Jing-Lin

    2015-01-01

    AIM To determine the effect of different concentrations of the acetylcholinesterase (AChE) inhibitors tacrine and donepezil on retinal protection in AChE+/− mice (AChE knockout mice) of various ages. METHODS Cultured ARPE-19 cells were treated with hydrogen peroxide (H2O2) at concentrations of 0, 250, 500, 1000 and 2000 µmol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil (0.1 mg/mL, 0.2 mg/mL and 0.4 mg/mL) were respectively given to AChE+/− mice aged 2mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline (PBS) was administered to the control group. The mice were sacrificed after 30d by in vitro cardiac perfusion and retinal samples were taken. AChE-deficient mice were identified by polymerase chain reaction (PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website. H&E staining, immunofluorescence and Western blot were performed to observe AChE protein expression changes in the retinal pigment epithelial (RPE) cell layer. RESULTS Different concentrations of H2O2 induced AChE expression during RPE cell apoptosis. AChE+/− mice retina were thinner than those in wild-type mice (P<0.05); the retinal structure was still intact at 2mo but became thinner with increasing age (P<0.05); furthermore, AChE+/− mice developed more slowly than wild-type mice (P<0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology (P>0.05). CONCLUSION In vivo, tacrine and donepezil can inhibit the expression of AChE; the decrease of AChE expression in the retina is beneficial for the development of the retina. PMID:26558196

  13. Learning, memory and long-term potentiation are altered in Nedd4 heterozygous mice.

    PubMed

    Camera, Daria; Coleman, Harold A; Parkington, Helena C; Jenkins, Trisha A; Pow, David V; Boase, Natasha; Kumar, Sharad; Poronnik, Philip

    2016-04-15

    The consolidation of short-term memory into long-term memory involves changing protein level and activity for the synaptic plasticity required for long-term potentiation (LTP). AMPA receptor trafficking is a key determinant of LTP and recently ubiquitination by Nedd4 has been shown to play an important role via direct action on the GluA1 subunit, although the physiological relevance of these findings are yet to be determined. We therefore investigated learning and memory in Nedd4(+/-) mice that have a 50% reduction in levels of Nedd4. These mice showed decreased long-term spatial memory as evidenced by significant increases in the time taken to learn the location of and subsequently find a platform in the Morris water maze. In contrast, there were no significant differences between Nedd4(+/+) and Nedd4(+/-) mice in terms of short-term spatial memory in a Y-maze test. Nedd4(+/-) mice also displayed a significant reduction in post-synaptic LTP measured in hippocampal brain slices. Immunofluorescence of Nedd4 in the hippocampus confirmed its expression in hippocampal neurons of the CA1 region. These findings indicate that reducing Nedd4 protein by 50% significantly impairs LTP and long-term memory thereby demonstrating an important role for Nedd4 in these processes.

  14. Resistance of R-Ras knockout mice to skin tumour induction

    PubMed Central

    May, Ulrike; Prince, Stuart; Vähätupa, Maria; Laitinen, Anni M.; Nieminen, Katriina; Uusitalo-Järvinen, Hannele; Järvinen, Tero A. H.

    2015-01-01

    The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment. PMID:26133397

  15. Hmga1/Hmga2 double knock-out mice display a “superpygmy” phenotype

    PubMed Central

    Federico, Antonella; Forzati, Floriana; Esposito, Francesco; Arra, Claudio; Palma, Giuseppe; Barbieri, Antonio; Palmieri, Dario; Fedele, Monica; Pierantoni, Giovanna Maria; De Martino, Ivana; Fusco, Alfredo

    2014-01-01

    ABSTRACT The HMGA1 and HMGA2 genes code for proteins belonging to the High Mobility Group A family. Several genes are negatively or positively regulated by both these proteins, but a number of genes are specifically regulated by only one of them. Indeed, knock-out of the Hmga1 and Hmga2 genes leads to different phenotypes: cardiac hypertrophy and type 2 diabetes in the former case, and a large reduction in body size and amount of fat tissue in the latter case. Therefore, to better elucidate the functions of the Hmga genes, we crossed Hmga1-null mice with mice null for Hmga2. The Hmga1−/−/Hmga2−/− mice showed reduced vitality and a very small size (75% smaller than the wild-type mice); they were even smaller than pygmy Hmga2-null mice. The drastic reduction in E2F1 activity, and consequently in the expression of the E2F-dependent genes involved in cell cycle regulation, likely accounts for some phenotypic features of the Hmga1−/−/Hmga2−/− mice. PMID:24728959

  16. Doublecortin knockout mice show normal hippocampal-dependent memory despite CA3 lamination defects.

    PubMed

    Germain, Johanne; Bruel-Jungerman, Elodie; Grannec, Gael; Denis, Cécile; Lepousez, Gabriel; Giros, Bruno; Francis, Fiona; Nosten-Bertrand, Marika

    2013-01-01

    Mutations in the human X-linked doublecortin gene (DCX) cause major neocortical disorganization associated with severe intellectual disability and intractable epilepsy. Although Dcx knockout (KO) mice exhibit normal isocortical development and architecture, they show lamination defects of the hippocampal pyramidal cell layer largely restricted to the CA3 region. Dcx-KO mice also exhibit interneuron abnormalities. As well as the interest of testing their general neurocognitive profile, Dcx-KO mice also provide a relatively unique model to assess the effects of a disorganized CA3 region on learning and memory. Based on its prominent anatomical and physiological features, the CA3 region is believed to contribute to rapid encoding of novel information, formation and storage of arbitrary associations, novelty detection, and short-term memory. We report here that Dcx-KO adult males exhibit remarkably preserved hippocampal- and CA3-dependant cognitive processes using a large battery of classical hippocampus related tests such as the Barnes maze, contextual fear conditioning, paired associate learning and object recognition. In addition, we show that hippocampal adult neurogenesis, in terms of proliferation, survival and differentiation of granule cells, is also remarkably preserved in Dcx-KO mice. In contrast, following social deprivation, Dcx-KO mice exhibit impaired social interaction and reduced aggressive behaviors. In addition, Dcx-KO mice show reduced behavioral lateralization. The Dcx-KO model thus reinforces the association of neuropsychiatric behavioral impairments with mouse models of intellectual disability.

  17. Doublecortin Knockout Mice Show Normal Hippocampal-Dependent Memory Despite CA3 Lamination Defects

    PubMed Central

    Grannec, Gael; Denis, Cécile; Lepousez, Gabriel; Giros, Bruno; Francis, Fiona; Nosten-Bertrand, Marika

    2013-01-01

    Mutations in the human X-linked doublecortin gene (DCX) cause major neocortical disorganization associated with severe intellectual disability and intractable epilepsy. Although Dcx knockout (KO) mice exhibit normal isocortical development and architecture, they show lamination defects of the hippocampal pyramidal cell layer largely restricted to the CA3 region. Dcx-KO mice also exhibit interneuron abnormalities. As well as the interest of testing their general neurocognitive profile, Dcx-KO mice also provide a relatively unique model to assess the effects of a disorganized CA3 region on learning and memory. Based on its prominent anatomical and physiological features, the CA3 region is believed to contribute to rapid encoding of novel information, formation and storage of arbitrary associations, novelty detection, and short-term memory. We report here that Dcx-KO adult males exhibit remarkably preserved hippocampal- and CA3-dependant cognitive processes using a large battery of classical hippocampus related tests such as the Barnes maze, contextual fear conditioning, paired associate learning and object recognition. In addition, we show that hippocampal adult neurogenesis, in terms of proliferation, survival and differentiation of granule cells, is also remarkably preserved in Dcx-KO mice. In contrast, following social deprivation, Dcx-KO mice exhibit impaired social interaction and reduced aggressive behaviors. In addition, Dcx-KO mice show reduced behavioral lateralization. The Dcx-KO model thus reinforces the association of neuropsychiatric behavioral impairments with mouse models of intellectual disability. PMID:24073232

  18. Decreased pulmonary inflammation after ethanol exposure and burn injury in intercellular adhesion molecule-1 knockout mice.

    PubMed

    Bird, Melanie D; Morgan, Michelle O; Ramirez, Luis; Yong, Sherri; Kovacs, Elizabeth J

    2010-01-01

    Clinical and laboratory evidence suggests that alcohol consumption dysregulates immune function. Burn patients who consume alcohol before their injuries demonstrate higher rates of morbidity and mortality, including acute respiratory distress syndrome, than patients without alcohol at the time of injury. Our laboratory observed higher levels of proinflammatory cytokines and leukocyte infiltration in the lungs of mice after ethanol exposure and burn injury than with either insult alone. To understand the mechanism of the increased pulmonary inflammatory response in mice treated with ethanol and burn injury, we investigated the role of intercellular adhesion molecule (ICAM)-1. Wild-type and ICAM-1 knockout (KO) mice were treated with vehicle or ethanol and subsequently given a sham or burn injury. Twenty-four hours postinjury, lungs were harvested and analyzed for indices of inflammation. Higher numbers of neutrophils were observed in the lungs of wild-type mice after burn and burn with ethanol treatment. This increase in pulmonary inflammatory cell accumulation was significantly lower in the KO mice. In addition, levels of KC, interleukin-1beta, and interleukin-6 in the lung were decreased in the ICAM-1 KO mice after ethanol exposure and burn injury. Interestingly, no differences were observed in serum or lung tissue content of soluble ICAM-1 24 hours postinjury. These data suggest that upregulation of adhesion molecules such as ICAM-1 on the vascular endothelium may play a critical role in the excessive inflammation seen after ethanol exposure and burn injury.

  19. Hmga1/Hmga2 double knock-out mice display a "superpygmy" phenotype.

    PubMed

    Federico, Antonella; Forzati, Floriana; Esposito, Francesco; Arra, Claudio; Palma, Giuseppe; Barbieri, Antonio; Palmieri, Dario; Fedele, Monica; Pierantoni, Giovanna Maria; De Martino, Ivana; Fusco, Alfredo

    2014-04-11

    The HMGA1 and HMGA2 genes code for proteins belonging to the High Mobility Group A family. Several genes are negatively or positively regulated by both these proteins, but a number of genes are specifically regulated by only one of them. Indeed, knock-out of the Hmga1 and Hmga2 genes leads to different phenotypes: cardiac hypertrophy and type 2 diabetes in the former case, and a large reduction in body size and amount of fat tissue in the latter case. Therefore, to better elucidate the functions of the Hmga genes, we crossed Hmga1-null mice with mice null for Hmga2. The Hmga1(-/-)/Hmga2(-/-) mice showed reduced vitality and a very small size (75% smaller than the wild-type mice); they were even smaller than pygmy Hmga2-null mice. The drastic reduction in E2F1 activity, and consequently in the expression of the E2F-dependent genes involved in cell cycle regulation, likely accounts for some phenotypic features of the Hmga1(-/-)/Hmga2(-/-) mice.

  20. Maneb causes pro-oxidant effects in the hippocampus of Nrf2 knockout mice.

    PubMed

    Kurzatkowski, Daniela M; Trombetta, Louis D

    2013-09-01

    The effects of maneb were investigated in C57BL/6 Nrf2 wildtype and knockout mice. Treated KO mice showed significant weight loss as compared to WT counterparts. ICPAAS analysis demonstrated a significant increase in manganese concentration in the tissues of treated KO mice as compared to WT. Biochemical analysis revealed significant decreases of antioxidants including glutathione, glutathione reductase and heme oxygenase-1. Levels of TBARS were significantly increased in hippocampal tissue in Nrf2 KO mice at the 30 and 60mg doses. qPCR demonstrated that the only gene mediated by the Nrf2 transcription pathway that was significantly modulated by at least 1.5 fold was glutathione peroxidase 4. GPX4 was significantly upregulated in Nrf2 WT mice treated with 30mg/kg maneb and significantly downregulated in Nrf2 KO mice treated with the same dose. Microscopy revealed neuronal pyknosis and eosinophilia of the cytoplasm in the hippocampi of both WT and KO animals treated with 60mg/kg maneb. PMID:23764462

  1. Ibuprofen partially attenuates neurodegenerative symptoms in presenilin conditional double-knockout mice.

    PubMed

    Dong, Z; Yan, L; Huang, G; Zhang, L; Mei, B; Meng, B

    2014-06-13

    Ibuprofen is a widely used nonsteroidal anti-inflammatory drug that reportedly reduces the risk of Alzheimer's disease (AD) development. The anti-inflammatory effect of ibuprofen occurred via inhibition of cyclooxygenases and anti-amyloidogenesis through modulation of γ-secretase. Presenilin 1 and 2 conditional double-knockout (cDKO) mice exhibited age-dependent memory impairment and forebrain degeneration without elevation of amyloid β deposition. Therefore, cDKO mice can be an ideal animal model on which to independently test the effects of ibuprofen anti-inflammatory properties on the prevention of AD. Three- and six-month-old cDKO mice were fed diet containing 375 ppm ibuprofen for six months. After multiple, well-validated behavioral tests, treatment with ibuprofen improved cognition-related behavioral performance, and drug efficacy was correlated with the timing of administration. Ibuprofen was more effective on six-month-old than on three-month-old cDKO mice. Biochemical analysis demonstrated that the effects of ibuprofen on glial fibrillary acidic protein and CD68 expression levels were uneven in different brain regions of cDKO mice and that age also influenced such effects. Tau hyperphosphorylation and the cleavage of caspase-3 decreased after ibuprofen treatment, and this effect was more significant in the older than the younger group of mice, which was consistent with the results of behavioral tests.

  2. Claudin 4 knockout mice: normal physiological phenotype with increased susceptibility to lung injury

    PubMed Central

    Kage, Hidenori; Flodby, Per; Gao, Danping; Kim, Yong Ho; Marconett, Crystal N.; DeMaio, Lucas; Kim, Kwang-Jin; Crandall, Edward D.

    2014-01-01

    Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly upregulated in wild-type mice, whereas Cldn4 KO mice showed greater degrees of lung injury. RNA sequencing, in conjunction with differential expression and upstream analysis after ventilator-induced lung injury, suggested Egr1, Tnf, and Il1b as potential mediators of increased lung injury in Cldn4 KO mice. These results demonstrate that claudin 4 has little effect on normal lung physiology but may function to protect against acute lung injury. PMID:25106430

  3. Maneb causes pro-oxidant effects in the hippocampus of Nrf2 knockout mice.

    PubMed

    Kurzatkowski, Daniela M; Trombetta, Louis D

    2013-09-01

    The effects of maneb were investigated in C57BL/6 Nrf2 wildtype and knockout mice. Treated KO mice showed significant weight loss as compared to WT counterparts. ICPAAS analysis demonstrated a significant increase in manganese concentration in the tissues of treated KO mice as compared to WT. Biochemical analysis revealed significant decreases of antioxidants including glutathione, glutathione reductase and heme oxygenase-1. Levels of TBARS were significantly increased in hippocampal tissue in Nrf2 KO mice at the 30 and 60mg doses. qPCR demonstrated that the only gene mediated by the Nrf2 transcription pathway that was significantly modulated by at least 1.5 fold was glutathione peroxidase 4. GPX4 was significantly upregulated in Nrf2 WT mice treated with 30mg/kg maneb and significantly downregulated in Nrf2 KO mice treated with the same dose. Microscopy revealed neuronal pyknosis and eosinophilia of the cytoplasm in the hippocampi of both WT and KO animals treated with 60mg/kg maneb.

  4. Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues.

    PubMed

    Hewitt, Sylvia Curtis; Korach, Kenneth S

    2003-02-01

    Oestrogen is an essential component of female reproduction, with well-characterized functions in the uterus, ovaries, mammary gland and hypothalamic-pituitary axis. The mechanism of oestrogen action involves mediation of the rate of transcription by nuclear-localized oestrogen receptor molecules. Two oestrogen receptors are present in mouse tissues, oestrogen receptors alpha and beta. Each receptor exhibits differential tissue expression patterns. Mouse models with genetically engineered disruption or 'knockout' of the oestrogen receptors have been developed. Characterization of the resulting defects in reproductive tissues as well as alterations in physiological and genomic responses has given insight into the receptor-mediated effects of oestrogen in reproduction. Oestrogen receptor alpha knockout females are infertile because they are anovulatory, have disruption in LH regulation and have uteri that are insensitive to oestrogen. In contrast, oestrogen receptor beta knockout females are sub-fertile and primarily lack efficient ovulatory function. Mice with deletion of both oestrogen receptors alpha and beta are similar to those lacking oestrogen receptor alpha only, but exhibit a unique ovarian pathology. These observed phenotypes elucidate the relative roles of the oestrogen receptors in reproductive functions of female rodents.

  5. Theca-specific estrogen receptor-alpha knockout mice lose fertility prematurely.

    PubMed

    Lee, Sungeun; Kang, Dong-Wook; Hudgins-Spivey, Susan; Krust, Andree; Lee, Eun-Young; Koo, Youngbum; Cheon, Yongpil; Gye, Myung Chan; Chambon, Pierre; Ko, ChemYong

    2009-08-01

    Estrogen receptor-alpha (Esr1) mediates estrogen action in regulating at all levels of the hypothalamic-pituitary-ovarian axis. Whereas the importance of Esr1 in hypothalamus and pituitary has been demonstrated by loss of fertility in the neuron- and pituitary-specific Esr1 knockout mice, whether Esr1 plays a critical role in the ovary remains to be determined. In the ovary, Esr1 is mainly expressed in the theca/interstitial cells and germinal epithelium and thus is believed to mediate estrogen action in these cells. In this study, we assessed the importance of Esr1 in the ovarian theca cells in regulating female reproduction. The Cre-LoxP approach was used to selectively delete the Esr1 gene in the theca cells, and the reproductive consequence of the deletion was measured. Adolescent theca-specific Esr1 knockout (thEsr1KO) mice (<4 months of age) are fertile and cycling. However, they begin to display an erratic pattern of estrous cycles and become infertile before they reach the age of 6 months. The ovaries of thEsr1KOmice (>or=4 months) have fewer corpora lutea but more antral follicles than the age-matching wild-type mice. The numbers of 17-hydroxylase-expressing cells are largely increased in the interstitium of the thEsr1KO mouse ovary. Interestingly, whereas basal levels of serum testosterone and FSH were mildly elevated, LH level was either markedly lower or undetectable in the thEsr1KO mice. When superstimulated by exogenous gonadotropins, thEsr1KO mice released significantly fewer oocytes that wild-type littermates and developed multiple hemorrhagic cysts. Taken together, this study demonstrates that theca Esr1 plays a critical role in regulating female reproduction.

  6. Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System

    PubMed Central

    Zhang, Tao; Yin, Yajun; Liu, Huan; Du, Weili; Ren, Chonghua; Wang, Ling; Lu, Hongzhao; Zhang, Zhiying

    2016-01-01

    CRISPR/Cas9 system has become a new versatile technology for genome engineering in various species. To achieve targeted modifications at the same site in both human and mice genomes by a CRISPR/Cas9 nuclease, we designed two target sites in conserved regions of vitamin D receptor (VDR) gene, which cover more than 17 kb of chromosome region depending on the species. We first validated the efficacy of single sgRNA mediated gene specific modifications were 36% and 31% in HEK293T cells. Concurrently, targeted of the intervening genomic segments deletions were generated in chromosomes when two sgRNAs worked simultaneously. The large genomic DNA segments up to 23.4 Kb could be precisely deleted in human chromosomes. Subsequently, Cas9 mRNA and sgRNAs targeting VDRT1 and VDRT2 were co-microinjected into one-cell-stage embryos of C57BL/6 mice. Verified by T7E1 assay and DNA sequencing analysis, 12 mice showed VDR targeted disruption and 8 of which were biallelic knock-out, which demonstrated obvious phenotype of hair thinning. Furthermore, expression changes of Vitamin D metabolism genes in VDR-/-mice were detected. These results indicated that CRISPR/Cas9 mediated knock-out of VDR diminished its gene function in vivo. The off-target effects of CRISPR/Cas9 in VDR-/- founder mice were analyzed. Our results showed that CRISPR/Cas9 system could be employed to target the same sites in different species, when sgRNAs are designed within conserved regions, and therefore will be critically important and applicable for human disease model. PMID:27685656

  7. Generation of a conditional knockout allele for the NFAT5 gene in mice.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx) ) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre (+)) under the control of the ubiqitinC promoter. The resultant homozygous conditional knockout mice (Cre (+) NFAT5 (flx/flx) ) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions. PMID:25601839

  8. Compensatory network alterations upon onset of epilepsy in synapsin triple knock-out mice.

    PubMed

    Ketzef, M; Kahn, J; Weissberg, I; Becker, A J; Friedman, A; Gitler, D

    2011-08-25

    Adult synapsin triple-knockout mice exhibit epilepsy that manifests as generalized tonic-clonic seizures. Because in vitro recordings have shown a reduction in quantal release from inhibitory neurons, an inherent excitation-inhibition imbalance has been hypothesized as the direct culprit for epilepsy in these mice. We critically assessed this hypothesis by examining neurotransmission during the emergence of epilepsy. Using long-term video and telemetric EEG monitoring we found that synapsin triple-knockout mice exhibit an abrupt transition during early adulthood from a seizure-free presymptomatic latent state to a consistent symptomatic state of sensory-induced seizures. Electrophysiological recordings showed that during the latent period larger field responses could be elicited in slices from mutant mice. However, only after the transition to a symptomatic state in the adult mice did evoked epileptiform activity become prevalent. This state was characterized by resistance to the epileptiform-promoting effects of 4-aminopyridine, by marked hypersensitivity to blockage of GABAA receptors, and by the emergence of unresponsiveness to NMDA receptor antagonism, all of which were not observed during the latent period. Importantly, enhancement in inhibitory transmission was associated with upregulation of GAD67 expression without affecting the number of inhibitory neurons in the same brain areas where epileptiform activity was recorded. We therefore suggest that while deletion of the synapsins initially increases cortical network activity, this enhanced excitability is insufficient to elicit seizures. Rather, compensatory epileptogenic mechanisms are activated during the latent period that lead to an additional almost-balanced enhancement of both the excitatory and inhibitory components of the network, finally culminating in the emergence of epilepsy. PMID:21621590

  9. Subchronic exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice.

    PubMed

    Weng, Zuquan; Suda, Megumi; Ohtani, Katsumi; Mei, Nan; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

    2013-09-15

    Ethyl tertiary butyl ether (ETBE) is biofuel additive recently used in Japan and some other countries. Limited evidence shows that ETBE has low toxicity. Acetaldehyde (AA), however, as one primary metabolite of ETBE, is clearly genotoxic and has been considered to be a potential carcinogen. The aim of this study was to evaluate the effects of ALDH2 gene on ETBE-induced genotoxicity and metabolism of its metabolites after inhalation exposure to ETBE. A group of wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice were exposed to 500ppm ETBE for 1-6h, and the blood concentrations of ETBE metabolites, including AA, tert-butyl alcohol and 2-methyl-1,2-propanediol, were measured. Another group of mice of WT and KO were exposed to 0, 500, 1750, or 5000ppm ETBE for 6h/day with 5 days per weeks for 13 weeks. Genotoxic effects of ETBE in these mice were measured by the alkaline comet assay, 8-hydroxyguanine DNA-glycosylase modified comet assay and micronucleus test. With short-term exposure to ETBE, the blood concentrations of all the three metabolites in KO mice were significantly higher than the corresponding concentrations of those in WT mice of both sexes. After subchronic exposure to ETBE, there was significant increase in DNA damage in a dose-dependent manner in KO male mice, while only 5000ppm exposure significantly increased DNA damage in male WT mice. Overall, there was a significant sex difference in genetic damage in both genetic types of mice. These results showed that ALDH2 is involved in the detoxification of ETBE and lack of enzyme activity may greatly increase the sensitivity to the genotoxic effects of ETBE, and male mice were more sensitive than females. PMID:23810710

  10. Mood and memory-associated behaviors in neuropeptide Y5 knockout mice.

    PubMed

    Ito, Masanobu; Dumont, Yvan; Quirion, Remi

    2013-04-01

    Recent data led to suggest that in addition to Y1 and Y2 subtypes, Y5 receptors may be involved in mood-related behaviors (Morales-Medina et al., 2010). In the present study, using a battery of behavioral tests to assess anxiety and depression-like paradigms, as well as memory function, we evaluated the potential behavioral changes induced in mice devoid of Y5 receptors. Those paradigms were assessed using the open field (OF), elevated plus maze (EPM), forced swim test (FST), social interaction test (SI), object recognition test (ORT) and Morris water maze (MWM) in Y5 knockout (KO) mice and wild type (WT) animals. In the tests associated to anxiety related behaviors (OF, EPM and SI), no difference for locomotion and time spent in the lateral area of open field were observed between Y5 KO and WT mice. Similar results were observed for time and number of entries in open arms in EPM. Additionally, in SI test, Y5 KO mice spent same amount of time and number of entries in the stranger chamber as compared to WT animals. In the FST, as compared to WT mice, Y5 KO mice had similar immobility time on day 1. No memory dysfunction was observed in the MWM and ORT in Y5 KO mice, as compared to WT. Altogether these data suggest that under basal conditions Y5 KO and WT mice display similar mood behaviors and memory functions. However, as compared to WT, Y5 KO mice display increased grooming and rearing in the OF, lower ratio entries in open arms in the EPM and increased immobility time on the second day of the FST.

  11. Attenuation of lithium-induced natriuresis and kaliuresis in P2Y2 receptor knockout mice

    PubMed Central

    Zhang, Yue; Li, Lijun; Kohan, Donald E.; Ecelbarger, Carolyn M.

    2013-01-01

    Whole body knockout (KO) of the P2Y2 receptor (P2Y2R) results in enhanced vasopressin V2 receptor activity and increased renal Na+ conservation. We hypothesized that P2Y2R KO mice would be less sensitive to lithium-induced natriuresis and kaliuresis due to attenuated downregulation of one or more of the major renal Na+ or K+ transporter/channel proteins. KO and wild-type (WT) mice were fed a control or lithium-added diet (40 mmol/kg food) for 14 days. Lithium-induced natriuresis and kaliuresis were significantly (∼25%) attenuated in KO mice. The subunits of the epithelial Na+ channel (ENaC) were variably affected by lithium and genotype, but, overall, medullary levels were decreased substantially by lithium (15–60%) in both genotypes. In contrast, cortical, β-, and γ-ENaC were increased by lithium (∼50%), but only in WT mice. Moreover, an assessment of ENaC activity by benzamil sensitivity suggested that lithium increased ENaC activity in WT mice but in not KO mice. In contrast, medullary levels of Na+-K+-2Cl− cotransporter 2 and cortical levels of the renal outer medullary K+ channel were not downregulated by lithium and were significantly (15–76%) higher in KO mice under both dietary conditions. In addition, under control conditions, tissue osmolality of the inner medulla as well as furosemide sensitivity were significantly higher in KO mice versus WT mice. Therefore, we suggest that increased expression of these proteins, particularly in the control state, reduces Na+ delivery to the distal nephron and provides a buffer to attenuate collecting duct-mediated natriuresis and kaliuresis. Additional studies are warranted to explore the potential therapeutic benefits of purinergic antagonism. PMID:23739592

  12. Complexin 1 knockout mice exhibit marked deficits in social behaviours but appear to be cognitively normal.

    PubMed

    Drew, Cheney J G; Kyd, Rachel J; Morton, A Jennifer

    2007-10-01

    Complexins are presynaptic proteins that modulate neurotransmitter release. Abnormal expression of complexin 1 (Cplx1) is seen in several neurodegenerative and psychiatric disorders in which disturbed social behaviour is commonplace. These include Parkinsons's disease, Alzheimer's disease, schizophrenia, major depressive illness and bipolar disorder. We wondered whether changes in Cplx1 expression contribute to the psychiatric components of the diseases in which Cplx1 is dysregulated. To investigate this, we examined the cognitive and social behaviours of complexin 1 knockout mice (Cplx1(-/-)) mice. Cplx1(-/-) mice have a profound ataxia that limits their ability to perform co-ordinated motor tasks. Nevertheless, when we taught juvenile Cplx1(-/-) mice to swim, they showed no evidence of cognitive impairment in the two-choice swim tank. In contrast, although olfactory discrimination in Cplx1(-/-) mice was normal, Cplx1(-/-) mice failed in the social transmission of food preference task, another cognitive paradigm. This was due to abnormal social interactions rather than cognitive impairments, increased anxiety or neophobia. When we tested social behaviour directly, Cplx1(-/-) mice failed to demonstrate a preference for social novelty. Further, in a resident-intruder paradigm, male Cplx1(-/-) mice failed to show the aggressive behaviour that is typical of wild-type males towards an intruder mouse. Together our results show that in addition to the severe motor and exploratory deficits already described, Cplx1(-/-) mice have pronounced deficits in social behaviours. Abnormalities in complexin 1 levels in the brain may therefore contribute to the psycho-social aspects of human diseases in which this protein is dysregulated.

  13. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

    PubMed Central

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

  14. Salivary Gland Dysplasia in Fgf10 Heterozygous Mice: A New Mouse Model of Xerostomia

    PubMed Central

    May, A.J.; Chatzeli, L.; Proctor, G.B.; Tucker, A.S.

    2016-01-01

    Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia.

  15. Salivary Gland Dysplasia in Fgf10 Heterozygous Mice: A New Mouse Model of Xerostomia.

    PubMed

    May, A J; Chatzeli, L; Proctor, G B; Tucker, A S

    2015-01-01

    Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia. PMID:26321752

  16. Intragastric fat self-administration is impaired in GPR40/120 double knockout mice

    PubMed Central

    Sclafani, Anthony; Touzani, Khalid; Ackroff, Karen

    2015-01-01

    Mice acquire strong preferences for flavors paired with intragastric (IG) fat infusions. This IG fat conditioning is attenuated in double knockout (DoKO) mice missing GPR40 and GPR120 fatty acid receptors. Here we determined if GPR40/120 DoKO mice are also impaired in IG fat self-administration in an operant lick task. In daily 1-h sessions the mice were trained with a sipper spout that contained dry food pellets; licks on the spout triggered infusions of IG fat (Intralipid). The training sessions were followed by test sessions with an empty spout. GPR40/120 DoKO mice self-infused more 20% fat than wild type (WT) C57BL/6 mice in training with a food-baited spout (2.4 vs. 2.0 kcal/h) but self-infused less 20% fat than WT mice in empty spout tests (1.2 vs. 1.7 kcal/h). The DoKO mice also self-infused less 5% fat than WT mice (0.6 vs. 1.3 kcal/h) although both groups emitted more licks for 5% fat than 20% fat. The DoKO and WT mice did not differ, however, in their self-infusion of 12.5% glucose (1.5 vs. 1.6 kcal/h), which is isocaloric to 5% fat. A second 5% IL test showed that the DoKO mice reverted to a reduced self-infusion compared to WT mice. When the infusion was shifted to water, WT mice reduced licking in the first extinction session, whereas DoKO mice were less sensitive to the absence of infused fat. Our results indicate that post-oral GPR40/120 signaling is not required to process IG fat infusions in food-baited spout training sessions but contributes to post-oral fat reinforcement in empty spout tests and flavor conditioning tests. PMID:25911263

  17. Heterozygous Ambra1 Deficiency in Mice: A Genetic Trait with Autism-Like Behavior Restricted to the Female Gender

    PubMed Central

    Dere, Ekrem; Dahm, Liane; Lu, Derek; Hammerschmidt, Kurt; Ju, Anes; Tantra, Martesa; Kästner, Anne; Chowdhury, Kamal; Ehrenreich, Hannelore

    2014-01-01

    Autism-spectrum disorders (ASD) are heterogeneous, highly heritable neurodevelopmental conditions affecting around 0.5% of the population across cultures, with a male/female ratio of approximately 4:1. Phenotypically, ASD are characterized by social interaction and communication deficits, restricted interests, repetitive behaviors, and reduced cognitive flexibility. Identified causes converge at the level of the synapse, ranging from mutation of synaptic genes to quantitative alterations in synaptic protein expression, e.g., through compromised transcriptional or translational control. We wondered whether reduced turnover and degradation of synapses, due to deregulated autophagy, would lead to similar phenotypical consequences. Ambra1, strongly expressed in cortex, hippocampus, and striatum, is a positive regulator of Beclin1, a principal player in autophagosome formation. While homozygosity of the Ambra1 null mutation causes embryonic lethality, heterozygous mice with reduced Ambra1 expression are viable, reproduce normally, and lack any immediately obvious phenotype. Surprisingly, comprehensive behavioral characterization of these mice revealed an autism-like phenotype in Ambra1+/− females only, including compromised communication and social interactions, a tendency of enhanced stereotypies/repetitive behaviors, and impaired cognitive flexibility. Reduced ultrasound communication was found in adults as well as pups, which achieved otherwise normal neurodevelopmental milestones. These features were all absent in male Ambra1+/− mice. As a first hint explaining this gender difference, we found a much stronger reduction of Ambra1 protein in the cortex of Ambra1+/− females compared to males. To conclude, Ambra1 deficiency can induce an autism-like phenotype. The restriction to the female gender of autism-generation by a defined genetic trait is unique thus far and warrants further investigation. PMID:24904333

  18. Pituitary gonadotrophic hormone synthesis, secretion, subunit gene expression and cell structure in normal and follicle-stimulating hormone β knockout, follicle-stimulating hormone receptor knockout, luteinising hormone receptor knockout, hypogonadal and ovariectomised female mice.

    PubMed

    Abel, M H; Widen, A; Wang, X; Huhtaniemi, I; Pakarinen, P; Kumar, T R; Christian, H C

    2014-11-01

    To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic-pituitary-gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle-stimulating hormone β knockout (FSHβKO), follicle-stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild-type or heterozygote female mice. Serum levels of LH were elevated in FSHβKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the FSHβ and LHβ subunit genes in FSHRKO female mice. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHβ and FSHβ mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin-releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were

  19. Gender-specific alteration of adrenergic responses in small femoral arteries from estrogen receptor-beta knockout mice.

    PubMed

    Luksha, Leonid; Poston, Lucilla; Gustafsson, Jan-Ake; Aghajanova, Lusine; Kublickiene, Karolina

    2005-11-01

    Estrogen receptor-beta knockout mice become hypertensive as they age, and males have a higher blood pressure than females. We hypothesized that the absence of estrogen receptor-beta may contribute to development of cardiovascular dysfunction by modification of adrenergic responsiveness in the peripheral vasculature. Small femoral arteries (internal diameter <200 microm) were isolated from estrogen receptor-beta knockout and wild-type mice and mounted on a wire myograph. Concentration-response curves to phenylephrine and norepinephrine were compared and the contribution of adrenoceptor subtypes established using specific agonists and antagonists. The involvement of endothelial factors in the modulation of resting tone was also investigated and immunohistochemical analysis used to confirm the presence or absence of estrogen receptor expression. Compared with wild type, arteries from estrogen receptor-beta knockout male, but not female, mice demonstrated gender-specific enhancement of the response to phenylephrine (alpha1-adrenoceptor agonist), which was accompanied by elevated basal tension attributable to endothelial factors. Contractile responses to the mixed adrenoceptor agonist norepinephrine did not differ significantly between estrogen receptor-beta knockout and wild type; however, beta-adrenoceptor inhibition unmasked an enhanced underlying alpha1-adrenoceptor responsiveness in estrogen receptor-beta knockout males. beta-adrenoceptor-mediated dilatation was also enhanced in estrogen receptor-beta knockout versus wild-type males. We suggest that estrogen receptor-beta modifies the adrenergic control of small artery tone in males but not in females.

  20. Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Iijima, Mikio; Li, Meng Xian; Kobayashi, Keiko; Horiuchi, Masahisa; Ushikai, Miharu; Okumura, Fumihiko; Meng, Xiao Jian; Inoue, Ituro; Tajima, Atsushi; Moriyama, Mitsuaki; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Tsui, Lap-Chee; Tsuji, Mihoko; Okano, Akira; Kobayashi, Tsuyoshi

    2007-08-24

    Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

  1. Only minor differences in renal osteodystrophy features between wild-type and sclerostin knockout mice with chronic kidney disease.

    PubMed

    Cejka, Daniel; Parada-Rodriguez, Diego; Pichler, Stefanie; Marculescu, Rodrig; Kramer, Ina; Kneissel, Michaela; Gross, Thomas; Reisinger, Andreas; Pahr, Dieter; Monier-Faugere, Marie-Claude; Haas, Martin; Malluche, Hartmut H

    2016-10-01

    Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice. PMID:27528549

  2. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    SciTech Connect

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  3. Ethanol metabolism in ALDH2 knockout mice--blood acetate levels.

    PubMed

    Kiyoshi, Ameno; Weihuan, Wang; Mostofa, Jamal; Mitsuru, Kumihashi; Toyoshi, Isse; Toshihiro, Kawamoto; Kyoko, Kitagawa; Keiichi, Nakayama; Iwao, Ijiri; Hiroshi, Kinoshita

    2009-04-01

    We described here blood acetate levels in aldehyde dehydrogenase 2 knockout (ALDH2 KO) male mice based on C57BL/6J strain after ethanol (EtOH) dosing (2 g/kg). Blood samples were collected at 30, 60, 90, 120 180, and 240 min after decapitation, and then EtOH, acetaldehyde (AcH) and acetate were determined by head-space gas chromatography. We found that blood acetate levels in ALDH2 KO mice were slightly lower than those in wild type (WT), whereas EtOH and AcH levels in ALDH2 KO were significantly higher than those in WT. These observations indicate that high EtOH, AcH and low acetate in the blood of ALDH2 KO are due to the deficient effect of ALDH2 enzyme activity. PMID:19356968

  4. Reduced ethanol consumption and preference in cocaine- and amphetamine-regulated transcript (CART) knockout mice.

    PubMed

    Salinas, Armando G; Nguyen, Chinh T Q; Ahmadi-Tehrani, Dara; Morrisett, Richard A

    2014-03-01

    Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide implicated in addiction to drugs of abuse. Several studies have characterized the role of CART in addiction to psychostimulants, but few have examined the role of CART in alcohol use disorders including alcoholism. The current study utilized a CART knockout (KO) mouse model to investigate the role of CART in ethanol appetitive behaviors. A two-bottle choice, unlimited-access paradigm was used to compare ethanol appetitive behaviors between CART wild type (WT) and KO mice. The mice were presented with an ethanol solution (3%-21%) and water, each concentration for 4 days, and their consumption was measured daily. Consumption of quinine (bitter) and saccharin (sweet) solutions was measured following the ethanol preference tests. In addition, ethanol metabolism rates and ethanol sensitivity were compared between genotypes. CART KO mice consumed and preferred ethanol less than their WT counterparts in both sexes. This genotype effect could not be attributed to differences in bitter or sweet taste perception or ethanol metabolism rates. There was also no difference in ethanol sensitivity in male mice; however, CART KO female mice showed a greater ethanol sensitivity than the WT females. Taken together, these data demonstrate a role for CART in ethanol appetitive behaviors and as a possible therapeutic drug target for alcoholism and abstinence enhancement.

  5. Apolipoprotein E knockout induced inflammatory responses related to microglia in neonatal mice brain via astrocytes

    PubMed Central

    Liu, Yimei; Xu, Xiaohua; Dou, Hongbo; Hua, Ying; Xu, Jinwen; Hui, Xu

    2015-01-01

    More and more evidences suggestted that ApoE plays an important role in modulating the systemic and central nervous inflammatory responses. However, there is a lack of exacted mechanism of ApoE. In this study, we aimed to investigate whether apolipoprotein E (ApoE) induced inflammatory responses and apoptosis in neonatal mice brain from ApoE deficient (ApoE-/-) and wildtype (WT). Compared to control group, the microglia cell from ApoE-/- mice showed more severe inflammation and cell death such as iNOS and IL-1β. Furthermore, anti-inflammatory such as TGF-β, IL-10 from microglia and astrocytes in ApoE-/- mice were decreased. On the other way, TGF-β from astrocytes can inhibit inflammation factors secretion from microglia. Our findings suggested that the anti- inflammation factor such as IL-10 mainly from microglia and TGF-β mainly from astrocyte is significant decreased after Loss of ApoE function in ApoE-/- mice which induced severe inflammation. Furthrtmore, anti- inflammation factor such as IL-10 and TGF-β Therefore, we conclude that apolipoprotein E knockout induced inflammatory responses related to microglia in neonatal mice brain via astrocytes. PMID:25785051

  6. Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

    PubMed Central

    Takao, Keizo; Tanda, Koichi; Nakamura, Kenji; Kasahara, Jiro; Nakao, Kazuki; Katsuki, Motoya; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Adachi, Minami; Umeda, Masahiro; Araki, Tsutomu; Fukunaga, Kohji; Kondo, Hisatake; Sakagami, Hiroyuki; Miyakawa, Tsuyoshi

    2010-01-01

    Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency. PMID:20209163

  7. Epilepsy in Dcx Knockout Mice Associated with Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

    PubMed Central

    Denis, Cécile; Germain, Johanne; Dinh Tuy, Françoise Phan; Verstraeten, Soraya; Alvarez, Chantal; Métin, Christine; Chelly, Jamel; Giros, Bruno; Miles, Richard; Depaulis, Antoine; Francis, Fiona

    2008-01-01

    Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities. PMID:18575605

  8. Serotonergic involvement in the amelioration of behavioral abnormalities in dopamine transporter knockout mice by nicotine.

    PubMed

    Uchiumi, Osamu; Kasahara, Yoshiyuki; Fukui, Asami; Hall, F Scott; Uhl, George R; Sora, Ichiro

    2013-01-01

    Dopamine transporter knockout (DAT KO) mice exhibit elevated extracellular dopamine levels in brain regions that include the striatum and the nucleus accumbens, but not the prefrontal cortex. DAT KO mice model some aspects of psychiatric disorders, including schizophrenia. Smoking is more common in patients with schizophrenia, suggesting that nicotine might ameliorate aspects of the behavioral abnormalities and/or treatment side effects seen in these individuals. We report nicotine-induced normalization of effects on locomotion and prepulse inhibition of acoustic startle (PPI) in DAT KO mice that require intact serotonin 5-HT1A systems. First, we observed that the marked hyperactivity displayed by DAT KO mice was reduced by administration of nicotine. This nicotine effect was blocked by pretreatment with the non-specific nicotinic acetylcholine (nACh) receptor antagonist mecamylamine, or the 5-HT1A antagonist WAY100635. Secondly, we examined the effects of nicotine on PPI in DAT KO mice. Treatment with nicotine significantly ameliorated the PPI deficits observed in DAT KO mice. The ameliorating action of nicotine on PPI deficits in DAT KO mice was blocked by mecamylamine, the α₇ nACh receptor antagonist methyllycaconitine or WAY100635, while the α₄β₂ nACh receptor antagonist dihydro-β-erythroidinehydrobromide (DHβE) produced only a non-significant trend toward attenuation of nicotine effects. Finally, we observed that administration of the 5-HT1A receptor agonist 8-OH-DPAT also ameliorated the deficit in PPI observed in DAT KO mice. This amelioration was antagonized by pretreatment with WAY100635. These data support the idea that nicotine might ameliorate some of the cognitive dysfunctions found in schizophrenia in a 5-HT1A-dependent fashion. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

  9. Impaired spatial learning and reduced adult hippocampal neurogenesis in histamine H1-receptor knockout mice.

    PubMed

    Ambrée, Oliver; Buschert, Jens; Zhang, Weiqi; Arolt, Volker; Dere, Ekrem; Zlomuzica, Armin

    2014-08-01

    The histamine H1-receptor (H1R) is expressed in wide parts of the brain including the hippocampus, which is involved in spatial learning and memory. Previous studies in H1R knockout (H1R-KO) mice revealed deficits in a variety of learning and memory tasks. It was also proposed that H1R activation is crucial for neuronal differentiation of neural progenitors. Therefore, the aim of this study was to investigate negatively reinforced spatial learning in the water-maze and to assess survival and neuronal differentiation of newborn cells in the adult hippocampus of H1R-KO mice. H1R-KO and wild-type (WT) mice were subjected to the following sequence of tests: (a) cued version, (b) place learning, (c) spatial probe, (d) long-term retention and (e) reversal learning. Furthermore hippocampal neurogenesis in terms of survival and differentiation was assessed in H1R-KO and WT mice. H1R-KO mice showed normal cued learning, but impaired place and reversal learning as well as impaired long-term retention performance. In addition, a marked reduction of newborn neurons in the hippocampus but no changes in differentiation of neural progenitors into neuronal and glial lineage was found in H1R-KO mice. Our data suggest that H1R deficiency in mice is associated with pronounced deficits in hippocampus-dependent spatial learning and memory. Furthermore, we herein provide first evidence that H1R deficiency in the mouse leads to a reduced neurogenesis. However, the exact mechanisms for the reduced number of cells in H1R-KO mice remain elusive and might be due to a reduced survival of newborn hippocampal neurons and/or a reduction in cell proliferation.

  10. Impaired striatum-dependent behavior in GASP-1-knock-out mice.

    PubMed

    Mathis, C; Bott, J-B; Candusso, M-P; Simonin, F; Cassel, J-C

    2011-04-01

    G protein-coupled receptor (GPCR) associated sorting protein-1 (GASP-1) is suspected to play a key role in recycling and degradation of several GPCRs. In a previous study, we have shown that GASP-1-knock-out (GASP-1-KO) mice displayed deficits in acquiring a cocaine self-administration task, associated with an exacerbated down-regulation of striatal dopaminergic and cholinergic receptors. Among several possibilities, GASP-1 deficiency could have impaired memory processes underlying the acquisition of the operant conditioning task. Therefore, the present study investigated cognitive performances of GASP-1-KO mice and their wild-type littermates (WT) in a broad variety of memory tasks. Consistent with a deficit in procedural memory, GASP-1-KO mice showed delayed acquisition of a food-reinforced bar-press task. During water-maze training in hidden- or visible-platform paradigms, mutant and WT mice acquired the tasks at the same rate. However, GASP-1 mice exhibited persistent thigmotaxic swimming, longer distance to the platform, and reduced swim speed. There was no deficit in several tasks requiring simple behavioral responses (Barnes maze, object recognition and passive avoidance tasks). Thus, the ability to acquire and/or express complex responses seems affected in GASP-1-deficient mice. Hippocampal functions were preserved, as the retention of an acquired memory in spatial tasks remained unaffected. The pattern of behavioral deficits observed in GASP-1-KO mice is coherent with current knowledge on the role of striatal GPCRs in acquisition/expression of skilled behavior and in motivation. Together with the previous findings, the so far established phenotype of GASP-1-KO mice makes them a potentially exciting tool to study striatal functions.

  11. Changes in attack behavior and activity in EphA5 knockout mice.

    PubMed

    Mamiya, Ping Chao; Hennesy, Zach; Zhou, Renping; Wagner, George C

    2008-04-18

    During development, Eph tyrosine kinase receptors and their ephrin ligands function as axon guidance molecules while, in adults, these molecules appear to be involved in the regulation of neural plasticity and emotion. The absence of EphA5 receptor mediated forward signaling may cause alterations in connectivity of neural networks and boundary formation during development, including central monoaminergic systems. In the present studies, we demonstrated altered aggressive responses by animals lacking functional EphA5 receptors. These behavioral changes were accompanied by altered concentrations of serotonin (5-HT) and the metabolite, 5-HIAA, in the hypothalamus. The changes of serotonin activity in hypothalamus also result in increase of body weight in EphA5 knockout mice. Furthermore, EphA5 knockout mice exhibited a significant decrease in activity levels following exposure to naïve intruders in their home cages. We conclude that the EphA5 receptor may be involved in mediation of aggressive behavior regulated, in part, by hypothalamic serotonin. PMID:18353288

  12. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice

    PubMed Central

    Belizário, José; Vieira-Cordeiro, Luiz; Enns, Sylvia

    2015-01-01

    Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca2+ overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain-like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice. PMID:26491219

  13. Retinal Ganglion Cell Loss is Delayed Following Optic Nerve Crush in NLRP3 Knockout Mice

    PubMed Central

    Puyang, Zhen; Feng, Liang; Chen, Hui; Liang, Peiji; Troy, John B.; Liu, Xiaorong

    2016-01-01

    The NLRP3 inflammasome, a sensor for a variety of pathogen- and host-derived threats, consists of the adaptor ASC (Apoptosis-associated Speck-like protein containing a Caspase Activation and Recruitment Domain (CARD)), pro-caspase-1, and NLRP3 (NOD-Like Receptor family Pyrin domain containing 3). NLRP3-induced neuroinflammation is implicated in the pathogenesis and progression of eye diseases, but it remains unclear whether activation of NLRP3 inflammasome contributes to retinal ganglion cell (RGC) death. Here we examined NLRP3-induced neuroinflammation and RGC survival following partial optic nerve crush (pONC) injury. We showed that NLRP3 was up-regulated in retinal microglial cells following pONC, propagating from the injury site to the optic nerve head and finally the entire retina within one day. Activation of NLRP3-ASC inflammasome led to the up-regulation of caspase-1 and a proinflammatory cytokine, interleukin-1β (IL-1β). In NLRP3 knockout mice, up-regulation of ASC, caspase-1, and IL-1β were all reduced, and, importantly, RGC and axon loss was substantially delayed following pONC injury. The average survival time of RGCs in NLRP3 knockout mice was about one week longer than for control animals. Taken together, our study demonstrated that ablating the NLRP3 gene significantly reduced neuroinflammation and delayed RGC loss after optic nerve crush injury. PMID:26893104

  14. Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice

    NASA Technical Reports Server (NTRS)

    Weil, M. M.; Kittrell, F. S.; Yu, Y.; McCarthy, M.; Zabriskie, R. C.; Ullrich, R. L.

    2001-01-01

    Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

  15. Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

    PubMed

    Osaki, Yoshinori; Nakagawa, Yoshimi; Miyahara, Shoko; Iwasaki, Hitoshi; Ishii, Akiko; Matsuzaka, Takashi; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Sone, Hirohito; Ohashi, Ken; Ishibashi, Shun; Yamada, Nobuhiro; Shimano, Hitoshi

    2015-10-23

    HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs.

  16. CD8 Knockout Mice Are Protected from Challenge by Vaccination with WR201, a Live Attenuated Mutant of Brucella melitensis

    PubMed Central

    Yingst, Samuel L.; Hoover, David L.

    2013-01-01

    CD8+ T cells have been reported to play an important role in defense against B. abortus infection in mouse models. In the present report, we use CD8 knockout mice to further elucidate the role of these cells in protection from B. melitensis infection. Mice were immunized orally by administration of B. melitensis WR201, a purine auxotrophic attenuated vaccine strain, then challenged intranasally with B. melitensis 16M. In some experiments, persistence of WR201 in the spleens of CD8 knockout mice was slightly longer than that in the spleens of normal mice. However, development of anti-LPS serum antibody, antigen-induced production of γ-interferon (IFN-γ) by immune splenic lymphocytes, protection against intranasal challenge, and recovery of nonimmunized animals from intranasal challenge were similar between normal and knockout animals. Further, primary Brucella infection was not exacerbated in perforin knockout and Fas-deficient mice and these animals' anti-Brucella immune responses were indistinguishable from those of normal mice. These results indicate that CD8+ T cells do not play an essential role as either cytotoxic cells or IFN-γ producers, yet they do participate in a specific immune response to immunization and challenge in this murine model of B. melitensis infection. PMID:24288554

  17. Analysis of development of lesions in mice with serine palmitoyltransferase (SPT) deficiency -Sptlc2 conditional knockout mice-.

    PubMed

    Ohta, Etsuko; Ohira, Takashi; Matsue, Kenta; Ikeda, Yuika; Fujii, Kenji; Ohwaki, Kenji; Osuka, Sou; Hirabayashi, Yoshio; Sasaki, Minoru

    2009-10-01

    Serine palmitoyltransferase (SPT) is the enzyme which catalyzes the first step of the biosynthesis of sphingolipids. However, the precise roles of SPT in vivo are not well understood, since complete knockout (KO) of genes which compose SPT results in a fetal lethal phenotype. A conditional KO (cKO) mouse of SPT long chain base 2 (Sptlc2) was therefore developed, and the effects of Sptlc2 deficiency were examined. Single cell necrosis in the epithelia of the crypts of the small and large intestines was observed as early as 24 h after induction of knockout. At 48 h after induction, decreases in spleen and thymus weights and decreases in numbers of reticulocytes and lymphocytes were observed in cKO mice, and single cell necrosis in the intestine became prominent. At 72 h after induction, decreases in body weight, spleen and thymus weights, and numbers of reticulocytes and lymphocytes became obvious in cKO mice. Histologically, atrophy of gastrointestinal mucosa and lymphoid necrosis as well as depletion of lymphoid and hematopoietic tissues were observed. These findings suggest that SPT plays important roles in the maintenance of the gastrointestinal mucosa, especially in the proliferation of the mucosal epithelial cells, and that deficiency of Sptlc2 induces necrotic lesions in gastrointestinal cells followed by atrophic change of the tissue in short term.

  18. Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1.

    PubMed

    Lynch, I Jeanette; Welch, Amanda K; Gumz, Michelle L; Kohan, Donald E; Cain, Brian D; Wingo, Charles S

    2015-12-15

    Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids. PMID:26400543

  19. Knockout Mice Reveal Key Roles for Claudin 18 in Alveolar Barrier Properties and Fluid Homeostasis

    PubMed Central

    Li, Guanglei; Flodby, Per; Luo, Jiao; Kage, Hidenori; Sipos, Arnold; Gao, Danping; Ji, Yanbin; Beard, LaMonta L.; Marconett, Crystal N.; DeMaio, Lucas; Kim, Yong Ho; Kim, Kwang-Jin; Laird-Offringa, Ite A.; Minoo, Parviz; Liebler, Janice M.; Zhou, Beiyun; Crandall, Edward D.

    2014-01-01

    Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability. PMID:24588076

  20. Acute behavioral effects of nicotine in male and female HINT1 knockout mice.

    PubMed

    Jackson, K J; Wang, J B; Barbier, E; Chen, X; Damaj, M I

    2012-11-01

    Human genetic association and brain expression studies, and mouse behavioral and molecular studies implicate a role for the histidine triad nucleotide-binding protein 1 (HINT1) in schizophrenia, bipolar disorder, depression and anxiety. The high comorbidity between smoking and psychiatric disorders, schizophrenia in particular, is well established. Associations with schizophrenia and HINT1 are also sex specific, with effects more predominant in males; however, it is unknown if sex differences associated with the gene extend to other phenotypes. Thus, in this study, using a battery of behavioral tests, we elucidated the role of HINT1 in acute nicotine-mediated behaviors using male and female HINT1 wild-type (+/+) and knockout (-/-) mice. The results show that male HINT1 -/- mice were less sensitive to acute nicotine-induced antinociception in the tail-flick, but not hot-plate test. At low nicotine doses, male and female HINT1 -/- mice were less sensitive to nicotine-induced hypomotility, although the effect was more pronounced in females. Baseline differences in locomotor activity observed in male HINT1 +/+ and -/- mice were absent in females. Nicotine did not produce an anxiolytic effect in male HINT1 -/- mice, but rather an anxiogenic response. Diazepam also failed to induce an anxiolytic response in these mice, suggesting a general anxiety phenotype not specific to nicotine. Differences in anxiety-like behavior were not observed in female mice. These results further support a role for HINT1 in nicotine-mediated behaviors and suggest that alterations in the gene may have differential effects on phenotype in males and females. PMID:22827509

  1. Impairment of Immune Response against Dematiaceous Fungi in Card9 Knockout Mice.

    PubMed

    Wu, Weiwei; Zhang, Ruijun; Wang, Xiaowen; Song, Yinggai; Liu, Zhengyang; Han, Wenling; Li, Ruoyu

    2016-10-01

    Dematiaceous fungi are a large group of pathogens that can cause a wide range of diseases in both immunocompetent and immunocompromised hosts. Based on our previous finding of caspase recruitment domain-containing protein 9 (CARD9) mutations in patients with subcutaneous phaeohyphomycosis caused by Phialophora verrucosa (P. verrucosa), we further investigated the exact role of CARD9 in the pathogenesis of phaeohyphomycosis using Card9 knockout (Card9 KO) mice. We showed that Card9 KO mice are profoundly susceptible to P. verrucosa infection compared with wild-type mice, reflected by significantly more severe footpad swelling, higher fungal burden, lower survival, and systemic dissemination. The inability of Card9 KO mice to control P. verrucosa infection was associated with lack of Th17 differentiation and reduction of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-17A levels in footpad homogenates. In vitro experiments showed a defect of fungal conidia killing and pro-inflammatory cytokine production in Card9 KO bone marrow-derived macrophages and dendritic cells. Furthermore, ex vivo coculture and in vitro T cell differentiation assay demonstrated that Card9 signaling pathway acts indispensably on differentiation of Th17 cells. In conclusion, our findings suggest that CARD9 mediate the innate immune and Th17-mediated adaptive immune responses against dematiaceous fungal infections at the early stage of infection.

  2. Increased sensitivity of apolipoprotein E knockout mice to copper-induced oxidative injury to the liver.

    PubMed

    Chen, Yuan; Li, Bin; Zhao, Ran-ran; Zhang, Hui-feng; Zhen, Chao; Guo, Li

    2015-04-10

    Apolipoprotein E (ApoE) genotypes are related to clinical presentations in patients with Wilson's disease, indicating that ApoE may play an important role in the disease. However, our understanding of the role of ApoE in Wilson's disease is limited. High copper concentration in Wilson's disease induces excessive generation of free oxygen radicals. Meanwhile, ApoE proteins possess antioxidant effects. We therefore determined whether copper-induced oxidative damage differ in the liver of wild-type and ApoE knockout (ApoE(-/-)) mice. Both wild-type and ApoE(-/-) mice were intragastrically administered with 0.2 mL of copper sulfate pentahydrate (200 mg/kg; a total dose of 4 mg/d) or the same volume of saline daily for 12 weeks, respectively. Copper and oxidative stress markers in the liver tissue and in the serum were assessed. Our results showed that, compared with the wild-type mice administered with copper, TBARS as a marker of lipid peroxidation, the expression of oxygenase-1 (HO-1), NAD(P)H dehydrogenase, and quinone 1 (NQO1) significantly increased in the ApoE(-/-) mice administered with copper, meanwhile superoxide dismutase (SOD) activity significantly decreased. Thus, it is concluded that ApoE may protect the liver from copper-induced oxidative damage in Wilson's disease.

  3. Heme oxygenase 1 protects ethanol-administered liver tissue in Aldh2 knockout mice.

    PubMed

    Matsumoto, Akiko; Thompson, David; Chen, Ying; Vasiliou, Vasilis; Kawamoto, Toshihiro; Ichiba, Masayoshi

    2016-05-01

    A genetic polymorphism of the aldehyde dehydrogenase 2 (​ALDH2) gene, ALDH2*2, encodes an enzymatically defective ALDH2 protein. Recent epidemiological studies suggest that possessing ALDH2*2 is a protective factor for liver tissue in healthy individuals, although these studies lack a mechanistic explanation. Our animal studies have shown the same trend: levels of serum alanine transaminase (ALT), hepatic malondialdehyde (MDA), and hepatic tumor necrosis factor alpha (TNF-α) were lower in Aldh2 knockout (Aldh2(-/-)) mice than in wild-type (Aldh2(+/+)) mice after ethanol administration. To propose a mechanistic hypothesis, residual liver specimens from the previous experiment were analyzed. An anti-oxidative protein, heme oxygenase 1 (HO-1), and an oxidative stress-producing protein, cytochrome P450 2E1 (CYP2E1), were detected at higher levels in Aldh2(-/-) mice than in Aldh2(+/+) mice, regardless of ethanol treatment. Other oxidative stress-related proteins and inflammatory cytokines did not show such a significant difference. To conclude, we propose a protective role of HO-1 in individuals with A​LDH2*2. Our continued studies support the epidemiological finding that possession of ALDH2*2 is a protective factor in the liver of the healthy individual. PMID:27139237

  4. Behavioral analysis of male and female Fmr1 knockout mice on C57BL/6 background.

    PubMed

    Ding, Qi; Sethna, Ferzin; Wang, Hongbing

    2014-09-01

    Fragile X syndrome (FXS) is a monogenic disease caused by mutations in the FMR1 gene. The Fmr1 knockout (KO) mice show many aspects of FXS-related phenotypes, and have been used as a major pre-clinical model for FXS. Although FXS occurs in both male and female patients, most studies on the mouse model use male animals. Few studies test whether gender affects the face validity of the mouse model. Here, we examined multiple behavioral phenotypes with male hemizygous and female homozygous Fmr1 KO mice on C57BL/6 background. For each behavioral paradigm, we examined multiple cohorts from different litters. We found that both male and female Fmr1 KO mice displayed significant audiogenic seizures, hyperactivity in the open field test, deficits in passive avoidance and contextual fear memory, and significant enhancement of PPI at low stimulus intensity. Male and female Fmr1 KO mice also showed more transitional movement between the lit and dark chambers in the light-dark tests. The lack of gender effects suggests that the Fmr1 KO mouse is a reasonable tool to test the efficacy of potential FXS therapies.

  5. Hypersomnolence and reduced activity in pan-leptin receptor knockout mice.

    PubMed

    Wang, Yuping; He, Junyun; Kastin, Abba J; Hsuchou, Hung; Pan, Weihong

    2013-11-01

    Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity.

  6. Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice.

    PubMed

    Pacini, Luca; Suffredini, Silvia; Ponti, Donatella; Coppini, Raffaele; Frati, Giacomo; Ragona, Giuseppe; Cerbai, Elisabetta; Calogero, Antonella

    2013-12-01

    Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-β) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.

  7. Hypersomnolence and reduced activity in pan-leptin receptor knockout mice

    PubMed Central

    Wang, Yuping; He, Junyun; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2013-01-01

    Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement (REM) sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity. PMID:23955775

  8. Enhanced insulin signaling in density-enhanced phosphatase-1 (DEP-1) knockout mice

    PubMed Central

    Krüger, Janine; Brachs, Sebastian; Trappiel, Manuela; Kintscher, Ulrich; Meyborg, Heike; Wellnhofer, Ernst; Thöne-Reineke, Christa; Stawowy, Philipp; Östman, Arne; Birkenfeld, Andreas L.; Böhmer, Frank D.; Kappert, Kai

    2015-01-01

    Objective Insulin resistance can be triggered by enhanced dephosphorylation of the insulin receptor or downstream components in the insulin signaling cascade through protein tyrosine phosphatases (PTPs). Downregulating density-enhanced phosphatase-1 (DEP-1) resulted in an improved metabolic status in previous analyses. This phenotype was primarily caused by hepatic DEP-1 reduction. Methods Here we further elucidated the role of DEP-1 in glucose homeostasis by employing a conventional knockout model to explore the specific contribution of DEP-1 in metabolic tissues. Ptprj−/− (DEP-1 deficient) and wild-type C57BL/6 mice were fed a low-fat or high-fat diet. Metabolic phenotyping was combined with analyses of phosphorylation patterns of insulin signaling components. Additionally, experiments with skeletal muscle cells and muscle tissue were performed to assess the role of DEP-1 for glucose uptake. Results High-fat diet fed-Ptprj−/− mice displayed enhanced insulin sensitivity and improved glucose tolerance. Furthermore, leptin levels and blood pressure were reduced in Ptprj−/− mice. DEP-1 deficiency resulted in increased phosphorylation of components of the insulin signaling cascade in liver, skeletal muscle and adipose tissue after insulin challenge. The beneficial effect on glucose homeostasis in vivo was corroborated by increased glucose uptake in skeletal muscle cells in which DEP-1 was downregulated, and in skeletal muscle of Ptprj−/− mice. Conclusion Together, these data establish DEP-1 as novel negative regulator of insulin signaling. PMID:25830095

  9. Alpha-asarone improves striatal cholinergic function and locomotor hyperactivity in Fmr1 knockout mice.

    PubMed

    Qiu, Guozhen; Chen, Shengqiang; Guo, Jialing; Wu, Jie; Yi, Yong-Hong

    2016-10-01

    Hyperactivity is a symptom found in several neurological and psychiatric disorders, including Fragile X syndrome (FXS). The animal model of FXS, fragile X mental retardation gene (Fmr1) knockout (KO) mouse, exhibits robust locomotor hyperactivity. Alpha (α)-asarone, a major bioactive component isolated from Acorus gramineus, has been shown in previous studies to improve various disease conditions including central nervous system disorders. In this study, we show that treatment with α-asarone alleviates locomotor hyperactivity in Fmr1 KO mice. To elucidate the mechanism underlying this improvement, we evaluated the expressions of various cholinergic markers, as well as acetylcholinesterase (AChE) activity and acetylcholine (ACh) levels, in the striatum of Fmr1 KO mice. We also analyzed the AChE-inhibitory activity of α-asarone. Striatal samples from Fmr1 KO mice showed decreased m1 muscarinic acetylcholine receptor (m1 mAChR) expression, increased AChE activity, and reduced ACh levels. Treatment with α-asarone improved m1 mAChR expression and ACh levels, and attenuated the increased AChE activity. In addition, α-asarone dose-dependently inhibited AChE activity in vitro. These results indicate that direct inhibition of AChE activity and up-regulation of m1 mAChR expression in the striatum might contribute to the beneficial effects of α-asarone on locomotor hyperactivity in Fmr1 KO mice. These findings might improve understanding of the neurobiological mechanisms responsible for locomotor hyperactivity.

  10. Increased sensitivity of apolipoprotein E knockout mice to copper-induced oxidative injury to the liver.

    PubMed

    Chen, Yuan; Li, Bin; Zhao, Ran-ran; Zhang, Hui-feng; Zhen, Chao; Guo, Li

    2015-04-10

    Apolipoprotein E (ApoE) genotypes are related to clinical presentations in patients with Wilson's disease, indicating that ApoE may play an important role in the disease. However, our understanding of the role of ApoE in Wilson's disease is limited. High copper concentration in Wilson's disease induces excessive generation of free oxygen radicals. Meanwhile, ApoE proteins possess antioxidant effects. We therefore determined whether copper-induced oxidative damage differ in the liver of wild-type and ApoE knockout (ApoE(-/-)) mice. Both wild-type and ApoE(-/-) mice were intragastrically administered with 0.2 mL of copper sulfate pentahydrate (200 mg/kg; a total dose of 4 mg/d) or the same volume of saline daily for 12 weeks, respectively. Copper and oxidative stress markers in the liver tissue and in the serum were assessed. Our results showed that, compared with the wild-type mice administered with copper, TBARS as a marker of lipid peroxidation, the expression of oxygenase-1 (HO-1), NAD(P)H dehydrogenase, and quinone 1 (NQO1) significantly increased in the ApoE(-/-) mice administered with copper, meanwhile superoxide dismutase (SOD) activity significantly decreased. Thus, it is concluded that ApoE may protect the liver from copper-induced oxidative damage in Wilson's disease. PMID:25749341

  11. The restructuring of muscarinic receptor subtype gene transcripts in c-fos knock-out mice.

    PubMed

    Benes, Jan; Mravec, Boris; Kvetnansky, Richard; Myslivecek, Jaromir

    2013-05-01

    Although c-Fos plays a key role in intracellular signalling, the disruption of the c-fos gene has only minor consequences on the central nervous system (CNS) function. As muscarinic receptors (MR) play important roles in many CNS functions (attention, arousal, and cognition), the c-fos knock-out might be compensated through MR changes. The aim of this study was to evaluate changes in the M1-M5 MR mRNA in selected CNS areas: frontal, parietal, temporal and occipital cortex, striatum, hippocampus, hypothalamus and cerebellum (FC, PC, TC, OC, stria, hip, hypo, and crbl, respectively). Knocking out the c-fos gene changed the expression of MR in FC (reduced M1R, M4R and M5R expression), TC (increased M4R expression), OC (decreased M2R and M3R expression) and hippocampus (reduced M3R expression). Moreover, gender differences were observed in WT mice: increased expression of all M1-M5R in the FC in males and M1-M4R in the striatum in females. A detailed analysis of MR transcripts showed pre-existing correlations in the amount of MR-mRNA between specific regions. WT mice showed three major types of cortico-cortical correlations: fronto-occipital, temporo-parietal and parieto-occipital. The cortico-subcortical correlations involved associations between the FC, PC, TC and striatum. In KO mice, a substantial rearrangement of the correlation pattern was observed: only a temporo-parietal correlation and correlations between the FC and striatum remained, and a new correlation between the hypothalamus and cerebellum appeared. Thus, in addition to the previously described dopamine receptor restructuring, the restructuring of MR mRNA correlations reveals an additional mechanism for adaptation to the c-fos gene knockout.

  12. Masticatory hypermuscularity is not related to reduced cranial volume in myostatin-knockout mice.

    PubMed

    Cray, James; Kneib, Jared; Vecchione, Lisa; Byron, Craig; Cooper, Gregory M; Losee, Joseph E; Siegel, Michael I; Hamrick, Mark W; Sciote, James J; Mooney, Mark P

    2011-07-01

    It has been suggested recently that masticatory muscle size reduction in humans resulted in greater encephalization through decreased compressive forces on the cranial vault. Following this logic, if masticatory muscle size were increased, then a reduction in brain growth should also occur. This study was designed to test this hypothesis using a myostatin (GDF-8) knockout mouse model. Myostatin is a negative regulator of skeletal muscle growth, and individuals lacking this gene show significant hypermuscularity. Sixty-two [32 wild-type (WT) and 30 GDF-8 -/- knockout], 1, 28, 56, and 180-day-old CD-1 mice were used. Body and masseter muscle weights were collected following dissection and standardized lateral and dorsoventral cephalographs were obtained. Cephalometric landmarks were identified on the radiographs and cranial volume was calculated. Mean differences were assessed using a two-way ANOVA. KO mice had significantly greater body and masseter weights beginning at 28 days compared with WT controls. No significant differences in cranial volumes were noted between KO and WT. Muscle weight was not significantly correlated with cranial volume in 1, 28, or 180-day-old mice. Muscle weights exhibited a positive correlation with cranial volume at 56 days. Results demonstrate that masticatory hypermuscularity is not associated with reduced cranial volume. In contrast, there is abundant data demonstrating the opposite, brain growth determines cranial vault growth and masticatory apparatus only affects ectocranial morphology. The results presented here do not support the hypothesis that a reduction in masticatory musculature relaxed compressive forces on the cranial vault allowing for greater encephalization.

  13. MKP-1 knockout does not prevent glucocorticoid-induced bone disease in mice.

    PubMed

    Conradie, Maria M; Cato, Andrew C B; Ferris, William F; de Wet, Heidi; Horsch, Kay; Hough, Stephen

    2011-09-01

    Glucocorticoid-induced osteoporosis (GCOP) is predominantly caused by inhibition of bone formation, resulting from a decrease in osteoblast numbers. Employing mouse (MBA-15.4) and human (MG-63) osteoblast cell lines, we previously found that the glucocorticoid (GC) dexamethasone (Dex) inhibits cellular proliferation as well as activation of the MAPK/ERK signaling pathway, essential for mitogenesis in these cells, and that both these effects could be reversed by the protein tyrosine phosphatase (PTP) inhibitor vanadate. In a rat model of GCOP, the GC-induced changes in bone formation, mass, and strength could be prevented by vanadate cotreatment, suggesting that the GC effects on bone were mediated by one or more PTPs. Employing phosphatase inhibitors, qRT-PCR, Western blotting, and overexpression/knockdown experiments, we concluded that MKP-1 was upregulated by Dex, that this correlated with the dephosphorylation of ERK, and that it largely mediated the in vitro effects of GCs on bone. To confirm the pivotal role of MKP-1 in vivo, we investigated the effects of the GC methylprednisolone on the quantitative bone histology of wild-type (WT) and MKP-1 homozygous knockout (MKP-1(-/-)) mice. In WT mice, static bone histology revealed that GC administration for 28 days decreased osteoid surfaces, volumes, and osteoblast numbers. Dynamic histology, following time-spaced tetracycline labeling, confirmed a significant GC-induced reduction in osteoblast appositional rate and bone formation rate. However, identical results were obtained in MKP-1 knockout mice, suggesting that in these animals upregulation of MKP-1 by GCs cannot be regarded as the sole mediator of the GC effects on bone. PMID:21698455

  14. CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory.

    PubMed

    Li, Yong; Kim, Jimok

    2016-01-01

    Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas.

  15. CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory

    PubMed Central

    Li, Yong; Kim, Jimok

    2016-01-01

    Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas. PMID:26819779

  16. What have we learned about GPER function in physiology and disease from knockout mice?

    PubMed Central

    Prossnitz, Eric R.; Hathaway, Helen J.

    2015-01-01

    Estrogens, predominantly 17β-estradiol, exert diverse effects throughout the body in both normal and patho-physiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERβ) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERβ KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also revealed roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer. PMID:26189910

  17. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

    PubMed

    Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

    2013-05-01

    Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs. PMID:23428971

  18. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

    PubMed

    Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

    2013-05-01

    Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs.

  19. Oral rapamycin inhibits growth of atherosclerotic plaque in apoE knock-out mice

    SciTech Connect

    Waksman, Ron; Pakala, Rajbabu; Burnett, Mary S.; Gulick, Cindy P.; Leborgne, Laurent; Fournadjiev, Jana; Wolfram, Roswitha; Hellinga, David

    2003-03-01

    Introduction: Inflammatory and immunological responses of vascular cells are known to play significant roles in atherosclerotic plaque development. Rapamycin with antiinflammatory, immunosuppressive and antiproliferative properties has been shown to reduce neointima formation when coated on stents. This study is designed to test the potential of oral rapamycin to inhibit atherosclerotic plaque development. Methods: Eight-week-old apoE knock-out mice were fed with 0.25% cholesterol supplemented diet (control diet), control diet containing 50 {mu}g/kg rapamycin (low-dose rapamycin) or 100 {mu}g/kg rapamycin (high-dose rapamycin) for 4 or 8 weeks. Subsets of mice from each group (n=10) were weighed and euthanized. Whole blood rapamycin levels were determined using HPLC-MS/MS, and histological analyses of atherosclerotic lesions in the aortic root were performed. Results: Mice fed with high-dose rapamycin did not gain weight (18.5{+-}1.5 vs. 20.6{+-}0.9 g, P=.01). Blood levels of rapamycin 117{+-}7 pg/ml were detected in the blood of mice fed with high-dose rapamycin for 8 weeks. The plaque area in mice fed with high dose oral rapamycin is significantly less as compared to control (0.168{+-}0.008 vs. 0.326{+-}0.013 mm{sup 2}, P=.001 at 4 weeks; 0.234{+-}0.013 vs. 0.447{+-}0.011 mm{sup 2}, P=.001 at 8 weeks). Lumen area was inversely proportional to the plaque area. Conclusions: The results indicate that oral rapamycin is effective in attenuating the progression of atherosclerotic plaque in the mice.

  20. Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes

    PubMed Central

    2014-01-01

    Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice. PMID:24758191

  1. 5'-Ectonucleotidase-knockout mice lack non-REM sleep responses to sleep deprivation.

    PubMed

    Zielinski, Mark R; Taishi, Ping; Clinton, James M; Krueger, James M

    2012-06-01

    Adenosine and extracellular adenosine triphosphate (ATP) have multiple physiological central nervous system actions including regulation of cerebral blood flow, inflammation and sleep. However, their exact sleep regulatory mechanisms remain unknown. Extracellular ATP and adenosine diphosphate are converted to adenosine monophosphate (AMP) by the enzyme ectonucleoside triphosphate diphosphohydrolase 1, also known as CD39, and extracellular AMP is in turn converted to adenosine by the 5'-ectonuleotidase enzyme CD73. We investigated the role of CD73 in sleep regulation. Duration of spontaneous non-rapid eye movement sleep (NREMS) was greater in CD73-knockout (KO) mice than in C57BL/6 controls whether determined in our laboratory or by others. After sleep deprivation (SD), NREMS was enhanced in controls but not CD73-KO mice. Interleukin-1 beta (IL1β) enhanced NREMS in both strains, indicating that the CD73-KO mice were capable of sleep responses. Electroencephalographic power spectra during NREMS in the 1.0-2.5 Hz frequency range was significantly enhanced after SD in both CD73-KO and WT mice; the increases were significantly greater in the WT mice than in the CD73-KO mice. Rapid eye movement sleep did not differ between strains in any of the experimental conditions. With the exception of CD73 mRNA, the effects of SD on various adenosine-related mRNAs were small and similar in the two strains. These data suggest that sleep is regulated, in part, by extracellular adenosine derived from the actions of CD73.

  2. Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age☆

    PubMed Central

    Sikka, Gautam; Miller, Karen L.; Steppan, Jochen; Pandey, Deepesh; Jung, Sung M.; Fraser, Charles D.; Ellis, Carla; Ross, Daniel; Vandegaer, Koenraad; Bedja, Djahida; Gabrielson, Kathleen; Walston, Jeremy D.; Berkowitz, Dan E.; Barouch, Lili A.

    2013-01-01

    Cardiovascular dysfunction is a primary independent predictor of age-related morbidity and mortality. Frailty is associated with activation of inflammatory pathways and fatigue that commonly presents and progresses with age. Interleukin 10 (IL-10), the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine produced by immune and non-immune cells. Homozygous deletion of IL-10 in mice yields a phenotype that is consistent with human frailty, including age-related increases in serum inflammatory mediators, muscular weakness, higher levels of IGF-1 at midlife, and early mortality. While emerging evidence suggests a role for IL-10 in vascular protection, a clear mechanism has not yet been elucidated. Methods In order to evaluate the role of IL-10 in maintenance of vascular function, force tension myography was utilized to access ex-vivo endothelium dependent vasorelaxation in vessels isolated from IL-10 knockout IL-10(tm/tm) and control mice. Pulse wave velocity ((PWV), index of stiffness) of vasculature was measured using ultrasound and blood pressure was measured using the tail cuff method. Echocardiography was used to elucidated structure and functional changes in the heart. Results Mean arterial pressures were significantly higher in IL-10(tm/tm) mice as compared to C57BL6/wild type (WT) controls. PWV was increased in IL-10(tm/tm) indicating stiffer vasculature. Endothelial intact aortic rings isolated from IL-10(tm/tm) mice demonstrated impaired vasodilation at low acetylcholine doses and vasoconstriction at higher doses whereas vasorelaxation responses were preserved in rings from WT mice. Cyclo-oxygenase (COX-2)/thromboxane A2 inhibitors improved endothelial dependent vasorelaxation and reversed vasoconstriction. Left ventricular end systolic diameter, left ventricular mass, isovolumic relaxation time, fractional shortening and ejection fraction were all significantly different in the aged IL-10(tm/tm) mice compared to WT mice. Conclusion Aged IL

  3. Flavor preference conditioning by different sugars in sweet ageusic Trpm5 knockout mice.

    PubMed

    Sclafani, Anthony; Ackroff, Karen

    2015-03-01

    Knockout (KO) mice missing the taste signaling protein Trpm5 have greatly attenuated sweetener preferences but develop strong preferences for glucose in 24-h tests, which is attributed to post-oral sugar conditioning. Trpm5 KO mice express mild preferences for galactose but no preferences for fructose in 24-h tests, which suggests that these sugars differ in their post-oral reinforcing effects. Here we investigated sugar-conditioned flavor preferences in Trpm5 KO and C57BL/6J wildtype (B6) mice. The mice were trained to consume a flavored (CS+, e.g. grape) 8% sugar solution and flavored (CS-, e.g., cherry) water on alternating days followed by two-bottle choice tests with CS+ vs. CS- flavors in water and with unflavored sugar vs. water. The KO mice displayed strong preferences (>80%) for the CS+ glucose and CS+ galactose but not for the CS+ fructose flavor. They also preferred glucose and galactose, but not fructose to water. In contrast, the B6 mice preferred all three CS+ flavors to the CS- flavor, and all three sugars to water. In tests with the non-metabolizable sugar α-methyl-d-glucopyranoside (MDG), the KO and B6 mice preferred 8% MDG to water but did not prefer the CS+ 8% MDG to CS-. However, they preferred a CS+ flavor mixed with 4% MDG over the CS- flavor. Trpm5 KO mice also preferred galactose and MDG to fructose in direct choice tests. The Trpm5 KO data indicate that glucose and, to a lesser extent, galactose and MDG have post-oral reinforcing actions that stimulate intake and preference while fructose has a much weaker effect. The CS+ flavor and sugar preferences of B6 mice may be mediated by the sweet taste and/or post-oral actions of the various sugars. Glucose, galactose, and MDG, but not fructose, are ligands for the sodium-glucose transporter 1 (SGLT1) which is implicated in post-oral sugar conditioning in B6 mice.

  4. Iron speciation study in Hfe knockout mice tissues: magnetic and ultrastructural characterisation.

    PubMed

    Gutiérrez, Lucía; Quintana, Carmen; Patiño, Cristina; Bueno, Javier; Coppin, Hélène; Roth, Marie P; Lázaro, Francisco J

    2009-06-01

    Liver, spleen and heart tissues of DBA/2 Hfe knockout mice have been characterised by low temperature AC magnetic susceptibility measurements together with Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction in order to investigate the chemical iron speciation in a murine model of iron overload diseases. With emphasis on ferritin-like species, the temperature dependent in-phase and out-of-phase susceptibility profiles agree with the elemental analysis in that, in this model, iron accumulation takes place in the hepatic tissue while in the spleen and heart tissues no differences have been observed between knockout and wild type animals. The comparison of the magnetic properties between perfused and non-perfused liver tissues has made it possible to estimate the magnetic contribution of usually present blood remains. The TEM observations reveal that, besides the isolated ferritins and ferritin-containing lysosomes-siderosomes present in the hepatocytes, other iron deposits, of heterogeneous size, morphology and crystalline structure (haematite and/or goethite), are present in the cytoplasm, near the membrane, and in extracellular spaces. PMID:19348938

  5. Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

    PubMed

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

    2016-02-15

    Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. PMID:26857489

  6. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition.

    PubMed

    Naruse, Mie; Ono, Ryuichi; Irie, Masahito; Nakamura, Kenji; Furuse, Tamio; Hino, Toshiaki; Oda, Kanako; Kashimura, Misho; Yamada, Ikuko; Wakana, Shigeharu; Yokoyama, Minesuke; Ishino, Fumitoshi; Kaneko-Ishino, Tomoko

    2014-12-01

    Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

  7. Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

    PubMed

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

    2016-02-15

    Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions.

  8. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition.

    PubMed

    Naruse, Mie; Ono, Ryuichi; Irie, Masahito; Nakamura, Kenji; Furuse, Tamio; Hino, Toshiaki; Oda, Kanako; Kashimura, Misho; Yamada, Ikuko; Wakana, Shigeharu; Yokoyama, Minesuke; Ishino, Fumitoshi; Kaneko-Ishino, Tomoko

    2014-12-01

    Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function. PMID:25468940

  9. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition

    PubMed Central

    Naruse, Mie; Ono, Ryuichi; Irie, Masahito; Nakamura, Kenji; Furuse, Tamio; Hino, Toshiaki; Oda, Kanako; Kashimura, Misho; Yamada, Ikuko; Wakana, Shigeharu; Yokoyama, Minesuke; Ishino, Fumitoshi; Kaneko-Ishino, Tomoko

    2014-01-01

    Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function. PMID:25468940

  10. Impaired neurogenesis in embryonic spinal cord of Phgdh knockout mice, a serine deficiency disorder model.

    PubMed

    Kawakami, Yuriko; Yoshida, Kazuyuki; Yang, Jung Hoon; Suzuki, Takeshi; Azuma, Norihiro; Sakai, Kazuhisa; Hashikawa, Tsutomu; Watanabe, Masahiko; Yasuda, Kaori; Kuhara, Satoru; Hirabayashi, Yoshio; Furuya, Shigeki

    2009-03-01

    Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novol-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G(0)-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency. PMID:19114063

  11. CXC receptor knockout mice: characterization of skeletal features and membranous bone healing in the adult mouse.

    PubMed

    Bischoff, David S; Sakamoto, Taylor; Ishida, Kenji; Makhijani, Nalini S; Gruber, Helen E; Yamaguchi, Dean T

    2011-02-01

    The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR(-/-) knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR(-/-) mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR(-/-) mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR(-/-) (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR(-/-). Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR(-/-) long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR(-/-) and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR(-/-). New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR(-/-). Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR(-/-) mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular

  12. Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

    SciTech Connect

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; O'Roark, Erin M.; Kenyon, Nicholas J.; Last, Jerold A.

    2010-01-01

    Arginase1 and nitric oxide synthase2 (NOS2) utilize L-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N{sup o}mega-hydroxy-nor-L-arginine (nor-NOHA) significantly increased total L-arginine content in the airway compartment. We hypothesized that such an increase in L-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that L-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2

  13. Pravastatin and Sarpogrelate Synergistically Ameliorate Atherosclerosis in LDLr-Knockout Mice.

    PubMed

    Park, Kyung-Yeon; Oh, Euichaul; Kwak, Mi-Kyoung; Jun, Hyun Sik; Heo, Tae-Hwe

    2016-01-01

    Pravastatin is a lipid-lowering agent that attenuates atherosclerosis. However, the multifactorial pathogenesis of atherosclerosis requires other drugs with different anti-atherogenic mechanisms. We chose sarpogrelate as an anti-platelet agent and a novel component of a complex drug with pravastatin due to its high potential but little information on its beneficial effects on atherosclerosis. Low-density lipoprotein receptor-knockout mice were fed a high-fat, high-cholesterol diet and treated with pravastatin alone, sarpogrelate alone, or a combination of both drugs. Although sarpogrelate alone did not significantly reduce atherosclerotic plaque areas, co-treatment with pravastatin significantly decreased aortic lesions compared to those of the pravastatin alone treated group. The combined therapy was markedly more effective than that of the single therapies in terms of foam cell formation, smooth muscle cell proliferation, and inflammatory cytokine levels. These results suggest that pravastatin and sarpogrelate combined therapy may provide a new therapeutic strategy for treating atherosclerosis.

  14. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice

    PubMed Central

    2015-01-01

    Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points. PMID:26445171

  15. Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice

    PubMed Central

    Cacciola, Giovanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-01-01

    Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone. More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model. PMID:24324492

  16. Cytoskeletal architecture and cell motility remain unperturbed in mouse embryonic fibroblasts from Plk3 knockout mice

    PubMed Central

    Michel, Daniel R; Mun, Kyu-Shik; Ho, Chia-Chi

    2016-01-01

    Polo-like kinase 3 (Plk3) is best known for its involvement in cell cycle checkpoint regulation following exposure to cytotoxicants or induction of DNA damage. Yet, Plk3 has also been implicated in roles beyond those of cellular responses to DNA damage. Here, we have investigated the proposition, suggested by the Plk literature, that Plk3 regulates cytoskeletal architecture and cell functions mediated by the cytoskeleton. To this end, we have assayed mouse embryonic fibroblasts (MEFs) generated from both Plk3 knockout and wild-type mice. In particular, we asked whether Plk3 is involved in actin fiber and microtubule integrity, cell migration, cell attachment, and/or cell invasion. Our results demonstrate that functional Plk3 is not critical for the regulation of cytoskeletal integrity, cell morphology, cell adhesion, or motility in MEFs. PMID:26843517

  17. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice.

    PubMed

    McClatchy, Daniel B; Ma, Yuanhui; Liu, Chao; Stein, Benjamin D; Martínez-Bartolomé, Salvador; Vasquez, Debbie; Hellberg, Kristina; Shaw, Reuben J; Yates, John R

    2015-11-01

    Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.

  18. Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice.

    PubMed

    Knudtson, Jennifer F; Tekmal, Rajeshwar R; Santos, Marlen Tellez; Binkley, Peter A; Krishnegowda, Naveen; Valente, Philip; Schenken, Robert S

    2016-01-01

    Previous studies have shown endometrial cell (EC) CD44 and peritoneal mesothelial cell (PMC)-associated hyaluronan (hyaluronic acid [HA]) are involved in the attachment of endometrial stroma and epithelial cells to peritoneal mesothelium. Here we assess the CD44-HA interaction in the formation of the early endometriotic lesion using CD44(-/-) (knockout) mice. Using an established murine model and crossover technique, endometrial tissue from donor mice (wild type [WT] and CD44(-/-)) was used to induce endometriosis in recipient mice (WT and CD44(-/-)). Endometriotic lesions were visualized by fluorescent microscopy and confirmed by hematoxylin and eosin staining. Early endometriotic lesions were decreased when CD44(-/-) endometrium was placed in WT recipients and when WT endometrium was placed in CD44(-/-) recipients (P = .002). Early endometriotic lesions were also significantly decreased when both peritoneal and endometrial tissues lacked CD44 expression (P < .01). These studies demonstrate that both EC and PMC CD44 play a role in the development of early endometriotic lesion. PMID:26169039

  19. Olive oils modulate fatty acid content and signaling protein expression in apolipoprotein E knockout mice brain.

    PubMed

    Alemany, Regina; Navarro, María A; Vögler, Oliver; Perona, Javier S; Osada, Jesús; Ruiz-Gutiérrez, Valentina

    2010-01-01

    Atherosclerosis contributes to disruption of neuronal signaling pathways by producing lipid-dependent modifications of brain plasma membranes, neuroinflammation and oxidative stress. We investigated whether long-term (11 weeks) consumption of refined- (ROO) and pomace- (POO) olive oil modulated the fatty acid composition and the levels of membrane signaling proteins in the brain of apolipoprotein E (apoE) knockout (KO) mice, an animal model of atherosclerosis. Both of these oils are rich in bioactive molecules with anti-inflammatory and antioxidant effects. ROO and POO long-term consumption increased the proportion of monounsaturated fatty acids (MUFAs), particularly of oleic acid, while reducing the level of the saturated fatty acids (SFAs) palmitic and stearic acid. As a result, the MUFA:SFA ratio was higher in apoE KO mice brain fed with ROO and POO. Furthermore, both oils reduced the level of arachidonic and eicosapentaenoic acid, suggesting a decrease in the generation of pro- and anti-inflammatory eicosanoids. Finally, ROO and POO induced an increase in the density of membrane proteins implicated in both the Galphas/PKA and Galphaq/PLCbeta1/PKCalpha signaling pathways. The combined effects of long-term ROO and POO consumption on fatty acid composition and the level of signaling proteins involved in PKA and PKC activation, suggest positive effects on neuroinflammation and brain function in apoE KO mice brain, and convert these oils into promising functional foods in diseases involving apoE deficiency.

  20. ChemR23 knockout mice display mild obesity but no deficit in adipocyte differentiation.

    PubMed

    Rouger, Laurie; Denis, G Raphaël; Luangsay, Souphalone; Parmentier, Marc

    2013-12-01

    Chemerin was initially described as a chemoattractant factor for leukocyte populations. More recently, the protein has also been reported to be an adipokine, regulating adipocyte differentiation in vitro via its receptor ChemR23, and to be correlated with BMI and other parameters of the metabolic syndrome in humans. The aim of this study was to investigate the role of the chemerin/ChemR23 axis in the regulation of metabolism in vivo, using a mouse knockout (KO) model for ChemR23 (Cmklr1) in a C57BL/6 genetic background. Body weight and adipose tissue mass did not differ significantly in young animals, but were significantly higher in ChemR23 KO mice aged above 12 months. Glucose tolerance was unaffected. No significant modifications in the levels of blood lipids were observed and no increase in the levels of inflammatory markers was observed in the adipose tissue of KO mice. A high-fat diet did not exacerbate the obese phenotype in ChemR23 KO mice. No obvious defect in adipocyte differentiation was detected, while a marker of lipogenic activity (GPD1 expression) was found to be elevated. In conclusion, the chemerin/ChemR23 system does not appear to play a major role in adipocyte differentiation in vivo, but it may be involved in adipose tissue homeostasis. PMID:24084834

  1. Chronic immobilization in the malpar1 knockout mice increases oxidative stress in the hippocampus.

    PubMed

    García-Fernández, María; Castilla-Ortega, Estela; Pedraza, Carmen; Blanco, Eduardo; Hurtado-Guerrero, Isaac; Barbancho, Miguel Angel; Chun, Jerold; Rodríguez-de-Fonseca, Fernando; Estivill-Torrús, Guillermo; Santín Núñez, Luis Javier

    2012-10-01

    The lysophosphatidic acid LPA₁ receptor has recently been involved in the adaptation of the hippocampus to chronic stress. The absence of LPA₁ receptor aggravates the chronic stress-induced impairment of both hippocampal neurogenesis and apoptosis that were accompanied with hippocampus-dependent memory deficits. Apoptotic death and neurogenesis in the hippocampus are regulated by oxidative stress. In the present work, we studied the involvement of LPA₁ receptor signaling pathway in the regulation of the hippocampal redox after chronic stress. To this end, we used malpar1 knockout (KO) and wild-type mice assigned to either chronic stress (21 days of restraint, 3 h/day) or control conditions. Lipid peroxidation, the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), as well as mitochondrial function stimulation, monitored through the activity of cytochrome c oxidase (COX), were studied in the hippocampus. Our results showed that chronic immobilization stress enhanced lipid peroxidation as well as the activity of the antioxidant enzymes studied (CAT, SOD, and GPX). This effect was only observed in absence of LPA₁ receptor. Furthermore, only malpar1 KO mice submitted to chronic stress exhibited a severe downregulation of the COX activity, suggesting the presence of mitochondrial damage. Altogether, these results suggest that malpar1 KO mice display enhanced oxidative stress in the hippocampus after chronic stress. This may be involved in the hippocampal abnormalities observed in this genotype after chronic immobilization, including memory, neurogenesis, and apoptosis.

  2. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  3. Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

    PubMed

    Kouno, Tetsuya; Akiyama, Nobuteru; Ito, Takahito; Okuda, Tomohiko; Nanchi, Isamu; Notoya, Mitsuru; Oka, Shogo; Yukioka, Hideo

    2016-02-01

    Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food.

  4. Effects of Estrogens on Adipokines and Glucose Homeostasis in Female Aromatase Knockout Mice

    PubMed Central

    Van Sinderen, Michelle L.; Steinberg, Gregory R.; Jørgensen, Sebastian B.; Honeyman, Jane; Chow, Jenny D.; Herridge, Kerrie A.; Winship, Amy L.; Dimitriadis, Evdokia; Jones, Margaret E. E.; Simpson, Evan R.; Boon, Wah Chin

    2015-01-01

    The maintenance of glucose homeostasis within the body is crucial for constant and precise performance of energy balance and is sustained by a number of peripheral organs. Estrogens are known to play a role in the maintenance of glucose homeostasis. Aromatase knockout (ArKO) mice are estrogen-deficient and display symptoms of dysregulated glucose metabolism. We aim to investigate the effects of estrogen ablation and exogenous estrogen administration on glucose homeostasis regulation. Six month-old female wildtype, ArKO, and 17β-estradiol (E2) treated ArKO mice were subjected to whole body tolerance tests, serum examination of estrogen, glucose and insulin, ex-vivo muscle glucose uptake, and insulin signaling pathway analyses. Female ArKO mice display increased body weight, gonadal (omental) adiposity, hyperinsulinemia, and liver triglycerides, which were ameliorated upon estrogen treatment. Tolerance tests revealed that estrogen-deficient ArKO mice were pyruvate intolerant hence reflecting dysregulated hepatic gluconeogenesis. Analyses of skeletal muscle, liver, and adipose tissues supported a hepatic-based glucose dysregulation, with a down-regulation of Akt phosphorylation (a key insulin signaling pathway molecule) in the ArKO liver, which was improved with E2 treatment. Concurrently, estrogen treatment lowered ArKO serum leptin and adiponectin levels and increased inflammatory adipokines such as tumour necrosis factor alpha (TNFα) and interleukin 6 (IL6). Furthermore, estrogen deficiency resulted in the infiltration of CD45 macrophages into gonadal adipose tissues, which cannot be reversed by E2 treatment. This study describes the effects of estrogens on glucose homeostasis in female ArKO mice and highlights a primary phenotype of hepatic glucose dysregulation and a parallel estrogen modified adipokine profile. PMID:26317527

  5. Blue-Green Algae Inhibit the Development of Atherosclerotic Lesions in Apolipoprotein E Knockout Mice.

    PubMed

    Ku, Chai Siah; Kim, Bohkyung; Pham, Tho X; Yang, Yue; Wegner, Casey J; Park, Young-Ki; Balunas, Marcy; Lee, Ji-Young

    2015-12-01

    Hyperlipidemia and inflammation contribute to the development of atherosclerotic lesions. Our objective was to determine antiatherogenic effect of edible blue-green algae (BGA) species, that is, Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), in apolipoprotein E knockout (ApoE(-/-)) mice, a well-established mouse model of atherosclerosis. Male ApoE(-/-) mice were fed a high-fat/high-cholesterol (HF/HC, 15% fat and 0.2% cholesterol by wt) control diet or a HF/HC diet supplemented with 5% (w/w) of NO or SP powder for 12 weeks. Plasma total cholesterol (TC) and triglycerides (TG) were measured, and livers were analyzed for histology and gene expression. Morphometric analysis for lesions and immunohistochemical analysis for CD68 were conducted in the aorta and the aortic root. NO supplementation significantly decreased plasma TC and TG, and liver TC, compared to control and SP groups. In the livers of NO-fed mice, less lipid droplets were present with a concomitant decrease in fatty acid synthase protein levels than the other groups. There was a significant increase in hepatic low-density lipoprotein receptor protein levels in SP-supplemented mice than in control and NO groups. Quantification of aortic lesions by en face analysis demonstrated that both NO and SP decreased aortic lesion development to a similar degree compared with control. While lesions in the aortic root were not significantly different between groups, the CD68-stained area in the aortic root was significantly lowered in BGA-fed mice than controls. In conclusion, both NO and SP supplementation decreased the development of atherosclerotic lesions, suggesting that they may be used as a natural product for atheroprotection. PMID:26566121

  6. Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice

    PubMed Central

    Correnti, Jason M.; Juskeviciute, Egle; Swarup, Aditi

    2014-01-01

    Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPAR-α) activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver and has been shown to reduce ceramide levels and activate AMPK and PPAR-α. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol-containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum alanine aminotransferase, and reduced liver TNF. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPAR-α DNA binding activity compared with control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting that alcohol-dependent deregulation of AMPK and PPAR-α in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis, and hepatomegaly. These data suggest that adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides, but its absence does not exacerbate alcohol-induced liver damage. PMID

  7. Decreased Neointimal Extracellular Matrix Formation in RAGE-Knockout Mice After Microvascular Denudation

    SciTech Connect

    Groezinger, Gerd Schmehl, Joerg Bantleon, Ruediger Kehlbach, Rainer; Mehra, Tarun; Claussen, Claus Wiesinger, Benjamin

    2012-12-15

    Purpose: To evaluate in vivo the role of RAGE (receptor for advanced glycated end products) in the development of restenosis and neointimal proliferation in RAGE-deficient knockout (KO) mice compared with wild-type (WT) mice in an animal model. Materials and Methods: Sixteen WT and 15 RAGE-deficient mice underwent microvascular denudation of the common femoral artery under general anaesthesia. Contralateral arteries underwent a sham operation and served as controls. Four weeks after the intervention, all animals were killed, and paraformaldehyde-fixed specimens of the femoral artery were analysed with different stains (hematoxylin and eosin and Elastica van Gieson) and several different types of immunostaining (proliferating cell nuclear antigen, {alpha}-actin, collagen, von Willebrand factor, RAGE). Luminal area, area of the neointima, and area of the media were measured in all specimens. In addition, colony-formation assays were performed, and collagen production by WT smooth muscle cells (SMCs) and RAGE-KO SMCs was determined. For statistical analysis, P < 0.05 was considered statistically significant. Results: Four weeks after denudation, WT mice showed a 49.6% loss of luminal area compared with 14.9% loss of luminal area in RAGE-deficient mice (sham = 0% loss) (P < 0.001). The neointima was 18.2 (*1000 {mu}m{sup 2} [n = 15) in the WT group compared with only 8.4 (*1000 {mu}m{sup 2} [n = 16]) in the RAGE-KO group. RAGE-KO SMCs showed significantly decreased proliferation activity and production of extracellular matrix protein. Conclusion: RAGE may be shown to play a considerable role in the formation of neointima leading to restenosis after vascular injury.

  8. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice.

    PubMed

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R; Ma, Runlin Z

    2015-11-30

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3(-/-)) and wild-type (AC3(+/+)) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3(-/-) mice was significantly altered, compared to AC3(+/+) mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3(-/-) and AC3(+/+) mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

  9. Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zinc transporter 7 (Znt7, Slc30a7) knockout (KO) mice display abnormalities in body weight gain and body adiposity. Regulation of body weight and fatness is complex, involving multiple genetic and environmental factors. To understand how zinc homeostasis influences body weight gain and fat deposit a...

  10. EFFECTS OF HEAT AND BROMOCHLOROACETIC ACID ON MALE REPRODUCTION IN HEAT SHOCK FACTOR-1 GENE KNOCKOUT MICE

    EPA Science Inventory

    Effects of heat and bromochloroacetic acid on male reproduction in heat shock factor-1 gene knockout mice.
    Luft JC1, IJ Benjamin2, JB Garges1 and DJ Dix1. 1Reproductive Toxicology Division, USEPA, RTP, NC, 27711 and 2Dept of Internal Medicine, Univ.of Texas Southwestern Med C...

  11. Troglitazone-induced hepatic mitochondrial proteome expression dynamics in heterozygous Sod2{sup +/-} mice: Two-stage oxidative injury

    SciTech Connect

    Lee, Y.H. |; Chung, Maxey C.M. | Lin Qingsong; Boelsterli, Urs A. ||

    2008-08-15

    The determinants of susceptibility to troglitazone-induced idiosyncratic liver injury have not yet been determined; however, troglitazone has been shown to target mitochondria and induce mitochondria-mediated hepatocellular injury in vitro. The aim of this study was to use a systems approach to analyze the dynamics of mitochondrial changes at the proteome level and more clearly define the mechanisms and time course of troglitazone hepatotoxicity by using a previously characterized mouse model that is highly sensitized to troglitazone hepatotoxicity. Mice heterozygous in mitochondrial superoxide dismutase-2 (Sod2{sup +/-}) were injected intraperitoneally with troglitazone (30 mg/kg/day) or vehicle daily for 2 or 4 weeks. Hepatic mitochondria were isolated, purified, and subjected to two-dimensional difference gel electrophoresis (2D-DIGE). We found that among the {approx} 1500 resolved hepatic mitochondrial proteins, 70 exhibited significantly altered abundance after troglitazone treatment. MALDI-TOF/TOF MS/MS analysis revealed that early changes (2 weeks) included increased levels of heat shock protein family members (mortalin, HSP7C), Lon protease, and catalase, indicating induction of a mitochondrial stress response. In contrast, after 4 weeks, a number of critical proteins including ATP synthase {beta}-subunit, aconitase-2, and catalase exhibited decreased abundance, and total protein carbonyls were significantly increased, suggesting uncompensated oxidative damage. Aconitase-2 (ACO2) was decreased at both time points, making this protein a potential sensitive and early biomarker for mitochondrial oxidant stress. These results show that, in this murine model of underlying clinically silent mitochondrial stress, superimposed troglitazone induces a two-stage response: an initial adaptive response, followed by a toxic response involving oxidant injury to mitochondrial proteins.

  12. [Effect of P2X7 receptor knock-out on bone cancer pain in mice].

    PubMed

    Zhao, Xin; Liu, Hui-Zhu; Zhang, Yu-Qiu

    2016-06-25

    Cancer pain is one of the most common symptoms in patients with late stage cancer. Lung, breast and prostate carcinoma are the most common causes of pain from osseous metastasis. P2X7 receptor (P2X7R) is one of the subtypes of ATP-gated purinergic ion channel family, predominately distributed in microglia in the spinal cord. Activation of P2X7Rs in the spinal dorsal horn has been associated with release of proinflammatory cytokines from glial cells, causing increased neuronal excitability and exaggerated nociception. Mounting evidence implies a critical role of P2X7R in inflammatory and neuropathic pain. However, whether P2X7R is involved in cancer pain remains controversial. Here we established a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6J wild-type mice (C57 WT mice) and P2X7R knockout mice (P2rx7(-/-) mice) to explore the role of P2X7R in bone cancer pain. Following intrafemur carcinoma inoculation, robust mechanical allodynia and thermal hyperalgesia in C57 WT mice were developed on day 7 and 14, respectively, and persisted for at least 28 days in the ipsilateral hindpaw of the affected limb. CatWalk gait analysis showed significant decreases in the print area and stand phase, and a significant increase in swing phase in the ipsilateral hindpaw on day 21 and 28 after carcinoma cells inoculation. Histopathological sections (hematoxylin and eosin stain) showed that the bone marrow of the affected femur was largely replaced by invading tumor cells, and the femur displayed medullary bone loss and bone destruction on day 28 after inoculation. Unexpectedly, no significant changes in bone cancer-induced hypersensitivity of pain behaviors were found in P2rx7(-/-) mice, and the changes of pain-related values in CatWalk gait analysis even occurred earlier in P2rx7(-/-) mice, as compared with C57 WT mice. Together with our previous study in rats that blockade of P2X7R significantly alleviated bone cancer

  13. Liver-Specific Commd1 Knockout Mice Are Susceptible to Hepatic Copper Accumulation

    PubMed Central

    Vonk, Willianne I. M.; Bartuzi, Paulina; de Bie, Prim; Kloosterhuis, Niels; Wichers, Catharina G. K.; Berger, Ruud; Haywood, Susan; Klomp, Leo W. J.; Wijmenga, Cisca; van de Sluis, Bart

    2011-01-01

    Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1Δhep mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1Δhep mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1Δhep mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable

  14. p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

    SciTech Connect

    Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho; Williams, Stuart; Chen, Qin M.

    2011-11-15

    Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

  15. Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

    PubMed

    Thomsen, Morgane; Caine, Simon Barak

    2016-04-01

    Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. PMID:26874213

  16. Benzo[a]pyrene potentiates the pathogenesis of abdominal aortic aneurysms in apolipoprotein E knockout mice.

    PubMed

    Prins, Petra A; Perati, Prudhvidhar R; Kon, Valentina; Guo, Zhongmao; Ramesh, Aramandla; Linton, MacRae F; Fazio, Sergio; Sampson, Uchechukwu K

    2012-01-01

    The objective of this study was to determine the effect of benzo[a]pyrene (BaP), an abundant environmental polycyclic aromatic hydrocarbon compound, on the pathogenesis of abdominal aortic aneurysms (AAA). Earlier studies have shown that BaP promotes vasculopathy, including atherosclerosis, a predisposing factor for AAA development. In two experimental arms, 203 apolipoprotein E knockout (ApoE-/-) mice were evaluated in 4 groups: BaP, angiotensin II (AngII), BaP+AngII and control. Mice in the first arm were exposed to 5mg/kg/week of BaP for 42 days, and in the second arm to 0.71mg/kg daily for 60 days. In arm one, AAA incidence was higher in the BaP+AngII (14/28) versus AngII (8/27) group (p < 0.05), rupture (n=3) was observed only in BaP+AngII treated mice (p < 0.05). In the second arm, AAA incidence did not differ between AngII (17/30) and BaP+AngII (16/29) groups. However, intact AAA diameter was larger in the BaP+AngII (2.3 ± 0.1mm) versus AngII (1.9 ± 0.1mm) group (p < 0.05), but AAA rupture did not differ (p=NS). In both experimental arms, BaP+AngII mice showed increased expression of tumor necrosis factor alpha (TNF-α), cyclophilin A (Cyp A), and matrix metalloproteinase-9 (MMP9) (p < 0.05). No AAA occurred in control or BaP groups. These findings suggest the role of BaP exposure in potentiating AAA pathogenesis, which may have potential public health significance.

  17. Reduced Bone Density and Cortical Bone Indices in Female Adiponectin-Knockout Mice.

    PubMed

    Naot, Dorit; Watson, Maureen; Callon, Karen E; Tuari, Donna; Musson, David S; Choi, Ally J; Sreenivasan, Dharshini; Fernandez, Justin; Tu, Pao Ting; Dickinson, Michelle; Gamble, Greg D; Grey, Andrew; Cornish, Jillian

    2016-09-01

    A positive association between fat and bone mass is maintained through a network of signaling molecules. Clinical studies found that the circulating levels of adiponectin, a peptide secreted from adipocytes, are inversely related to visceral fat mass and bone mineral density, and it has been suggested that adiponectin contributes to the coupling between fat and bone. Our study tested the hypothesis that adiponectin affects bone tissue by comparing the bone phenotype of wild-type and adiponectin-knockout (APN-KO) female mice between the ages of 8-37 weeks. Using a longitudinal study design, we determined body composition and bone density using dual energy x-ray absorptiometry. In parallel, groups of animals were killed at different ages and bone properties were analyzed by microcomputed tomography, dynamic histomorphometry, 3-point bending test, nanoindentation, and computational modelling. APN-KO mice had reduced body fat and decreased whole-skeleton bone mineral density. Microcomputed tomography analysis identified reduced cortical area fraction and average cortical thickness in APN-KO mice in all the age groups and reduced trabecular bone volume fraction only in young APN-KO mice. There were no major differences in bone strength and material properties between the 2 groups. Taken together, our results demonstrate a positive effect of adiponectin on bone geometry and density in our mouse model. Assuming adiponectin has similar effects in humans, the low circulating levels of adiponectin associated with increased fat mass are unlikely to contribute to the parallel increase in bone mass. Therefore, adiponectin does not appear to play a role in the coupling between fat and bone tissue. PMID:27384302

  18. NFAT is required for spontaneous pulmonary hypertension in superoxide dismutase 1 knockout mice.

    PubMed

    Ramiro-Diaz, Juan Manuel; Nitta, Carlos H; Maston, Levi D; Codianni, Simon; Giermakowska, Wieslawa; Resta, Thomas C; Gonzalez Bosc, Laura V

    2013-05-01

    Elevated reactive oxygen species are implicated in pulmonary hypertension (PH). Superoxide dismutase (SOD) limits superoxide bioavailability, and decreased SOD activity is associated with PH. A decrease in SOD activity is expected to increase superoxide and reduce hydrogen peroxide levels. Such an imbalance of superoxide/hydrogen peroxide has been implicated as a mediator of nuclear factor of activated T cells (NFAT) activation in epidermal cells. We have shown that NFATc3 is required for chronic hypoxia-induced PH. However, it is unknown whether NFATc3 is activated in the pulmonary circulation in a mouse model of decreased SOD1 activity and whether this leads to PH. Therefore, we hypothesized that an elevated pulmonary arterial superoxide/hydrogen peroxide ratio activates NFATc3, leading to PH. We found that SOD1 knockout (KO) mice have elevated pulmonary arterial wall superoxide and decreased hydrogen peroxide levels compared with wild-type (WT) littermates. Right ventricular systolic pressure (RVSP) was elevated in SOD1 KO and was associated with pulmonary arterial remodeling. Vasoreactivity to endothelin-1 was also greater in SOD1 KO vs. WT mice. NFAT activity and NFATc3 nuclear localization were increased in pulmonary arteries from SOD1 KO vs. WT mice. Administration of A-285222 (selective NFAT inhibitor) decreased RVSP, arterial wall thickness, vasoreactivity, and NFAT activity in SOD1 KO mice to WT levels. The SOD mimetic, tempol, also reduced NFAT activity, NFATc3 nuclear localization, and RVSP to WT levels. These findings suggest that an elevated superoxide/hydrogen peroxide ratio activates NFAT in pulmonary arteries, which induces vascular remodeling and increases vascular reactivity leading to PH.

  19. Benzo[a]pyrene Potentiates the Pathogenesis of Abdominal Aortic Aneurysms in Apolipoprotein E Knockout Mice

    PubMed Central

    Prins, Petra A.; Perati, Prudhvidhar R.; Kon, Valentina; Guo, Zhongmao; Ramesh, Aramandla; Linton, MacRae F.; Fazio, Sergio; Sampson, Uchechukwu K.

    2012-01-01

    The objective of this study was to determine the effect of benzo[a]pyrene (BaP), an abundant environmental polycyclic aromatic hydrocarbon compound, on the pathogenesis of abdominal aortic aneurysms (AAA). Earlier studies have shown that BaP promotes vasculopathy, including atherosclerosis, a predisposing factor for AAA development. In two experimental arms, 203 apolipoprotein E knockout (ApoE-/-) mice were evaluated in 4 groups: BaP, angiotensin II (AngII), BaP+AngII and control. Mice in the first arm were exposed to 5mg/kg/week of BaP for 42 days, and in the second arm to 0.71mg/kg daily for 60 days. In arm one, AAA incidence was higher in the BaP+AngII (14/28) versus AngII (8/27) group (p < 0.05), rupture (n=3) was observed only in BaP+AngII treated mice (p < 0.05). In the second arm, AAA incidence did not differ between AngII (17/30) and BaP+AngII (16/29) groups. However, intact AAA diameter was larger in the BaP+AngII (2.3 ± 0.1mm) versus AngII (1.9 ± 0.1mm) group (p < 0.05), but AAA rupture did not differ (p=NS). In both experimental arms, BaP+AngII mice showed increased expression of tumor necrosis factor alpha (TNF-α), cyclophilin A (Cyp A), and matrix metalloproteinase-9 (MMP9) (p < 0.05). No AAA occurred in control or BaP groups. These findings suggest the role of BaP exposure in potentiating AAA pathogenesis, which may have potential public health significance. PMID:22415081

  20. Systemic and cerebral iron homeostasis in ferritin knock-out mice.

    PubMed

    Li, Wei; Garringer, Holly J; Goodwin, Charles B; Richine, Briana; Acton, Anthony; VanDuyn, Natalia; Muhoberac, Barry B; Irimia-Dominguez, Jose; Chan, Rebecca J; Peacock, Munro; Nass, Richard; Ghetti, Bernardino; Vidal, Ruben

    2015-01-01

    Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.

  1. Delayed procedural learning in α7-nicotinic acetylcholine receptor knockout mice

    PubMed Central

    Young, J. W.; Meves, J. M.; Tarantino, I. S.; Caldwell, S.; Geyer, M. A.

    2014-01-01

    The α7-nicotinic acetylcholine receptor (nAChR) has long been a procognitive therapeutic target to treat schizophrenia. Evidence on the role of this receptor in cognition has been lacking, however, in part due to the limited availability of suitable ligands. The behavior of α7-nAChR knockout (KO) mice has been examined previously, but cognitive assessments using tests with cross-species translatability have been limited to date. Here, we assessed the cognitive performance of α7-nAChR KO and wild-type (WT) littermate mice in the attentional set-shifting task of executive functioning, the radial arm maze test of spatial working memory span capacity and the novel object recognition test of short-term memory. The reward motivation of these mutants was assessed using the progressive ratio breakpoint test. In addition, we assessed the exploratory behavior and sensorimotor gating using the behavioral pattern monitor and prepulse inhibition, respectively. α7-nAChR KO mice exhibited normal set-shifting, but impaired procedural learning (rule acquisition) in multiple paradigms. Spatial span capacity, short-term memory, motivation for food, exploration and sensorimotor gating were all comparable to WT littermates. The data presented here support the notion that this receptor is important for such procedural learning, when patterns in the environment become clear and a rule is learned. In combination with the impaired attention observed previously in these mice, this finding suggests that agonist treatments should be examined in clinical studies of attention and procedural learning, perhaps in combination with cognitive behavioral therapy. PMID:21679297

  2. Safrole-2',3'-oxide induces atherosclerotic plaque vulnerability in apolipoprotein E-knockout mice.

    PubMed

    Su, Le; Zhang, Haiyan; Zhao, Jing; Zhang, Shangli; Zhang, Yun; Zhao, Baoxiang; Miao, Junying

    2013-02-27

    Safrole-2',3'-oxide (SFO) is the major electrophilic metabolite of safrole (4-allyl-1, 2-methylenedioxybenzene), a natural plant constituent found in essential oils of numerous edible herbs and spices and in food containing these herbs, such as pesto sauce, cola beverages and bologna sausages. The effects of SFO in mammalian systems, especially the cardiovascular system, are little known. Disruption of vulnerable atherosclerotic plaques in atherosclerosis, a chronic inflammatory disease, is the main cause of cardiovascular events. In this study, we investigated SFO-induced atherosclerotic plaque vulnerability (possibility of rupture) in apolipoprotein E-knockout (apoE(-/-)) mice. Lipid area in vessel wall reached 59.8% in high dose SFO (SFO-HD) treated group, which is only 31.2% in control group. SFO treatment changed the lesion composition to an unstable phenotype, increased the number of apoptotic cells in plaque and the endothelium in plaques was damaged after SFO treatment. Furthermore, compared with control groups, the plaque endothelium level of p75(NTR) was 3-fold increased and the liver level of p75(NTR) was 17.4-fold increased by SFO-HD. Meanwhile, the serum level of KC (a functional homolog of IL-8 and the main proinflammatory alpha chemokine in mice) in apoE(-/-) mice was up to 357pg/ml in SFO-HD treated group. Thus, SFO contributes to the instability of atherosclerotic plaque in apoE(-/-) mice through activating p75(NTR) and IL-8 and cell apoptosis in plaque.

  3. Heterozygous deletion of ATG5 in Apc(Min/+) mice promotes intestinal adenoma growth and enhances the antitumor efficacy of interferon-gamma.

    PubMed

    Wang, Lu; Wang, Yan; Lu, Yuyin; Zhang, Qianyun; Qu, Xianjun

    2015-01-01

    Autophagy related gene 5 (ATG5) was lost in 23% of the patients with colorectal cancer (CRC) and the role of loss of ATG5 in the pathogenesis of CRC remains unclear. Knockdown of ATG5 in cancer cells enhances the antitumor efficacy of lots of chemotherapeutic agents. However, there is still no animal model to validate these in vitro observations in vivo. In this study, we found that heterozygous deletion of ATG5 in Apc(Min/+) mice increased the number and size of adenomas as compared with those in Apc(Min/+)ATG5(+/+) mice. To investigate whether ATG5 deficiency could sensitize tumors to chemotherapies, we compared the antitumor effects of Interferon-gamma (IFN-γ) between Apc(Min/+)ATG5(+/+) and Apc(Min/+)ATG5(+/-) mice, as IFN-γ is a potential tumor suppressor for CRC and has been used clinically as an efficient adjuvant to chemotherapy of cancer. We revealed that heterozygous deletion of ATG5 significantly enhanced the antitumor efficacy of IFN-γ. Early treatment of Apc(Min/+)ATG5(+/-) mice with IFN-γ decreased tumor incidence rate to 16.7% and reduced the number of adenomas by 95.5% and late treatment led to regression of tumor. Moreover, IFN-γ treatment did not cause any evident toxic reaction. Mechanistic analysis revealed that heterozygous deletion of ATG5 activated EGFR/ERK1/2 and Wnt/β-catenin pathways in adenomas of Apc(Min/+) mice and enhanced the effects of IFN-γ-dependent inhibition of these 2 pathways. Our results demonstrate that ATG5 plays important roles in intestinal tumor growth and combination of IFN-γ and ATG5 deficiency or ATG5-targeted inhibition is a promising strategy for prevention and treatment of CRC. PMID:25695667

  4. Complex, multimodal behavioral profile of the Homer1 knockout mouse.

    PubMed

    Jaubert, P J; Golub, M S; Lo, Y Y; Germann, S L; Dehoff, M H; Worley, P F; Kang, S H; Schwarz, M K; Seeburg, P H; Berman, R F

    2007-03-01

    Proteins of the Homer1 immediate early gene family have been associated with synaptogenesis and synaptic plasticity suggesting broad behavioral consequences of loss of function. This study examined the behavior of male Homer1 knockout (KO) mice compared with wild-type (WT) and heterozygous mice using a battery of 10 behavioral tests probing sensory, motor, social, emotional and learning/memory functions. KO mice showed mild somatic growth retardation, poor motor coordination, enhanced sensory reactivity and learning deficits. Heterozygous mice showed increased aggression in social interactions with conspecifics. The distribution of mGluR5 and N-methyl-D-aspartate receptors (NMDA) receptors appeared to be unaltered in the hippocampus (HIP) of Homer1 KO mice. The results indicate an extensive range of disrupted behaviors that should contribute to the understanding of the Homer1 gene in brain development and behavior.

  5. GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting.

    PubMed

    Nanjidsuren, Tsevelmaa; Park, Chae-Won; Sim, Bo-Woong; Kim, Sun-Uk; Chang, Kyu-Tae; Kang, Myung-Hwa; Min, Kwan-Sik

    2016-10-01

    Transcription activator-like effector nucleases (TALENs) are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout (KO) of the G protein-coupled receptor kinase (GRK) 5 gene by microinjection of TALEN mRNA. TALEN vectors were designed to target exons 1, 3, and 5 of the mouse GRK5 gene. Flow cytometry showed that the activity of the TALEN mRNAs targeted to exons 1, 3, and 5 was 8.7%, 9.7%, and 12.7%, respectively. The TALEN mRNA for exon 5 was injected into the cytoplasm of 180 one-cell embryos. Of the 53 newborns, three (5.6%) were mutant founders (F0) with mutations. Two clones from F028 showed a 45-bp deletion and F039 showed the same biallelic non-frame-shifting 3-bp deletions. Three clones from F041 were shown to possess a combination of frame-shifting 2-bp deletions. All of the mutations were transmitted through the germline but not to all progenies (37.5%, 37.5%, and 57.1% for the F028, F039, and F041 lines, respectively). The homozygote GRK5-KO mice for 28 and 41 lines created on F3 progenies and the homozygous genotype was confirmed by PCR, T7E1 assay and sequencing. PMID:27565865

  6. Prostanoids and inflammation: a new concept arising from receptor knockout mice.

    PubMed

    Narumiya, Shuh

    2009-10-01

    Prostanoids including various types of prostaglandins and thromboxanes are arachidonate metabolites produced and released in response to a variety of physiological and pathological stimuli and function to maintain the body homeostasis. Since cyclooxygenase, the enzyme initiating their biosynthesis, is inhibited by aspirin-like antipyretic, anti-inflammatory, and analgesic drugs, contribution of prostanoids to acute inflammation such as fever generation, pain sensitization, and inflammatory swelling has been recognized very early. On the other hand, since aspirin-like drugs generally show little effects on allergy and immunity, it has been believed that prostanoids play little roles in these processes. Prostanoids act on a family of G-protein-coupled receptors designated PGD receptor, PGE receptor subtypes EP1-EP4, PGF receptor, PGI receptor, and TX receptor to elicit their actions. Studies using mice deficient in each of these receptors have revealed that prostanoids indeed function in the above aspirin-sensitive processes. However, these studies have also revealed that prostanoids exert both pro-inflammatory and anti-inflammatory actions not only by acting as mediators of acute inflammation but also by regulating gene expression in mesenchymal and epithelial cells at inflammatory site. Such dual actions of prostanoids are frequently seen in immune and allergic reactions, where different type of prostanoids and their receptors often exert opposite actions in a single process. Thus, a new concept on the role of prostanoids in inflammation has arisen from studies using the receptor knockout mice. PMID:19609495

  7. Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

    PubMed

    Kirchner, Liselotte; Weitzdoerfer, Rachel; Hoeger, Harald; Url, Angelika; Schmidt, Peter; Engelmann, Mario; Villar, Santiago Rosell; Fountoulakis, Michael; Lubec, Gert; Lubec, Barbara

    2004-12-01

    Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

  8. Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin.

    PubMed

    Stachowicz, Aneta; Suski, Maciej; Olszanecki, Rafał; Madej, Józef; Okoń, Krzysztof; Korbut, Ryszard

    2012-12-21

    Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. Metformin, a widely known anti-diabetic drug, used for patients with type 2 diabetes mellitus, is also claimed to be useful in treatment of NAFLD. However, both the clinical efficacy and the putative mechanisms underlying the clinical effects of metformin in treating NAFLD are unclear. Adenosine monophosphate-activated protein kinase (AMPK), the primary molecular target for metformin, is a known regulator of mitochondrial function. Thus, we used a proteomic approach to investigate the effect of metformin on liver mitochondria of apolipoprotein E knockout (apoE(-/-)) mice, an animal model of NAFLD. Two-dimensional electrophoresis coupled with mass spectrometry was applied to study the changes in liver mitochondrial protein expression in 6-month old metformin-treated apoE(-/-) mice as compared to non-treated animals. Collectively, 25 differentially expressed proteins were indentified upon metformin treatment including proteins related to metabolism, oxidative stress and cellular respiration. The most up-regulated protein was glycine N-methyltransferase (GNMT) - an enzyme, whose deficiency was shown to be directly related to the development of NAFLD. Our results clearly point to the strong mitochondrial action of metformin in NAFLD. Up-regulation of GNMT may represent an important mechanism of beneficial action of metformin in NAFLD treatment.

  9. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

    PubMed

    White, Carl W; Choong, Yan-Ting; Short, Jennifer L; Exintaris, Betty; Malone, Daniel T; Allen, Andrew M; Evans, Richard J; Ventura, Sabatino

    2013-12-17

    Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive. PMID:24297884

  10. [The role of tetrodotoxin-resistant sodium channels in pain sensation studied on sns-knockout mice].

    PubMed

    Ogata, N; Yamamoto, M; Maruyama, H

    2001-09-01

    Nociceptive dorsal root ganglion neurons express sensory neuron-specific tetrodotoxin (TTX)-resistant voltage-gated sodium channel(SNS). The role of SNS in nociception has been studied by constructing sns-knockout mice. The sns-knockout mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, demonstrating that the slow TTX-resistant currents are mediated by the sns gene. The mutant mice were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive sodium channels demonstrated compensatory up-regulation of TTX-sensitive currents in DRG neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain sensation. PMID:11554037

  11. Impaired neuronal migration and endochondral ossification in Pex7 knockout mice: a model for rhizomelic chondrodysplasia punctata.

    PubMed

    Brites, Pedro; Motley, Alison M; Gressens, Pierre; Mooyer, Petra A W; Ploegaert, Ingrid; Everts, Vincent; Evrard, Philippe; Carmeliet, Peter; Dewerchin, Mieke; Schoonjans, Luc; Duran, Marinus; Waterham, Hans R; Wanders, Ronald J A; Baes, Myriam

    2003-09-15

    Rhizomelic chondrodysplasia punctata is a human autosomal recessive disorder characterized by skeletal, eye and brain abnormalities. The disorder is caused by mutations in the PEX7 gene, which encodes the receptor for a class of peroxisomal matrix enzymes. We describe the generation and characterization of a Pex7 mouse knockout (Pex7(-/-)). Pex7(-/-) mice are born severely hypotonic and have a growth impairment. Mortality in Pex7(-/-) mice is highest in the perinatal period although some Pex7(-/-) mice survived beyond 18 months. Biochemically Pex7(-/-) mice display the abnormalities related to a Pex7 deficiency, i.e. a severe depletion of plasmalogens, impaired alpha-oxidation of phytanic acid and impaired beta-oxidation of very-long-chain fatty acids. In the intermediate zone of the developing cerebral cortex Pex7(-/-) mice have an increase in neuronal density. In vivo neuronal birthdating revealed that Pex7(-/-) mice have a delay in neuronal migration. Analysis of bone ossification in newborn Pex7(-/-) mice revealed a defect in ossification of distal bone elements of the limbs as well as parts of the skull and vertebrae. These findings demonstrate that Pex7 knockout mice provide an important model to study the role of peroxisomal functioning in the pathogenesis of the human disorder.

  12. Stress-induced dura vascular permeability does not develop in mast cell-deficient and neurokinin-1 receptor knockout mice.

    PubMed

    Kandere-Grzybowska, Kristiana; Gheorghe, Daniela; Priller, Josef; Esposito, Pamela; Huang, Man; Gerard, Norma; Theoharides, Theoharis C

    2003-08-01

    Migraine headaches are often precipitated by stress and seem to involve neurogenic inflammation (NI) of the dura mater associated with the sensation of throbbing pain. Trigeminal nerve stimulation had been reported to activate rat dura mast cells and increase vascular permeability, effects inhibited by neonatal pretreatment with capsaicin implicating sensory neuropeptides, such as substance P (SP). The aim of the present study was to investigate NI, assessed by extravasation of 99-Technetium-gluceptate (99Tc-G), as well as the role of mast cells, SP and its receptor (NK-1R) in dura mater of mice in response to acute stress. Restraint stress for thirty min significantly increased 99Tc-G extravasation in the dura mater of C57BL mice. This effect was absent in W/W(v) mast cell-deficient mice and NK-1 receptor knockout mice (NK-1R-/-), but was unaltered in SP knockout mice (SP-/-). Acute restraint stress also resulted in increased dura mast cell activation in C57BL mice, but not in NK-1R-/- mice. These data demonstrate for the first time that acute stress triggers NI and mast cell activation in mouse dura mater through the activation of NK-1 receptors. The fact that SP-/- mice had intact vascular permeability response to stress indicates that some other NK-1 receptor agonist may substitute for SP. These results may help explain initial events in pathogenesis of stress-induced migraines.

  13. β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice

    PubMed Central

    Propping, Stefan; Lorenz, Kristina; Michel, Martin C.; Wirth, Manfred P.; Ravens, Ursula

    2016-01-01

    Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological

  14. Heterozygous deficiency of hypoxia-inducible factor–2α protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia

    PubMed Central

    Brusselmans, Koen; Compernolle, Veerle; Tjwa, Marc; Wiesener, Michael S.; Maxwell, Patrick H.; Collen, Désiré; Carmeliet, Peter

    2003-01-01

    Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor–1α (HIF-1α), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2α is a homologue of HIF-1α and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2α–deficient (Hif2α+/–) mice after exposure to 10% O2 for 4 weeks. In contrast to WT mice, Hif2α+/– mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2α in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2α+/– mice after hypoxia, suggesting that HIF-2α–mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling. PMID:12750401

  15. Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice*

    PubMed Central

    Simmer, James P.; Hu, Yuanyuan; Lertlam, Rangsiyakorn; Yamakoshi, Yasuo; Hu, Jan C.-C.

    2009-01-01

    Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining demonstrated expression of β-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals. PMID:19578120

  16. Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

    PubMed

    Dubey, J P; Verma, S K; Dunams, D; Calero-Bernal, R; Rosenthal, B M

    2015-11-01

    The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri. PMID:26303093

  17. Temporal migration of gonadotrophin-releasing hormone-1 neurones is modified in GAD67 knockout mice.

    PubMed

    Lee, J M; Tiong, J; Maddox, D M; Condie, B G; Wray, S

    2008-01-01

    Gonadotrophin-releasing hormone (GnRH-1) neurones reside in the forebrain and regulate gonadal function via the hypothalamic-pituitary-gonadal axis. Disruption of this axis results in reproductive dysfunction. During embryonic development, GnRH-1 neurones migrate from the nasal pit through the nasal/forebrain junction (NFJ) into the developing brain. Prenatally gamma-aminobutyric acid (GABA) is excitatory and has been shown to play a role in nervous system development. Both in vivo and in vitro experiments suggest that GABA inhibits migration of GnRH-1 neurones. The present study examines the migration of GnRH-1 neurones in GAD67 knockout (KO) mice to further elucidate the role of GABA on GnRH-1 neuronal development. Three stages were examined, embryonic day (E)12.5, E14.5 and E17.5. GnRH-1 cell number and location were analysed by immunocytochemistry and in situ hybridisation histochemistry. The total number of GnRH-1 immunopositive cells was similar between wild-type (WT) and KO mice. However, significant differences were found in the overall distribution of GnRH-1 immunopositive cells in GAD67 KO compared to WT mice at all stages. Subsequent analysis by area revealed differences occurred at the NFJ with an increase in GnRH-1 cells in GAD67 KO at E14.5 and a decrease in GnRH-1 cells in GAD67 KO at E17.5. Comparable counts for cells expressing GnRH-1 transcript and protein were obtained. These data indicate that attenuated levels of GABA accelerate GnRH-1 cell migration in nasal areas as well as movement of GnRH-1 cells into the central nervous system at the NFJ.

  18. Increased susceptibility to pulmonary Pseudomonas infection in Splunc1 knockout mice.

    PubMed

    Liu, Yanyan; Di, Marissa E; Chu, Hong Wei; Liu, Xinyu; Wang, Ling; Wenzel, Sally; Di, Y Peter

    2013-10-15

    The airway epithelium is the first line of host defense against pathogens. The short palate, lung, and nasal epithelium clone (SPLUNC)1 protein is secreted in respiratory tracts and is a member of the bacterial/permeability increasing (BPI) fold-containing protein family, which shares structural similarities with BPI-like proteins. On the basis of its homology with BPIs and restricted expression of SPLUNC1 in serous cells of submucosal glands and surface epithelial cells of the upper respiratory tract, SPLUNC1 is thought to possess antimicrobial activity in host defense. SPLUNC1 is also reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to determine the in vivo functions of SPLUNC1 following Pseudomonas aeruginosa infection and to elucidate the underlying mechanism by using a knockout (KO) mouse model with a genetic ablation of Splunc1. Splunc1 KO mice showed accelerated mortality and increased susceptibility to P. aeruginosa infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels as compared with those of their wild-type littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated chemokine levels, including Cxcl1, Cxcl2, and Ccl20. Furthermore, the expression of several epithelial secretory proteins and antimicrobial molecules was considerably suppressed in the lungs of Splunc1 KO mice. The deficiency of Splunc1 in mouse airway epithelium also results in increased biofilm formation of P. aeruginosa. Taken together, our results support that the ablation of Splunc1 in mouse airways affects the mucociliary clearance, resulting in decreased innate immune response during Pseudomonas-induced respiratory infection. PMID:24048904

  19. Kisspeptin regulation of arcuate neuron excitability in kisspeptin receptor knockout mice.

    PubMed

    Liu, Xinhuai; Herbison, Allan

    2015-05-01

    The G protein-coupled receptor 54 (GPR54) is critical for kisspeptin to activate GnRH neurons to modulate fertility. However, the often mismatching distribution of kisspeptin and GPR54 in the brain suggests that kisspeptin may also act on other receptors. The arcuate nucleus (ARN) is one brain region with a very high density of kisspeptin fibers but only limited evidence for the expression of GPR54. Using acute brain slice electrophysiology in combination with Gpr54 knockout (GPR54KO) mouse models, we examined whether actions of kisspeptin in the ARN were dependent upon GPR54. Cell-attached recordings from unidentified ARN neurons in wild-type mice revealed that approximately one third of neurons were either excited or inhibited by kisspeptin in a dose-dependent manner. The responses of ARN neurons to kisspeptin were exactly the same in GPR54KO mice despite effects of kisspeptin on GnRH neurons being abolished. To evaluate whether kisspeptin may be acting through neuropeptide FF receptors, the effects of an agonist RFamide-related peptide 3 (RFRP-3) and antagonists RF9 and BIBP-3226 were evaluated. Both the excitatory and inhibitory effects of kisspeptin were mimicked by the agonist RFRP-3. RF9 itself activated ARN neurons and suppressed only the inhibitory actions of kisspeptin. BIBP-3226 suppressed kisspeptin actions in 50% of neurons. Whole-cell recordings in GPR54KO mice demonstrated that both kisspeptin and RFRP-3 acted directly on the same ARN neurons and activated the same ion channels. Together, these studies demonstrate that kisspeptin can act partly through neuropeptide FF receptors to modulate neuronal activity independent of GPR54 in the mouse brain.

  20. Reduced anxiety-like and depression-related behavior in neuropeptide Y Y4 receptor knockout mice.

    PubMed

    Painsipp, E; Wultsch, T; Edelsbrunner, M E; Tasan, R O; Singewald, N; Herzog, H; Holzer, P

    2008-07-01

    Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4-/-) mice with control and germ line Y2-/- animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4-/- and Y2-/- mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4-/- but reduced in Y2-/- mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4-/- and Y2-/- mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4-/- and Y2-/- mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2-/- but not in Y4-/- mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.

  1. Altered photic and non-photic phase shifts in 5-HT(1A) receptor knockout mice.

    PubMed

    Smith, V M; Sterniczuk, R; Phillips, C I; Antle, M C

    2008-12-01

    The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT(1A) receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT(1A) knockout animals. Finally, non-photic phase shifts to (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT(1A) receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.

  2. Improved efficacy of fluoxetine in increasing hippocampal 5-hydroxytryptamine outflow in 5-HT(1B) receptor knock-out mice.

    PubMed

    Malagié, Isabelle; David, Denis J; Jolliet, Pascale; Hen, René; Bourin, Michel; Gardier, Alain M

    2002-05-17

    To test for the contribution of the 5-HT(1B) receptor subtype in mediating the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), we used intracerebral in vivo microdialysis in awake, freely moving 5-HT(1B) receptor knock-out mice. We show that a single systemic administration of fluoxetine (1, 5 or 10 mg/kg, i.p.) increased extracellular serotonin levels [5-HT](ext) in the ventral hippocampus and frontal cortex of wild-type and mutant mice. However, in the ventral hippocampus, fluoxetine, at the three doses studied, induced a larger increase in [5-HT](ext) in knock-out than in wild-type mice. In the frontal cortex, the effect of fluoxetine did not differ between the two genotypes. The region-dependent response to fluoxetine described here in mutants confirms data we recently reported for another SSRI, paroxetine. These data suggest that 5-HT(1B) autoreceptors limit the effects of selective serotonin reuptake inhibitors on dialysate 5-HT levels at serotonergic nerve terminals located mainly in the ventral hippocampus. Alternative mechanisms, e.g., changes in 5-HT transporter and/or 5-HT(1A) receptor density in 5-HT(1B) receptor knock-out mice could also explain these findings.

  3. Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays.

    PubMed

    Kavaliers, M; Agmo, A; Choleris, E; Gustafsson, J A; Korach, K S; Muglia, L J; Pfaff, D W; Ogawa, S

    2004-08-01

    Social behavior involves both the recognition and pro-duction of social cues. Mice with selective deletion(knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -c or -B display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knock-outs IKO: OTKO, alphaERKO, betaERKO) from the odors of their wildtype littermates (WT: OTWT, alphaERWT, betaERWT). Females showed marked initial choices of the urine odors of OTWT and betaERWT males over those of OTKOand PERKO males, and alphaERKO males over alphaERWT males. The odors of OTKO and betaERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the alphaERWT andalphaERKO males induced aversive, analgesic responses,with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females dis-playing significantly greater responses to the odors of stressed OTKO and betaERKO males than their WTs, and significantly lower analgesia to the odors of stressedalphaERKO than alphaERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners'modified responses to their odors.

  4. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only.

    PubMed

    Pagani, J H; Williams Avram, S K; Cui, Z; Song, J; Mezey, É; Senerth, J M; Baumann, M H; Young, W S

    2015-02-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to 8 of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care.

  5. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only

    PubMed Central

    Pagani, Jerome H.; Williams Avram, Sarah K.; Cui, Zhenzhong; Song, June; Mezey, Éva; Senerth, Julia M.; Baumann, Michael H.; Young, W. Scott

    2015-01-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically-mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to eight of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. PMID:25677455

  6. Basal Bone Phenotype and Increased Anabolic Responses to Intermittent Parathyroid Hormone in Healthy Male COX-2 Knockout Mice

    PubMed Central

    Xu, Manshan; Choudhary, Shilpa; Voznesensky, Olga; Gao, Qi; Adams, Douglas; Diaz-Doran, Vilmaris; Wu, Qian; Goltzman, David; Raisz, Lawrence G.; Pilbeam, Carol C.

    2011-01-01

    Cyclooxygenase-2 (COX-2) knockout (KO) mice in inbred strains can have renal dysfunction with secondary hyperparathyroidism (HPTH), making direct effects of COX-2 KO on bone difficult to assess. COX-2 KO mice in an outbred CD-1 background did not have renal dysfunction but still had two-fold elevated PTH compared to wild type (WT) mice. Compared to WT mice, KO mice had increased serum markers of bone turnover, decreased femoral bone mineral density (BMD) and cortical bone thickness, but no differences in trabecular bone volume by μCT or dynamic histomorphometry. Because PTH is a potent inducer of COX-2 and prostaglandin (PG) production, we examined effects of COX-2 KO on bone responses after three weeks of intermittent PTH. Intermittent PTH increased femoral BMD and cortical bone area more in KO mice than in WT mice and increased trabecular bone volume in the distal femur in both WT and KO mice. Although not statistically significant, PTH-stimulated increases in trabecular bone tended to be greater in KO mice than in WT mice. PTH increased serum markers of bone formation and resorption more in KO than in WT mice but increased the ratio of osteoblastic surface to osteoclastic surface only in KO mice. PTH also increased femoral mineral apposition rates and bone formation rates in KO mice more than in WT mice. Acute mRNA responses to PTH of genes that might mediate some anabolic and catabolic effects of PTH tended to be greater in KO than WT mice. We conclude that (1) the basal bone phenotype in male COX-2 KO mice might reflect HPTH, COX-2 deficiency or both, and (2) increased responses to intermittent PTH in COX-2 KO mice, despite the presence of chronic HPTH, suggest that absence of COX-2 increased sensitivity to PTH. It is possible that manipulation of endogenous PGs could have important clinical implications for anabolic therapy with PTH. PMID:20471507

  7. Increased frequencies of micronucleated reticulocytes and T-cell receptor mutation in Aldh2 knockout mice exposed to acetaldehyde.

    PubMed

    Kunugita, Naoki; Isse, Toyohi; Oyama, Tsunehiro; Kitagawa, Kyoko; Ogawa, Masanori; Yamaguchi, Tetsunosuke; Kinaga, Tsuyoshi; Kawamoto, Toshihiro

    2008-02-01

    Aldehyde dehydrogenase-2 (ALDH2) metabolizes acetaldehyde produced from ethanol into acetate and plays a major role in the oxidation of acetaldehyde in vivo. About half of all Japanese people have inactive ALDH2. We generated homozygous Aldh2 null (Aldh2-/-) mice by gene targeting knockout as a model of ALDH2-deficient humans. To investigate the mutagenicity of acetaldehyde, a micronucleus assay and a T-cell receptor (TCR) gene mutation assay were performed in Aldh2-/- mice and wild-type (Aldh2 +/+) mice exposed to acetaldehyde. The mice were continuously exposed to 125 and 500 ppm of acetaldehyde vapor for 2 weeks. Another group was orally administered 100 mg/kg once a day for 2 weeks continuously. The mice were killed after 2 weeks of exposure to acetaldehyde, and the frequency of micronucleated reticulocytes was measured by flow cytometry. We also observed the incidence of TCR gene mutations in T-lymphocytes by measuring the variant CD3(-CD4+) expression by flow cytometry. The frequency of micronucleated reticulocytes induced by acetaldehyde was significantly increased in Aldh2 -/- mice, but not in Aldh2 +/+ mice. TCR mutant frequency was also associated with acetaldehyde exposure in Aldh2-/ - mice, especially after oral administration; however, it was not associated with acetaldehyde exposure in Aldh2 +/+ mice. In conclusion, Aldh2 -/- mice showed high sensitivity in the micronuclei and TCR mutation assays compared with Aldh2 +/+ mice after exposure to acetaldehyde. PMID:18303182

  8. Role of Olfaction in the Conditioned Sucrose Preference of Sweet-Ageusic T1R3 Knockout Mice

    PubMed Central

    Zukerman, Steven; Touzani, Khalid; Margolskee, Robert F.

    2009-01-01

    Prior work has shown that sweet taste–deficient T1R3 knockout (KO) mice developed significant sucrose preferences when given long-term sugar versus water tests. The current study investigated the role of olfaction in this experience-conditioned sucrose preference. T1R3 KO and C57BL/6 wild-type (WT) mice were given 24-h sugar versus water tests with ascending concentrations of sucrose (0.5–32%), after which the mice received olfactory bulbectomy (OBx) or sham surgery. When retested with sucrose, the Sham-KO mice preferred all sugar solutions to water, although their intake and preference were less than those of the Sham-WT mice. The OBx-KO mice, in contrast, showed no or weak preferences for dilute sucrose solutions (0.5–8%) although they strongly preferred concentrated sugar solutions (16–32%). OBx-WT mice displayed only a partial reduction in their sucrose preference. Although the OBx mice of both genotypes underconsumed dilute sucrose solutions relative to Sham mice, they overconsumed concentrated sucrose. These results indicate that olfaction plays a critical role in the conditioned preference of T1R3 KO mice for dilute sugar solutions. Further, the fact that OBx-KO mice preferred concentrated sucrose solutions in the absence of normal sweet taste and olfactory sensations underscores the potency of postoral nutritive signals in promoting ingestion. PMID:19736224

  9. Profound defects in pancreatic beta-cell function in mice with combined heterozygous mutations in Pdx-1, Hnf-1alpha, and Hnf-3beta.

    PubMed

    Shih, David Q; Heimesaat, Markus; Kuwajima, Satoru; Stein, Roland; Wright, Christopher V E; Stoffel, Markus

    2002-03-19

    Defects in pancreatic beta-cell function contribute to the development of type 2 diabetes, a polygenic disease that is characterized by insulin resistance and compromised insulin secretion. Hepatocyte nuclear factors (HNFs) -1alpha, -3beta, -4alpha, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in beta-cell development and function. In mice, a heterozygous mutation in Pdx-1 alone, but not Hnf-1alpha(+/-), Hnf-3beta(+/-), or Hnf-4alpha(+/-), causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on beta-cell activity in vivo, we generated mice with the following combined heterozygous mutations: Pdx-1(+/-)/Hnf-1alpha(+/-), Pdx-1(+/-)/Hnf-3beta(+/-), Pdx-1(+/-)/Hnf-4alpha(+/-), Hnf-1alpha(+/-)/Hnf-4alpha(+/-), and Hnf-3beta(+/-)/Hnf-4alpha(+/-). The greatest loss in function was in combined heterozygous null alleles of Pdx-1 and Hnf-1alpha (Pdx-1(+/-)/Hnf-1alpha(+/-)), or Pdx-1 and Hnf-3beta (Pdx-1(+/-)/Hnf-3beta(+/-)). Both double mutants develop progressively impaired glucose tolerance and acquire a compromised first- and second-phase insulin secretion profile in response to glucose compared with Pdx-1(+/-) mice alone. The loss in beta-cell function in Pdx-1(+/-)/Hnf-3beta(+/-) mice was associated with decreased expression of Nkx-6.1, glucokinase (Gck), aldolase B (aldo-B), and insulin, whereas Nkx2.2, Nkx-6.1, Glut-2, Gck, aldo-B, the liver isoform of pyruvate kinase, and insulin expression was reduced in Pdx-1(+/-)/Hnf-1alpha(+/-) mice. The islet cell architecture was also abnormal in Pdx-1(+/-)/Hnf-3beta(+/-) and Pdx-1(+/-)/Hnf-1alpha(+/-) mice, with glucagon-expressing cells scattered throughout the islet, a defect that may be connected to decreased E-cadherin expression. Our data suggest that functional interactions between key islet regulatory factors play an important role in maintaining islet architecture and

  10. Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice

    PubMed Central

    Poulos, Michael G.; Batra, Ranjan; Li, Moyi; Yuan, Yuan; Zhang, Chaolin; Darnell, Robert B.; Swanson, Maurice S.

    2013-01-01

    The muscleblind-like (MBNL) genes encode alternative splicing factors that are essential for the postnatal development of multiple tissues, and the inhibition of MBNL activity by toxic C(C)UG repeat RNAs is a major pathogenic feature of the neuromuscular disease myotonic dystrophy. While MBNL1 controls fetal-to-adult splicing transitions in muscle and MBNL2 serves a similar role in the brain, the function of MBNL3 in vivo is unknown. Here, we report that mouse Mbnl3, which encodes protein isoforms that differ in the number of tandem zinc-finger RNA-binding motifs and subcellular localization, is expressed primarily during embryonic development but also transiently during injury-induced adult skeletal muscle regeneration. Mbnl3 expression is required for normal C2C12 myogenic differentiation and high-throughput sequencing combined with cross-linking/immunoprecipitation analysis indicates that Mbnl3 binds preferentially to the 3′ untranslated regions of genes implicated in cell growth and proliferation. In addition, Mbnl3ΔE2 isoform knockout mice, which fail to express the major Mbnl3 nuclear isoform, show age-dependent delays in injury-induced muscle regeneration and impaired muscle function. These results suggest that Mbnl3 inhibition by toxic RNA expression may be a contributing factor to the progressive skeletal muscle weakness and wasting characteristic of myotonic dystrophy. PMID:23660517

  11. Repair of dentin defects from DSPP knockout mice by PILP mineralization

    PubMed Central

    Nurrohman, H.; Saeki, K.; Carneiro, K.; Chien, Y.C.; Djomehri, S.; Ho, S.P.; Qin, C.; Marshall, S.J.; Gower, L.B.; Marshall, G.W.; Habelitz, S.

    2016-01-01

    Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp−/−) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the “polymer-induced liquid-precursor” (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp−/− and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp−/− dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair. PMID:27239097

  12. Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice.

    PubMed

    Ohsawa, Ikuroh; Nishimaki, Kiyomi; Yamagata, Kumi; Ishikawa, Masahiro; Ohta, Shigeo

    2008-12-26

    Oxidative stress is implicated in atherogenesis; however most clinical trials with dietary antioxidants failed to show marked success in preventing atherosclerotic diseases. We have found that hydrogen (dihydrogen; H(2)) acts as an effective antioxidant to reduce oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K. Katsura, Y. Katayama, S, Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688-694]. Here, we investigated whether drinking H(2)-dissolved water at a saturated level (H(2)-water) ad libitum prevents arteriosclerosis using an apolipoprotein E knockout mouse (apoE(-/-)), a model of the spontaneous development of atherosclerosis. ApoE(-/-) mice drank H(2)-water ad libitum from 2 to 6 month old throughout the whole period. Atherosclerotic lesions were significantly reduced by ad libitum drinking of H(2)-water (p=0.0069) as judged by Oil-Red-O staining series of sections of aorta. The oxidative stress level of aorta was decreased. Accumulation of macrophages in atherosclerotic lesions was confirmed. Thus, consumption of H(2)-dissolved water has the potential to prevent arteriosclerosis. PMID:18996093

  13. Pravastatin and Sarpogrelate Synergistically Ameliorate Atherosclerosis in LDLr-Knockout Mice.

    PubMed

    Park, Kyung-Yeon; Oh, Euichaul; Kwak, Mi-Kyoung; Jun, Hyun Sik; Heo, Tae-Hwe

    2016-01-01

    Pravastatin is a lipid-lowering agent that attenuates atherosclerosis. However, the multifactorial pathogenesis of atherosclerosis requires other drugs with different anti-atherogenic mechanisms. We chose sarpogrelate as an anti-platelet agent and a novel component of a complex drug with pravastatin due to its high potential but little information on its beneficial effects on atherosclerosis. Low-density lipoprotein receptor-knockout mice were fed a high-fat, high-cholesterol diet and treated with pravastatin alone, sarpogrelate alone, or a combination of both drugs. Although sarpogrelate alone did not significantly reduce atherosclerotic plaque areas, co-treatment with pravastatin significantly decreased aortic lesions compared to those of the pravastatin alone treated group. The combined therapy was markedly more effective than that of the single therapies in terms of foam cell formation, smooth muscle cell proliferation, and inflammatory cytokine levels. These results suggest that pravastatin and sarpogrelate combined therapy may provide a new therapeutic strategy for treating atherosclerosis. PMID:26950217

  14. Decreased BMP2 signal in GIT1 knockout mice slows bone healing

    PubMed Central

    Fan, Jin; Zhou, Hao; Zuscik, Michael J.; Xie, Chao; Yin, Guoyong; Berk, Bradford C.

    2015-01-01

    Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor b (TGFb) superfamily plays important roles and comprises TGFbs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFbs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFb signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFb1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFb1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site. PMID:25138700

  15. Selective Degeneration of Central Photoreceptors after Hyperbaric Oxygen in Normal and Metallothionein-Knockout Mice

    PubMed Central

    Nachman-Clewner, Michele; Giblin, Frank J.; Kathleen Dorey, C.; Blanks, Robert H. I.; Dang, Loan; Dougherty, Christopher J.; Blanks, Janet C.

    2009-01-01

    Purpose Metallothioneins (MTs) in the brain and retina are believed to bind metals and reduce free radicals, thereby protecting neurons from oxidative damage. This study was undertaken to investigate whether retinal photoreceptor (PR) cells lacking MTs are more susceptible to hyperbaric oxygen (HBO)–induced cell death in vivo. Methods Wild-type (WT) and MT-knockout (MT-KO) mice lacking metallothionein (MT)-1 and MT-2 were exposed to three atmospheres of 100% oxygen for 3 hours, 3 times per week for 1, 3, or 5 weeks. The control animals were not exposed. Histologic analysis of PR viability was performed by counting rows of nuclei in the outer nuclear layer (ONL). Ultrastructure studies verified PR damage. Results HBO exposure produced a major loss of PR cells in the central retinas of WT and MT-KO mice, with no effect on the peripheral retina even at the longest (5 weeks) exposures. The degree of PR damage and cell death increased with duration of HBO exposure. One week of HBO exposure was insufficient to cause PR death, but tissue damage was observed in the inner and outer segments. At 3 weeks, the rows of PR nuclei in the central retina were significantly reduced by 38% in WT and 28% in MT-KO animals. At 5 weeks, PR loss was identical in WT (34%) and MT-KO (34%) animals and was comparable to that in WT at 3 weeks. Conclusions The data suggest that MT-1 and -2 alone are not sufficient for protecting PRs against HBO-induced cell death. The selective degeneration of central PRs may provide clues to mechanisms of oxidative damage in retinal disease. PMID:18579766

  16. Cadium pathways during gestation and lactation in control vs. metallothionein 1,2-knockout mice.

    SciTech Connect

    Brako, E. E.; Wilson, A. K.; Jonah, M. M.; Blum, C. A.; Cerny, E. A.; Williams, K. L.; Bhattacharyya, M. H.; Winona State Univ.; Benedictine Univ.; Dominican Univ.

    2003-01-01

    Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of {sup 109}CdCl{sub 2} (0.15 ng Cd/ml; 0.074 {mu}Ci {sup 109}Cd/ml). {sup 109}Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of {sup 109}Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific {sup 109}Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental {sup 109}Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal {sup 109}Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of {sup 109}Cd transferred to pups via milk; furthermore, 85--90% of total pup {sup 109}Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal {sup 109}Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.

  17. Acceleration of bone development and regeneration through the Wnt/β-catenin signaling pathway in mice heterozygously deficient for GSK-3β

    SciTech Connect

    Arioka, Masaki; Takahashi-Yanaga, Fumi; Sasaki, Masanori; Yoshihara, Tatsuya; Morimoto, Sachio; Takashima, Akihiko; Mori, Yoshihide; Sasaguri, Toshiyuki

    2013-11-01

    Highlights: •The Wnt/β-catenin signaling pathway was activated in GSK-3β{sup +/−} mice. •The cortical and trabecular bone volumes were increased in GSK-3β{sup +/−} mice. •Regeneration of a partial bone defect was accelerated in GSK-3β{sup +/−} mice. -- Abstract: Glycogen synthase kinase (GSK)-3β plays an important role in osteoblastogenesis by regulating the Wnt/β-catenin signaling pathway. Therefore, we investigated whether GSK-3β deficiency affects bone development and regeneration using mice heterozygously deficient for GSK-3β (GSK-3β{sup +/−}). The amounts of β-catenin, c-Myc, cyclin D1, and runt-related transcription factor-2 (Runx2) in the bone marrow cells of GSK-3β{sup +/−} mice were significantly increased compared with those of wild-type mice, indicating that Wnt/β-catenin signals were enhanced in GSK-3β{sup +/−} mice. Microcomputed tomography of the distal femoral metaphyses demonstrated that the volumes of both the cortical and trabecular bones were increased in GSK-3β{sup +/−} mice compared with those in wild-type mice. Subsequently, to investigate the effect of GSK-3β deficiency on bone regeneration, we established a partial bone defect in the femur and observed new bone at 14 days after surgery. The volume and mineral density of the new bone were significantly higher in GSK-3β{sup +/−} mice than those in wild-type mice. These results suggest that bone formation and regeneration in vivo are accelerated by inhibition of GSK-3β, probably through activation of the Wnt/β-catenin signaling pathway.

  18. Cadherin 13: Human cis-Regulation and Selectively Altered Addiction Phenotypes and Cerebral Cortical Dopamine in Knockout Mice

    PubMed Central

    Drgonova, Jana; Walther, Donna; Hartstein, G Luke; Bukhari, Mohammad O; Baumann, Michael H; Katz, Jonathan; Hall, F Scott; Arnold, Elizabeth R; Flax, Shaun; Riley, Anthony; Rivero, Olga; Lesch, Klaus-Peter; Troncoso, Juan; Ranscht, Barbara; Uhl, George R

    2016-01-01

    The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and connections of brain circuits that modulate addiction, locomotion and cognition, including those that involve midbrain dopamine neurons. Human CDH13 mRNA expression differs by more than 80% in postmortem cerebral cortical samples from individuals with different CDH13 genotypes, supporting examination of mice with altered CDH13 expression as models for common human variation at this locus. Constitutive CDH13 knockout mice display evidence for changed cocaine reward: shifted dose response relationship in tests of cocaine-conditioned place preference using doses that do not alter cocaine-conditioned taste aversion. Reduced adult CDH13 expression in conditional knockouts also alters cocaine reward in ways that correlate with individual differences in cortical CDH13 mRNA levels. In control and comparison behavioral assessments, knockout mice display modestly quicker acquisition of rotarod and water maze tasks, with a trend toward faster acquisition of 5-choice serial reaction time tasks that otherwise displayed no genotype-related differences. They display significant differences in locomotion in some settings, with larger effects in males. In assessments of brain changes that might contribute to these behavioral differences, there are selective alterations of dopamine levels, dopamine/metabolite ratios, dopaminergic fiber densities and mRNA encoding the activity dependent transcription factor npas4 in cerebral cortex of knockout mice. These novel data and previously reported human associations of CDH13 variants with addiction, individual differences in responses to stimulant administration and attention deficit hyperactivity disorder (ADHD) phenotypes suggest that levels of CDH13 expression, through mechanisms likely to include effects on mesocortical dopamine, influence stimulant reward and may contribute modestly to cognitive and locomotor phenotypes relevant to ADHD.

  19. Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice.

    PubMed

    Gardier, A M; David, D J; Jego, G; Przybylski, C; Jacquot, C; Durier, S; Gruwez, B; Douvier, E; Beauverie, P; Poisson, N; Hen, R; Bourin, M

    2003-07-01

    The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in

  20. Targeted Disruption of the Meprin β Gene in Mice Leads to Underrepresentation of Knockout Mice and Changes in Renal Gene Expression Profiles

    PubMed Central

    Norman, Lourdes P.; Jiang, Weiping; Han, Xiaoli; Saunders, Thomas L.; Bond, Judith S.

    2003-01-01

    Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded α and/or β subunits. Homooligomers of meprin α are secreted; oligomers containing meprin β are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin β is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin β null mice were generated by targeted disruption of the meprin β gene on mouse chromosome 18q12. Analyses of meprin β knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin β knockout mice lack membrane-associated meprin α in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin β allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin α in kidney and intestine. PMID:12556482

  1. Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

    PubMed

    Westbrook, Reyhan; Bonkowski, Michael S; Arum, Oge; Strader, April D; Bartke, Andrzej

    2014-01-01

    Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice. PMID:23833202

  2. Sex-related neurogenesis decrease in hippocampal dentate gyrus with depressive-like behaviors in sigma-1 receptor knockout mice.

    PubMed

    Sha, Sha; Hong, Juan; Qu, Wei-Jun; Lu, Zi-Hong; Li, Lin; Yu, Wen-Feng; Chen, Ling

    2015-08-01

    Male sigma-1 receptor knockout (σ1R(-/-)) mice showed depressive-like phenotype with deficit in the survival of newly generated neuronal cells in the hippocampal dentate gyrus (DG), but female σ1R(-/-) mice did not. The level of serum estradiol (E2) at proestrus or diestrus did not differ between female σ1R(-/-) mice and wild-type (WT) mice. Ovariectomized (OVX) female σ1R(-/-) mice, but not WT mice, presented the same depressive-like behaviors and neurogenesis decrease as male σ1R(-/-) mice. Treatment of male σ1R(-/-) mice with E2 could alleviate the depressive-like behaviors and rescue the neurogenesis decrease. In addition, E2 could correct the decline in the density of NMDA-activated current (INMDA) in granular cells of DG and the phosphorylation of NMDA receptor (NMDAr) subtype 2B (NR2B) in male σ1R(-/-) mice, which was associated with the elevation of Src phosphorylation. The neuroprotection and antidepressant effects of E2 in male σ1R(-/-) mice were blocked by the inhibitor of Src or NR2B. The NMDAr agonist showed also the neuroprotection and antidepressant effects in male σ1R(-/-) mice, which were insensitive to the Src inhibitor. On the other hand, either the deprivation of E2 or the inhibition of Src in female σ1R(-/-) mice rather than WT mice led to a distinct decline in INMDA and NR2B phosphorylation. Similarly, the Src inhibitor could cause neurogenesis decrease and depressive-like behaviors in female σ1R(-/-) mice, but not in WT mice. These results indicate that the σ1R deficiency impairs neurogenesis leading to a depressive-like phenotype, which is alleviated by the neuroprotection of E2.

  3. Generation of knockout mice expressing a GFP-reporter under the control of the Lmx1a locus.

    PubMed

    Griesel, Gundula; Krug, Christian; Yurlova, Larisa; Diaconu, Mihaela; Mansouri, Ahmed

    2011-01-01

    Lmx1a is a member of the LIM homeodomain containing transcription factors and plays an important role during embryonic development. Specifically, it is required for the proper formation of several structures in the central nervous system, such as the roof plate, the cerebellum, and the inner ear. All these defects may contribute to the neurological phenotype observed in dreher mice, lacking functional Lmx1a protein. Interestingly, this factor was also found to promote midbrain dopaminergic neuron fate. We have introduced Green fluorescent protein (GFP) coding sequences into the Lmx1a locus by homologous recombination, and created knockout mice where GFP recapitulates the Lmx1a endogenous expression pattern.

  4. Animal models of depression in dopamine, serotonin, and norepinephrine transporter knockout mice: prominent effects of dopamine transporter deletions.

    PubMed

    Perona, Maria T G; Waters, Shonna; Hall, Frank Scott; Sora, Ichiro; Lesch, Klaus-Peter; Murphy, Dennis L; Caron, Marc; Uhl, George R

    2008-09-01

    Antidepressant drugs produce therapeutic actions and many of their side effects via blockade of the plasma membrane transporters for serotonin (SERT/SLC6A2), norepinephrine (NET/SLC6A1), and dopamine (DAT/SLC6A3). Many antidepressants block several of these transporters; some are more selective. Mouse gene knockouts of these transporters provide interesting models for possible effects of chronic antidepressant treatments. To examine the role of monoamine transporters in models of depression DAT, NET, and SERT knockout (KO) mice and wild-type littermates were studied in the forced swim test (FST), the tail suspension test, and for sucrose consumption. To dissociate general activity from potential antidepressant effects three types of behavior were assessed in the FST: immobility, climbing, and swimming. In confirmation of earlier reports, both DAT KO and NET KO mice exhibited less immobility than wild-type littermates whereas SERT KO mice did not. Effects of DAT deletion were not simply because of hyperactivity, as decreased immobility was observed in DAT+/- mice that were not hyperactive as well as in DAT-/- mice that displayed profound hyperactivity. Climbing was increased, whereas swimming was almost eliminated in DAT-/- mice, and a modest but similar effect was seen in NET KO mice, which showed a modest decrease in locomotor activity. Combined increases in climbing and decreases in immobility are characteristic of FST results in antidepressant animal models, whereas selective effects on swimming are associated with the effects of stimulant drugs. Therefore, an effect on climbing is thought to more specifically reflect antidepressant effects, as has been observed in several other proposed animal models of reduced depressive phenotypes. A similar profile was observed in the tail suspension test, where DAT, NET, and SERT knockouts were all found to reduce immobility, but much greater effects were observed in DAT KO mice. However, to further determine whether these

  5. Animal models of depression in dopamine, serotonin and norepinephrine transporter knockout mice: prominent effects of dopamine transporter deletions

    PubMed Central

    Perona, Maria T.G.; Waters, Shonna; Hall, F. Scott; Sora, Ichiro; Lesch, Klaus-Peter; Murphy, Dennis L.; Caron, Marc; Uhl, George R.

    2008-01-01

    Antidepressant drugs produce therapeutic actions and many of their side effects via blockade of the plasma membrane transporters for serotonin (SERT/SLC6A2), norepinephrine (NET/SLC6A1) and dopamine (DAT/SLC6A3). Many antidepressants block several ofthese transporters; some are more selective. Mouse gene knockouts of these transporters provide interesting models for possible effects of chronic antidepressant treatments. To examine the role of monoamine transporters in models of depression DAT, NET and SERT KO mice and wildtype littermates were studied in the forced swim test (FST), the tail suspension test (TST) and for sucrose consumption. In order to dissociate general activity from the potential antidepressant effects three types of behavior were assessed in the FST: immobility, climbing and swimming. In confirmation of previous reports, both DAT KO and NET KO mice exhibited less immobility than wildtype littermates while SERT KO mice did not. Effects of DAT deletion were not simply due to hyperactivity as decreased immobility was observed in DAT +/- mice that were not hyperactive as well as in DAT -/- mice that displayed profound hyperactivity. Climbing was increased, while swimming was almost eliminated in DAT -/-mice, while a modest but similar effect was seen in NET KO mice, which showed a modest decrease in locomotor activity. Combined increases in climbing and decreases in immobility are characteristic of forced swim test results in antidepressant animal models, while selective effects on swimming are associated with the effects of stimulant drugs. Therefore, an effect on climbing is thought to more specifically reflect antidepressant effects, as has been observed in several other proposed animal models of reduced depressive phenotypes. A similar profile was observed in the TST, where DAT, NET and SERT knockouts were all found to reduce immobility, but much greater effects were observed in DAT KO mice. However, to further determine whether these effects of

  6. Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

    SciTech Connect

    Wang, Yuehai; Lu, Huixia; Huang, Ziyang; Lin, Huili; Lei, Zhenmin; Tang, Mengxiong; Gao, Fei; Dong, Mei; Li, Rongda; Lin, Ling

    2014-07-18

    Highlights: • Titers of ANA and anti-dsDNA antibodies were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • The spleen weights and glomerular areas were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • Expressions of IgG and C3 in glomeruli were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • IgG, C3 and macrophage infiltration in aortic plaques were found in ApoE{sup −/−} mice. - Abstract: Background: Apolipoprotein E-knockout (ApoE{sup −/−}) mice is a classic model of atherosclerosis. We have found that ApoE{sup −/−} mice showed splenomegaly, higher titers of serum anti-nuclear antibody (ANA) and anti-dsDNA antibody compared with C57B6/L (B6) mice. However, whether ApoE{sup −/−} mice show autoimmune injury remains unclear. Methods and results: Six females and six males in each group, ApoE{sup −/−}, Fas{sup −/−} and B6 mice, were used in this study. The titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein were measured by ELISA after 4 months of high-fat diet. The spleen weight and the glomerular area were determined. The expressions of IgG, C3 and macrophage in kidney and atherosclerotic plaque were detected by immunostaining followed by morphometric analysis. Similar to the characteristics of Fas{sup −/−} mice, a model of systemic lupus erythematosus (SLE), ApoE{sup −/−} mice, especially female, displayed significant increases of spleen weight and glomerular area when compared to B6 mice. Also, elevated titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein. Moreover, the expressions of IgG, C3 and macrophage in glomeruli and aortic plaques were found in ApoE{sup −/−} mice. In addition, the IgG and C3 expressions in glomeruli and plaques significantly increased (or a trend of increase) in female ApoE{sup −/−} mice compared with males. Conclusions: Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta.

  7. Rescue of heart lipoprotein lipase-knockout mice confirms a role for triglyceride in optimal heart metabolism and function.

    PubMed

    Khan, Raffay S; Lin, Yan; Hu, Yunying; Son, Ni-Huiping; Bharadwaj, Kalyani G; Palacios, Carla; Chokshi, Aalap; Ji, Ruiping; Yu, Shuiqing; Homma, Sunichi; Schulze, P Christian; Tian, Rong; Goldberg, Ira J

    2013-12-01

    Hearts utilize fatty acids as a primary source of energy. The sources of those lipids include free fatty acids and lipoprotein triglycerides. Deletion of the primary triglyceride-hydrolyzing enzyme lipoprotein lipase (LPL) leads to cardiac dysfunction. Whether heart LPL-knockout (hLPL0) mice are compromised due a deficiency in energetic substrates is unknown. To test whether alternative sources of energy will prevent cardiac dysfunction in hLPL0 mice, two different models were used to supply nonlipid energy. 1) hLPL0 mice were crossed with mice transgenically expressing GLUT1 in cardiomyocytes to increase glucose uptake into the heart; this cross-corrected cardiac dysfunction, reduced cardiac hypertrophy, and increased myocardial ATP. 2) Mice were randomly assigned to a sedentary or training group (swimming) at 3 mo of age, which leads to increased skeletal muscle production of lactate. hLPL0 mice had greater expression of the lactate transporter monocarboxylate transporter-1 (MCT-1) and increased cardiac lactate uptake. Compared with hearts from sedentary hLPL0 mice, hearts from trained hLPL0 mice had adaptive hypertrophy and improved cardiac function. We conclude that defective energy intake and not the reduced uptake of fat-soluble vitamins or cholesterol is responsible for cardiac dysfunction in hLPL0 mice. In addition, our studies suggest that adaptations in cardiac metabolism contribute to the beneficial effects of exercise on the myocardium of patients with heart failure. PMID:24085031

  8. Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator knockout mice

    PubMed Central

    Ianowski, Juan P; Choi, Jae Young; Wine, Jeffrey J; Hanrahan, John W

    2007-01-01

    Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild-type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftrm1UNC/Cftrm1UNC; CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP- and forskolin-stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO3− by Hepes reduced the carbachol response by ∼50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients. PMID:17204498

  9. Exposure to diesel exhaust upregulates COX-2 expression in ApoE knockout mice

    PubMed Central

    Bai, Ni; Tranfield, Erin M.; Kavanagh, Terrance J.; Kaufman, Joel D.; Rosenfeld, Michael E.; van Eeden, Stephan F.

    2015-01-01

    Introduction We have shown that diesel exhaust (DE) inhalation caused progression of atherosclerosis; however, the mechanisms are not fully understood. We hypothesize that exposure to DE upregulates cyclooxygenase (COX) expression and activity, which could play a role in DE-induced atherosclerosis. Methods ApoE knockout mice (30-week old) fed with regular chow were exposed to DE (at 200 μg/m3 of particulate matter) or filtered air (control) for 7 weeks (6 h/day, 5 days/week). The protein and mRNA expression of COX-1 and COX-2 were evaluated by immunohistochemistry analysis and quantitative real-time PCR, respectively. To examine COX activity, thoracic aortae were mounted in a wire myograph, and phenylephrine (PE)-stimulated vasoconstriction was measured with and without the presence of COX antagonists (indomethacin). COX-2 activity was further assessed by urine 2,3-dinor-6-keto PGF1α level, a major metabolite of prostacyclin I2 (PGI2). Results Immunohistochemistry analysis demonstrates that DE exposure enhanced COX-2 expression in both thoracic aorta (p < 0.01) and aortic root (p < 0.03), with no modification of COX-1 expression. The increased COX-2 expression was positively correlated with smooth muscle cell content in aortic lesions (R2 = 0.4081, p < 0.008). The fractional changes of maximal vasoconstriction in the presence of indomethacin was attenuated by 3-fold after DE exposure (p < 0.02). Urine 2,3-dinor-6-keto PGF1α level was 15-fold higher in DE group than the control (p < 0.007). The mRNA expression of COX-2 (p < 0.006) and PGI synthase (p < 0.02), but not COX-1, was significantly augmented after DE exposure. Conclusion We show that DE inhalation enhanced COX-2 expression, which is also associated with phenotypic changes of aortic lesion. PMID:22746401

  10. Prolonging the survival of Tsc2 conditional knockout mice by glutamine supplementation.

    PubMed

    Rozas, Natalia S; Redell, John B; McKenna, James; Moore, Anthony N; Gambello, Michael J; Dash, Pramod K

    2015-02-20

    The genetic disease tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by loss of function mutations in either TSC1 (hamartin) or TSC2 (tuberin), which serve as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1) activity. TSC patients exhibit developmental brain abnormalities and tuber formations that are associated with neuropsychological and neurocognitive impairments, seizures and premature death. Mechanistically, TSC1 and TSC2 loss of function mutations result in abnormally high mTORC1 activity. Thus, the development of a strategy to inhibit abnormally high mTORC1 activity may have therapeutic value in the treatment of TSC. mTORC1 is a master regulator of growth processes, and its activity can be reduced by withdrawal of growth factors, decreased energy availability, and by the immunosuppressant rapamycin. Recently, glutamine has been shown to alter mTORC1 activity in a TSC1-TSC2 independent manner in cells cultured under amino acid- and serum-deprived conditions. Since starvation culture conditions are not physiologically relevant, we examined if glutamine can regulate mTORC1 in non-deprived cells and in a murine model of TSC. Our results show that glutamine can reduce phosphorylation of S6 and S6 kinase, surrogate indicators of mTORC1 activity, in both deprived and non-deprived cells, although higher concentrations were required for non-deprived cultures. When administered orally to TSC2 knockout mice, glutamine reduced S6 phosphorylation in the brain and significantly prolonged their lifespan. Taken together, these results suggest that glutamine supplementation can be used as a potential treatment for TSC.

  11. Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice

    NASA Technical Reports Server (NTRS)

    Roman, Brian B.; Meyer, Ronald A.; Wiseman, Robert W.

    2002-01-01

    Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P < 0.001). Initial PCr/ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.

  12. Tgfbeta2 -/- Tgfbeta3 -/- double knockout mice display severe midline fusion defects and early embryonic lethality.

    PubMed

    Dünker, Nicole; Krieglstein, Kerstin

    2002-12-01

    Given all known biological activities, it is anticipated that transforming growth factors beta (TGF-betas) play important roles in many different developmental processes. As all three TGF-beta isoforms display overlapping expression patterns, deletion of one TGF-beta isoform might be compensated for by another. In the present study, targeted disruption of both Tgfbeta2 and Tgfbeta3 genes was undertaken to circumvent this problem and determine the essential roles of TGF-beta2 and TGF-beta3 in vivo. Tgfbeta2(-/-) Tgfbeta3(-/-) double knockout mice and their three-allelic Tgfbeta2(-/-) Tgfbeta3(+/-) littermates display a lack of distal parts of the rib, a lack of sternal primordia, and failure in ventral body wall closure, leading to an extrathoracic position of the heart and extrusion of the liver. In addition, abnormalities in connective tissue composition and an early embryonic lethality [around embryonic day (E) 15.5] are seen. In contrast, Tgfbeta2 (+/-) Tgfbeta3 (-/-) littermates show normal rib and sternum development, normal anterior body wall fusion, and are still alive on E18.5. TGF-beta2 is already known to play a role in skeletal and craniofacial development. The results presented here show that beyond this: (a). TGF-betas obviously play a fundamental role in midline fusion and (b). the Tgfbeta2 gene seems to play a more important role in mediating developmental processes than the Tgfbeta3 gene, since Tgfbeta2 (+/-) Tgfbeta3 (-/-) mutants - in contrast to their Tgfbeta2(-/-) Tgfbeta3 (+)(/-) littermates - do not display severe malformations.

  13. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy

    PubMed Central

    Chadi, Sead; Polyte, Jacqueline; Lefevre, Lucas; Castille, Johan; Ehanno, Aude; Laubier, Johann; Jaffrézic, Florence; Le Provost, Fabienne

    2016-01-01

    R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1’s partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function. PMID:27611670

  14. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy.

    PubMed

    Chadi, Sead; Polyte, Jacqueline; Lefevre, Lucas; Castille, Johan; Ehanno, Aude; Laubier, Johann; Jaffrézic, Florence; Le Provost, Fabienne

    2016-01-01

    R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1's partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function.

  15. Imaging colon cancer development in mice: IL-6 deficiency prevents adenoma in azoxymethane-treated Smad3 knockouts

    NASA Astrophysics Data System (ADS)

    Harpel, Kaitlin; Leung, Sarah; Faith Rice, Photini; Jones, Mykella; Barton, Jennifer K.; Bommireddy, Ramireddy

    2016-02-01

    The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

  16. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy.

    PubMed

    Chadi, Sead; Polyte, Jacqueline; Lefevre, Lucas; Castille, Johan; Ehanno, Aude; Laubier, Johann; Jaffrézic, Florence; Le Provost, Fabienne

    2016-01-01

    R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1's partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function. PMID:27611670

  17. Circadian rhythms of clock gene expression in the cerebellum of serotonin-deficient Pet-1 knockout mice.

    PubMed

    Paulus, Erin V; Mintz, Eric M

    2016-01-01

    Serotonin plays an important role in the central regulation of circadian clock function. Serotonin levels are generally higher in the brain during periods of high activity, and these periods are in turn heavily regulated by the circadian clock located in the suprachiasmatic nucleus. However, the role of serotonin as a regulator of circadian rhythms elsewhere in the brain has not been extensively examined. In this study, we examined circadian rhythms of clock gene expression in the cerebellum in mice lacking the Pet-1 transcription factor, which results in a developed brain that is deficient in serotonin neurons. If serotonin helps to synchronize rhythms in brain regions other than the suprachiasmatic nucleus, we would expect to see differences in clock gene expression in these serotonin deficient mice. We found minor differences in the expression of Per1 and Per2 in the knockout mice as compared to wild type, but these differences were small and of questionable functional importance. We also measured the response of cerebellar clocks to injections of the serotonin agonist 8-OH-DPAT during the early part of the night. No effect on clock genes was observed, though the immediate-early gene Fos showed increased expression in wild type mice but not the knockouts. These results suggest that serotonin is not an important mediator of circadian rhythms in the cerebellum in a way that parallels its regulation of the circadian clock in the suprachiasmatic nucleus.

  18. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  19. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

    PubMed

    Tu, Lan N; Morohaku, Kanako; Manna, Pulak R; Pelton, Susanne H; Butler, W Ronald; Stocco, Douglas M; Selvaraj, Vimal

    2014-10-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo(-/-)) mice. Contrary to the early report, Tspo(-/-) mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo(-/-) mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo(-/-) mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo(-/-) mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.

  20. Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu, Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

    PubMed Central

    Ibrahim, Osama M. A.; Dogru, Murat; Matsumoto, Yukihiro; Igarashi, Ayako; Kojima, Takashi; Wakamatsu, Tais Hitomi; Inaba, Takaaki; Shimizu, Takahiko; Shimazaki, Jun; Tsubota, Kazuo

    2014-01-01

    Purpose The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1−/−) mouse. Methods Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed. Results Corneal vital staining scores in the Sod1−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1−/− mice. Conclusions Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations. PMID:25036096

  1. ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice.

    PubMed

    Han, Jung-Youn; Hwang, Meeyul; Hwa, Sung-Yong; Park, Jin-Kyu; Ki, Mi-Ran; Hong, Il-Hwa; Kim, Ah-Young; Lee, Eun-Mi; Lee, Eun-Joo; Min, Chang-Woo; Kang, Kyung-Ku; Lee, Myeong-Mi; Sung, Soo-Eun; Jeong, Kyu-Shik

    2014-06-01

    ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

  2. TASK-3 knockout mice exhibit exaggerated nocturnal activity, impairments in cognitive functions, and reduced sensitivity to inhalation anesthetics.

    PubMed

    Linden, Anni-Maija; Sandu, Cristina; Aller, M Isabel; Vekovischeva, Olga Y; Rosenberg, Per H; Wisden, William; Korpi, Esa R

    2007-12-01

    The TASK-3 channel is an acid-sensitive two-pore-domain K+ channel, widely expressed in the brain and probably involved in regulating numerous neuronal populations. Here, we characterized the behavioral and pharmacological phenotypes of TASK-3 knockout (KO) mice. Circadian locomotor activity measurements revealed that the nocturnal activity of the TASK-3 KO mice was increased by 38% (P < 0.01) compared with wild-type littermate controls, light phase activity being similar. Although TASK-3 channels are abundant in cerebellar granule cells, the KO mice performed as well as the wild-type mice in walking on a rotating rod or along a 1.2-cm-diameter beam. However, they fell more frequently from a narrower 0.8-cm beam. The KO mice showed impaired working memory in the spontaneous alternation task, with the alternation percentage being 62 +/- 3% for the wild-type mice and 48 +/- 4% (P < 0.05) for the KO mice. Likewise, during training for the Morris water-maze spatial memory task, the KO mice were slower to find the hidden platform, and in the probe trial, the female KO mice visited fewer times the platform quadrant than the male KO and wild-type mice. In pharmacological tests, the TASK-3 KO mice showed reduced sensitivity to the inhalation anesthetic halothane and the cannabinoid receptor agonist WIN55212-2 mesylate [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] but unaltered responses to the alpha2 adrenoceptor agonist dexmedetomidine, the i.v. anesthetic propofol, the opioid receptor agonist morphine, and the local anesthetic lidocaine. Overall, our results suggest important contributions of TASK-3 channels in the neuronal circuits regulating circadian rhythms, cognitive functions, and mediating specific pharmacological effects.

  3. Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice.

    PubMed

    Irimia, Jose M; Meyer, Catalina M; Peper, Caron L; Zhai, Lanmin; Bock, Cheryl B; Previs, Stephen F; McGuinness, Owen P; DePaoli-Roach, Anna; Roach, Peter J

    2010-04-23

    Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

  4. Low-dose nicotine facilitates spatial memory in ApoE-knockout mice in the radial arm maze.

    PubMed

    Sultana, Ruby; Ameno, Kiyoshi; Jamal, Mostofa; Miki, Takanori; Tanaka, Naoko; Ono, Junichiro; Kinoshita, Hiroshi; Nakamura, Yu

    2013-06-01

    Here, we investigated the effects of nicotine on spatial memory in ApoE-knockout (ApoE-KO) and wild-type (WT) mice in a radial arm maze. Training occurred on three consecutive days and the test was performed on day 4, with one trial per day. Then on day 4, animals were administered nicotine (0.1, 0.25, 0.5, and 1.0 mg/kg) or the antagonist of nicotinic receptors (nAChRs) mecamylamine (MEC 2 mg/kg) alone or together with 0.1 mg/kg nicotine. The number of errors in the first eight choices was recorded. The results were that 0.1 mg/kg nicotine decreased errors in ApoE-KO mice, while 0.1 and 0.25 mg/kg nicotine reduced errors in WT mice, indicating that lower doses of nicotine elicit a memory improvement. In contrast, 1.0 mg/kg nicotine increased errors in WT mice, but not in ApoE-KO mice. MEC alone had no noticeable effect on errors in either strain of mice. However, co-administration of 0.1 mg/kg nicotine and MEC increased errors and reduced the effects of nicotine in WT mice, but not in ApoE-KO mice. Our study found a biphasic effect of nicotine in WT mice: it improves spatial memory at lower doses and impairs it at a higher dose. In ApoE-KO mice, nicotine improves memory at a low dose and has no effect at a higher dose, suggesting that the ApoE deficiency may influence the efficacy of nicotine. Moreover, a reversal of nicotinic effects with MEC was seen in WT mice, indicating the likelihood of the involvement of nAChRs in the spatial-memory response to nicotine.

  5. TASK-3 knockout mice exhibit exaggerated nocturnal activity, impairments in cognitive functions, and reduced sensitivity to inhalation anesthetics.

    PubMed

    Linden, Anni-Maija; Sandu, Cristina; Aller, M Isabel; Vekovischeva, Olga Y; Rosenberg, Per H; Wisden, William; Korpi, Esa R

    2007-12-01

    The TASK-3 channel is an acid-sensitive two-pore-domain K+ channel, widely expressed in the brain and probably involved in regulating numerous neuronal populations. Here, we characterized the behavioral and pharmacological phenotypes of TASK-3 knockout (KO) mice. Circadian locomotor activity measurements revealed that the nocturnal activity of the TASK-3 KO mice was increased by 38% (P < 0.01) compared with wild-type littermate controls, light phase activity being similar. Although TASK-3 channels are abundant in cerebellar granule cells, the KO mice performed as well as the wild-type mice in walking on a rotating rod or along a 1.2-cm-diameter beam. However, they fell more frequently from a narrower 0.8-cm beam. The KO mice showed impaired working memory in the spontaneous alternation task, with the alternation percentage being 62 +/- 3% for the wild-type mice and 48 +/- 4% (P < 0.05) for the KO mice. Likewise, during training for the Morris water-maze spatial memory task, the KO mice were slower to find the hidden platform, and in the probe trial, the female KO mice visited fewer times the platform quadrant than the male KO and wild-type mice. In pharmacological tests, the TASK-3 KO mice showed reduced sensitivity to the inhalation anesthetic halothane and the cannabinoid receptor agonist WIN55212-2 mesylate [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] but unaltered responses to the alpha2 adrenoceptor agonist dexmedetomidine, the i.v. anesthetic propofol, the opioid receptor agonist morphine, and the local anesthetic lidocaine. Overall, our results suggest important contributions of TASK-3 channels in the neuronal circuits regulating circadian rhythms, cognitive functions, and mediating specific pharmacological effects. PMID:17875609

  6. Temporal evolution of neurophysiological and behavioral features of synapsin I/II/III triple knock-out mice.

    PubMed

    Cambiaghi, Marco; Cursi, Marco; Monzani, Elena; Benfenati, Fabio; Comi, Giancarlo; Minicucci, Fabio; Valtorta, Flavia; Leocani, Letizia

    2013-02-01

    Deletion of one or more synapsin genes in mice results in a spontaneous epilepsy. In these animals, seizures can be evoked by opening or moving the cage. Aim of the present study was to characterize the evolution of the epileptic phenotype by neurophysiological examination and behavioral observation in synapsin triple knock-out (Syn-TKO) mice. Syn-TKO mice were studied from 20 postnatal days (PND) up to 6 months of age by video-EEG recording and behavioral observation. Background EEG spectral analysis was performed and data were compared to WT animals. Syn-TKO revealed rare spontaneous seizures and increased susceptibility to evoked seizures in mice from 60 to 100 PND. Spontaneous and evoked seizures presented similar duration and morphology. At times, seizures were followed by a post-ictal phase characterized by a 4 Hz rhythmic activity and immobility of the animal. Spectral analysis of background EEG evidenced a slowing of the theta-alpha peak in Syn-TKO mice compared to WT mice within the period from PND 40 to 100. These data indicate that Syn-TKO mice do not exhibit a linear progression of the epileptic phenotype, with the period corresponding to a higher susceptibility to evoked seizures characterized by background EEG slowing. This aspect might be connected to brain dysfunction often associated to epilepsy in the interictal period.

  7. Overexpression of Dmp1 fails to rescue the bone and dentin defects in Fam20C knockout mice.

    PubMed

    Wang, Xiaofang; Wang, Jingya; Yuan, Baozhi; Lu, Yongbo; Feng, Jian Q; Qin, Chunlin

    2014-08-01

    FAM20C is a kinase phosphorylating the small-integrin-binding ligand, N-linked glycoproteins (SIBLINGs), a group of extracellular matrix proteins that are essential for bone and dentin formation. Previously, we showed that Sox2-Cre;Fam20Cfl/fl mice had bone and dentin defects, along with hypophosphatemia and significant downregulation of dentin matrix protein 1 (DMP1). While the assumed phosphorylation failure of the SIBLINGs is likely associated with the defects in the Fam20C-deficient mice, it remains unclear if the downregulation of Dmp1 contributes to these phenotypes. In this study, we crossed 3.6 kb Col1-Dmp1 transgenic mice with 3.6 kb Col1-Cre;Fam20Cfl/fl mice to overexpress Dmp1 in the mineralized tissues of Fam20C conditional knockout (cKO) mice. X-ray, micro-computed tomography, serum biochemistry and histology analyses showed that expressing the Dmp1 transgene failed to rescue the bone and dentin defects, as well as the serum levels of FGF23 and phosphate in the Fam20C-cKO mice. These results indicated that the downregulation of Dmp1 may not directly associate with, or significantly contribute to the bone and dentin defects in the Fam20C-cKO mice.

  8. Mildly Reduced Brain Swelling and Improved Neurological Outcome in Aquaporin-4 Knockout Mice following Controlled Cortical Impact Brain Injury

    PubMed Central

    Uchida, Kazuyoshi; Papadopoulos, Marios C.; Zador, Zsolt; Manley, Geoffrey T.; Verkman, Alan S.

    2015-01-01

    Abstract Brain edema following traumatic brain injury (TBI) is associated with considerable morbidity and mortality. Prior indirect evidence has suggested the involvement of astrocyte water channel aquaporin-4 (AQP4) in the pathogenesis of TBI. Here, focal TBI was produced in wild type (AQP4+/+) and knockout (AQP4−/−) mice by controlled cortical impact injury (CCI) following craniotomy with dura intact (parameters: velocity 4.5 m/sec, depth 1.7 mm, dwell time 150 msec). AQP4-deficient mice showed a small but significant reduction in injury volume in the first week after CCI, with a small improvement in neurological outcome. Mechanistic studies showed reduced intracranial pressure at 6 h after CCI in AQP4−/− mice, compared with AQP4+/+ control mice (11 vs. 19 mm Hg), with reduced local brain water accumulation as assessed gravimetrically. Transmission electron microscopy showed reduced astrocyte foot-process area in AQP4−/− mice at 24 h after CCI, with greater capillary lumen area. Blood–brain barrier disruption assessed by Evans blue dye extravasation was similar in AQP4+/+ and AQP4−/− mice. We conclude that the mildly improved outcome in AQP4−/− mice following CCI results from reduced cytotoxic brain water accumulation, though concurrent cytotoxic and vasogenic mechanisms in TBI make the differences small compared to those seen in disorders where cytotoxic edema predominates. PMID:25790314

  9. IL-1 receptor-antagonist (IL-1Ra) knockout mice show anxiety-like behavior by aging.

    PubMed

    Wakabayashi, Chisato; Numakawa, Tadahiro; Odaka, Haruki; Ooshima, Yoshiko; Kiyama, Yuji; Manabe, Toshiya; Kunugi, Hiroshi; Iwakura, Yoichiro

    2015-07-10

    Interleukin 1 (IL-1) plays a critical role in stress responses, and its mRNA is induced in the brain by restraint stress. Previously, we reported that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lacked IL-1Ra molecules that antagonize the IL-1 receptor, showed anti-depression-like behavior via adrenergic modulation at the age of 8 weeks. Here, we report that IL-1Ra KO mice display an anxiety-like phenotype that is induced spontaneously by aging in the elevated plus-maze (EPM) test. This anxiety-like phenotype was improved by the administration of diazepam. The expression of the anxiety-related molecule glucocorticoid receptor (GR) was significantly reduced in 20-week-old but not in 11-week-old IL-1Ra KO mice compared to wild-type (WT) littermates. The expression of the mineralocorticoid receptor (MR) was not altered between IL-1Ra KO mice and WT littermates at either 11 or 20 weeks old. Analysis of monoamine concentration in the hippocampus revealed that tryptophan, the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA), and the dopamine metabolite homovanillic acid (HVA) were significantly increased in 20-week-old IL-1Ra KO mice compared to littermate WT mice. These findings strongly suggest that the anxiety-like behavior observed in older mice was caused by the complicated alteration of monoamine metabolism and/or GR expression in the hippocampus.

  10. Enhanced response to mouse thyroid-stimulating hormone (TSH) receptor immunization in TSH receptor-knockout mice.

    PubMed

    Nakahara, Mami; Mitsutake, Norisato; Sakamoto, Hikaru; Chen, Chun-Rong; Rapoport, Basil; McLachlan, Sandra M; Nagayama, Yuji

    2010-08-01

    Graves-like hyperthyroidism is induced in BALB/c mice by immunization with adenovirus expressing the human TSH receptor (TSHR) A-subunit (amino acids 1-289). However, because of nonidentity between the human and mouse TSHR ( approximately 87% amino acid homology), we compared the responses of mice immunized with adenoviruses expressing either the mouse or the human TSHR A-subunit. Wild-type (wt) BALB/c mice immunized with the mouse A-subunit developed neither TSHR antibodies (measured by flow cytometry) nor thyroid lymphocytic infiltration. However, wt C57BL/6 mice developed sparse intrathyroidal lymphocyte infiltration without antibody production. Depletion of naturally occurring regulatory CD4(+)CD25(+) T cells had little effect. These results indicate the inability to break tolerance to the mouse TSHR in wt mice. In contrast, TSHR knockout (KO) BALB/c mice generated mouse TSHR antibodies in response to mouse A-subunit immunization and augmented human TSHR antibody response to human A-subunit immunization. Thyroid-stimulating antibody titers measured in a functional bioassay were comparable in human A-subunit immunized wt mice and in TSHR KO mice immunized with either the mouse or human A-subunit. In conclusion, immune response to the mouse TSHR is readily induced in TSHR KO but not in wt mice. Only in the former does immunization with adenovirus expressing the mouse A-subunit generate antibodies capable of activating the mouse TSHR. TSHR KO mice are, therefore, of value for future studies dissecting the autoimmune response to the mouse TSHR.

  11. Serum cholesterol and expression of ApoAI, LXRbeta and SREBP2 in vitamin D receptor knock-out mice.

    PubMed

    Wang, Jing-Huan; Keisala, Tiina; Solakivi, Tiina; Minasyan, Anna; Kalueff, Allan V; Tuohimaa, Pentti

    2009-02-01

    Vitamin D insufficiency has been reported to be associated with increased blood cholesterol concentrations. Here we used two strains of VDR knock-out (VDR-KO) mice to study whether a lack of vitamin D action has any effect on cholesterol metabolism. In 129S1 mice, both in male and female VDR-KO mice serum total cholesterol levels were significantly higher than those in wild type (WT) mice (20.7% (P=0.05) and 22.2% (P=0.03), respectively). In addition, the serum high-density lipoprotein-bound cholesterol (HDL-C) level was 22% (P=0.03), respectively higher in male VDR-KO mice than in WT mice. The mRNA expression levels of five cholesterol metabolism related genes in livers of 129S1 mice were studied using quantitative real-time PCR (QRT-PCR): ATP-binding cassette transporter A1 (ABCA1), regulatory element binding protein (SREBP2), apolipoprotein A-I (ApoAI), low-density lipoprotein receptor (LDLR) and liver X receptor beta (LXRbeta). In the mutant male mice, the mRNA level of ApoAI and LXRbeta were 49.2% (P=0.005) and 38.8% (P=0.034) higher than in the WT mice. These changes were not observed in mutant female mice, but the female mutant mice showed 52.5% (P=0.006) decrease of SREBP2 mRNA expression compared to WT mice. Because the mutant mice were fed with a special rescue diet, we wanted to test whether the increased cholesterol levels in mutant mice were due to the diet. Both the WT and mutant NMRI mice were given the same diet for 3 weeks before the blood sampling. No difference in cholesterol or in HDL-C between WT and mutant mice was found. The results suggest that the food, gender and genetic background have an effect on the cholesterol metabolism. Although VDR seems to regulate some of the genes involved in cholesterol metabolism, its role in the regulation of serum cholesterol seems to be minimal.

  12. Attenuated cardiovascular hypertrophy and oxidant generation in response to angiotensin II infusion in glutaredoxin-1 knockout mice

    PubMed Central

    Bachschmid, Markus M.; Xu, Shanqin; Maitland-Toolan, Karlene A.; Ho, Ye-Shih; Cohen, Richard A.; Matsui, Reiko

    2010-01-01

    Glutaredoxin-1 (Glrx) is a thioltransferase that regulates protein S-glutathiolation. To elucidate the role of endogenous Glrx in cardiovascular disease, Glrx knockout (KO) mice were infused with angiotensin II (Ang II) for 6 days. After Ang II infusion, body weight and blood pressure were similar between WT and Glrx KO mice. However, compared to WT mice, Glrx KO mice demonstrated (1) less cardiac and aortic medial hypertrophy, (2) less oxidant generation in aorta assessed by dihydroethidium staining and nitrotyrosine, (3) decreased phosphorylation of Akt in the heart, and (4) less expression of inducible NOS (iNOS) in aorta and heart. In cultured embryonic fibroblasts from Glrx KO mice, S-glutathiolation of actin was enhanced and actin depolymerization was impaired after hydrogen peroxide stimulation compared with WT cells. Furthermore, oxidant generation in phorbol ester-stimulated fibroblasts and RAW 264.7 macrophage-like cells was lower with Glrx siRNA knockdown. These data indicate that Ang II-induced oxidant production and hypertrophic responses were attenuated in Glrx KO mice, which may result from impaired NADPH oxidase activation. PMID:20638471

  13. Nrf2 knockout: The effect on neurological dysfunction and the activation of glial cells of mice after brain injury.

    PubMed

    Zhang, Dongfeng; Teng, Junfang

    2016-07-01

    To investigate the protective role and possible mechanisms of Nrf2 gene in cerebral trauma in mice. The types Nrf2(-/-) and Nrf2(+/+) mice were confirmed by PCR, and the model of closed head injury was established. The severity of injury and the effect of the injury on neurological status were assessed by Neurological Severity Score (NSS) and fatality rate, and the activated conditions of microglia and astrocyte around the injured area were observed by immunohistochemical method. Compared with Nrf2(+/+) mice, the nerve dysfunction of the Nrf2(-/-) mice was obviously more severe (P<0.01). On the first day after injury, the activation of microglia around the injured area increased significantly in Nrf2 (-/-) mice, the difference was more significant on the third day, and there was still statistical difference until the 7th day (P<0.05). Moreover, On the days 1, 3, 7 after injury, the activation of astrocyte around the injured area also increased in Nrf2(-/-) mice, however, there was statistical difference only on the 3rd day (P<0.05). Nrf2 gene knockout can aggravate the nerve dysfunction after cerebral trauma, and this effect is achieved, at least partly, possibly via the effect of Nrf2 on glial activation. PMID:27592473

  14. Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction

    PubMed Central

    Sclip, Alessandra; Bacaj, Taulant; Giam, Louise R.; Südhof, Thomas C.

    2016-01-01

    Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges. PMID:27348751

  15. UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

    PubMed

    Tanaka, K; Kamiuchi, S; Ren, Y; Yonemasu, R; Ichikawa, M; Murai, H; Yoshino, M; Takeuchi, S; Saijo, M; Nakatsu, Y; Miyauchi-Hashimoto, H; Horio, T

    2001-06-01

    Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP. PMID:11376684

  16. A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations

    PubMed Central

    Vasco, Chiara; Berríos, Soledad; Parra, María Teresa; Viera, Alberto; Rufas, Julio S.; Zuccotti, Maurizio; Garagna, Silvia; Fernández-Donoso, Raúl

    2009-01-01

    Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian

  17. Heterozygous disruption of activin receptor-like kinase 1 is associated with increased arterial pressure in mice

    PubMed Central

    González-Núñez, María; Riolobos, Adela S.; Castellano, Orlando; Fuentes-Calvo, Isabel; de los Ángeles Sevilla, María; Oujo, Bárbara; Pericacho, Miguel; Cruz-Gonzalez, Ignacio; Pérez-Barriocanal, Fernando; ten Dijke, Peter; López-Novoa, Jose M.

    2015-01-01

    ABSTRACT The activin receptor-like kinase 1 (ALK-1) is a type I cell-surface receptor for the transforming growth factor-β (TGF-β) family of proteins. Hypertension is related to TGF-β1, because increased TGF-β1 expression is correlated with an elevation in arterial pressure (AP) and TGF-β expression is upregulated by the renin-angiotensin-aldosterone system. The purpose of this study was to assess the role of ALK-1 in regulation of AP using Alk1 haploinsufficient mice (Alk1+/−). We observed that systolic and diastolic AP were significantly higher in Alk1+/− than in Alk1+/+ mice, and all functional and structural cardiac parameters (echocardiography and electrocardiography) were similar in both groups. Alk1+/− mice showed alterations in the circadian rhythm of AP, with higher AP than Alk1+/+ mice during most of the light period. Higher AP in Alk1+/− mice is not a result of a reduction in the NO-dependent vasodilator response or of overactivation of the peripheral renin-angiotensin system. However, intracerebroventricular administration of losartan had a hypotensive effect in Alk1+/− and not in Alk1+/+ mice. Alk1+/− mice showed a greater hypotensive response to the β-adrenergic antagonist atenolol and higher concentrations of epinephrine and norepinephrine in plasma than Alk1+/+ mice. The number of brain cholinergic neurons in the anterior basal forebrain was reduced in Alk1+/− mice. Thus, we concluded that the ALK-1 receptor is involved in the control of AP, and the high AP of Alk1+/− mice is explained mainly by the sympathetic overactivation shown by these animals, which is probably related to the decreased number of cholinergic neurons. PMID:26398936

  18. Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice

    PubMed Central

    Beck, Goichi; Shinzawa, Koei; Hayakawa, Hideki; Baba, Kousuke; Sumi-Akamaru, Hisae; Tsujimoto, Yoshihide; Mochizuki, Hideki

    2016-01-01

    Calcium-independent phospholipase A2β (iPLA2β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA2β-knockout (KO) mice. To clarify whether a deficiency of iPLA2β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA2β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA2β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA2β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA2β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA2β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA2β-KO mice. PMID:27078024

  19. Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice.

    PubMed

    Beck, Goichi; Shinzawa, Koei; Hayakawa, Hideki; Baba, Kousuke; Sumi-Akamaru, Hisae; Tsujimoto, Yoshihide; Mochizuki, Hideki

    2016-01-01

    Calcium-independent phospholipase A2β (iPLA2β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA2β-knockout (KO) mice. To clarify whether a deficiency of iPLA2β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA2β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA2β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA2β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA2β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA2β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA2β-KO mice. PMID:27078024

  20. Brief Report: Altered Social Behavior in Isolation-Reared "Fmr1" Knockout Mice

    ERIC Educational Resources Information Center

    Heitzer, Andrew M.; Roth, Alexandra K.; Nawrocki, Lauren; Wrenn, Craige C.; Valdovinos, Maria G.

    2013-01-01

    Social behavior abnormalities in Fragile X syndrome (FXS) are characterized by social withdrawal, anxiety, and deficits in social cognition. To assess these deficits, a model of FXS, the "Fmr1" knockout mouse ("Fmr1" KO), has been utilized. This mouse model has a null mutation in the fragile X mental retardation 1 gene ("Fmr1") and displays…

  1. Investigation of nasal CO₂ receptor transduction mechanisms in wild-type and GC-D knockout mice.

    PubMed

    Kenemuth, Jessica K; Hennessy, Shane P; Hanson, Ryan J; Hensler, Allison J; Coates, E Lee

    2013-11-01

    The main olfactory system of mice contains a small subset of olfactory sensory neurons (OSNs) that are stimulated by CO₂. The objective of this study was to record olfactory receptor responses to a range of CO₂ concentrations to further elucidate steps in the proposed CO₂ transduction pathway in mice. Electro-olfactograms (EOGs) were recorded before and after inhibiting specific steps in the CO₂ transduction pathway with topically applied inhibitors. Inhibition of extracellular carbonic anhydrase (CA) did not significantly affect EOG responses to CO₂ but did decrease EOG responses to several control odorants. Inhibition of intracellular CA or cyclic nucleotide-gated channels attenuated EOG responses to CO₂, confirming the role of these components in CO₂ sensing in mice. We also show that, like canonical OSNs, CO₂-sensitive OSNs depend on Ca²⁺-activated Cl⁻ channels for depolarization of receptor neurons. Lastly, we found that guanylyl cyclase-D knockout mice were still able to respond to CO₂, indicating that other pathways may exist for the detection of low concentrations of nasal CO₂. We discuss these findings as they relate to previous studies on CO₂-sensitive OSNs in mice and other animals.

  2. Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density

    PubMed Central

    Cawley, Niamh X.; Yanik, Tulin; Woronowicz, Alicja; Chang, Weizhong; Marini, Joan C.

    2010-01-01

    Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme, and mice bearing CPE mutations exhibit an obese and diabetic phenotype. Studies on CPE knockout (KO) mice revealed poor prohormone processing, resulting in deficiencies in peptide hormones/neuropeptides such as insulin, gonadotropin-releasing hormone, and cocaine- and amphetamine-regulated transcript (CART). Here, we show that CPE KO mice, an obese animal model, have low bone mineral density (BMD) accompanied by elevated plasma CTX-1 (carboxy-terminal collagen crosslinks), and osteocalcin, indicators of increased bone turnover. Receptor activator for NF-κB ligand (RANKL) expression was elevated ∼2-fold relative to osteoprotegerin in the femur of KO animals, suggesting increased osteoclastic activity in the KO mice. In the hypothalamus, mature CART, a peptide involved in eating behavior and implicated in bone metabolism, was undetectable. The melanocortin and neuropeptide Y (NPY) systems in the hypothalamus have also been implicated in bone remodeling, since MC4R KO and NPY KO mice have increased BMD. However, reduction of α-MSH, the primary ligand of MC4R by up to 94% and the lack of detectable NPY in the hypothalamus of CPE KO do not recapitulate the single-gene KO phenotypes. This study highlights the complex physiological interplay between peptides involved in energy metabolism and bone formation and furthermore suggests the possibility that patients, bearing CPE and CART mutations leading to inactive forms of these molecules, may be at a higher risk of developing osteoporosis. PMID:20460579

  3. Histamine responses of large neostriatal interneurons in histamine H1 and H2 receptor knock-out mice.

    PubMed

    Ogawa, Sachie; Yanai, Kazuhiko; Watanabe, Takeshi; Wang, Zhi-Ming; Akaike, Hironari; Ito, Yushi; Akaike, Norio

    2009-03-16

    Histamine (HA) is an important neuro-modulator, contributing to a variety of physiological responses in the mammalian central nervous system (CNS). However there is little information about the cell/signaling mechanism underlying its role. In the present study, we characterized HA responses in single large neostriatal neurons acutely dissociated from wild type (WT) and HA receptor knock-out (KO) mice, with a particular emphasis on identifying the role of HA receptor subtypes. HA (10 microM) and a selective H(2) receptor agonist dimaprit (1 microM) both evoked an inward current in H(1)-KO mice, and HA and a selective H(1) receptor agonist HTMT (10 microM) both evoked an inward current in H(2)-KO mice. In the H(1) and H(2) double (H(1/2)) KO mice, there was no response to either the application of HA or the selective H(1), H(2) receptor agonists. Hence we have confirmed that the targeted genes were indeed absent in these KO mice and that both receptor subtypes contribute to HA's excitatory actions. Furthermore the HA-induced inward currents were mediated by a decrease in current through K(+) channels. In addition, we observed the effects of methamphetamine (METH) on the locomotor activity of WT and HA receptor KO mice, and found that METH-induced behavioral sensitization is evident in H(1/2)-KO mice, but not in H(1)- or H(2)-KO mice. These observations suggest that suppressive roles of HA on methamphetamine-induced behavioral sensitization would be mediated through both H(1) and H(2) receptors in the CNS including neostriatum.

  4. Up-regulation of inducible nitric oxide synthase expression in cancer-prone p53 knockout mice.

    PubMed

    Ambs, S; Ogunfusika, M O; Merriam, W G; Bennett, W P; Billiar, T R; Harris, C C

    1998-07-21

    High concentrations of nitric oxide (NO) cause DNA damage and apoptosis in many cell types. Thus, regulation of NO synthase (NOS) activity is essential for minimizing effects of cytotoxic and genotoxic nitrogen oxide species. We have shown previously that NO-induced p53 protein accumulation down-regulates basal and cytokine-modulated inducible NOS (NOS2) expression in human cells in vitro. To further characterize the feedback loop between NOS2 and p53, we have investigated NO production, i.e., urinary nitrate plus nitrite excretion, and NOS2 expression in homozygous p53 knockout (KO) mice. We report here that untreated p53 KO mice excreted 70% more nitrite plus nitrate than mice with wild-type (wt) p53. NOS2 protein expression was constitutively detected in the spleen of untreated p53 KO mice, whereas it was undetectable in the spleen of wt p53 controls. Upon treatment with heat-inactivated Corynebacterium parvum, urinary nitrite plus nitrate excretion of p53 KO mice exceeded that of wt controls by approximately 200%. C. parvum treatment also induced p53 accumulation in the liver. Splenectomy reduced the NO output of C. parvum-treated p53 KO mice but not of wt p53 controls. Although NO production and NOS2 protein expression were increased similarly in KO and wt p53 mice 10 days after injection of C. parvum, NOS2 expression returned to baseline levels only in wt p53 controls while remaining up-regulated in p53 KO mice. These genetic and functional data indicate that p53 is an important transrepressor of NOS2 expression in vivo and attenuates excessive NO production in a regulatory negative feedback loop. PMID:9671763

  5. Hypothalamic orexin, OX1, alphaMSH, NPY and MCRs expression in dopaminergic D2R knockout mice.

    PubMed

    García-Tornadú, I; Díaz-Torga, G; Risso, G S; Silveyra, P; Cataldi, N; Ramirez, M C; Low, M J; Libertun, C; Becu-Villalobos, D

    2009-08-01

    In 5-month-old male and female dopamine receptor 2 (D2R) knockout mice food intake per animal was unaltered while food per g BW was increased. We wished to evaluate the effect of D2R disruption on different components of energy balance and food intake regulation. We determined hypothalamic orexin precursor (PPO) expression, its receptor OX1, serum leptin levels, hypothalamic leptin receptor (OBR), circulating and pituitary alpha MSH levels, as well as central MC3 and MC4 receptors and NPY mRNA in wildtype and D2R knockout mice (KO). Loss of D2R caused a marked increase in serum prolactin levels, to higher levels in females compared to male KO mice. On the other hand, it produced a female-specific increase in circulating alphaMSH, and hypothalamic alphaMSH content, while neurointermediate alphaMSH content was decreased in both sexes. No differences were found in hypothalamic NPY, MC3R or MC4R concentration. Hypothalamic PPO mRNA expression was significantly decreased only in female KOs, while OX1 mRNA was not different between genotypes. Serum leptin levels were also similar in both genotypes. Our results show that in female and not in male mice disruption of the D2R produces two potentially anorexigenic events: an increase in serum and hypothalamic alphaMSH, and a decrease in hypothalamic orexin expression. Very high prolactin levels, which are orexigenic, probably counterbalance these effects, so that food intake is slightly altered. In males, on the other hand, hypothalamic PPO, and serum or hypothalamic alphaMSH are not modified, and increased prolactin levels may account for increased food intake per g BW. These results suggest a sexually dimorphic participation of the D2R in food intake regulation.

  6. Animal model of autism using GSTM1 knockout mice and early post-natal sodium valproate treatment.

    PubMed

    Yochum, Carrie L; Bhattacharya, Prianka; Patti, Laryssa; Mirochnitchenko, Oleg; Wagner, George C

    2010-07-11

    Autism is a heterogeneous, behaviorally defined developmental disorder with unknown etiology but thought to be the result of environmental insult acting upon the developing brain of a genetically susceptible individual. Approximately 30% of individuals with autism have normal development up to the age of about 30 months after which they experience behavioral regression and lose previously acquired motor, cognitive and social skills. Early post-natal toxicant administration to mice has been used to model autistic regression. To test the hypothesis that genetically altered mice might be more sensitive to toxicant exposure early in life, mice with a deletion of glutathione-S-transferaseM1 (GSTM1; a gene associated with increased risk of autism that codes for an enzyme involved in the management of toxicant-induced oxidative stress) and wild-type controls were exposed to valproic acid (VPA; a toxicant known to cause autism-like behavioral deficits that, in part, are mediated through oxidative stress) on post-natal day 14. VPA treatment caused significant increases in apoptosis in granule cells of the hippocampus and cerebellum. There was a genotype by treatment by sex interaction with wild-type females exhibiting significantly fewer apoptotic cells in these regions compared to all other groups. VPA treatment also resulted in long-lasting deficits in social behaviors and significant alterations in brain chemistry. VPA-treated GSTM1 knockout animals performed significantly fewer crawl-under behaviors compared to saline-treated knockout animals as well as wild-type controls receiving either treatment. Collectively, these studies indicate that VPA-treatment causes cerebellar and hippocampal apoptosis and that having the wild-type GSTM1 genotype may confer protection against VPA-induced neuronal death in female mice.

  7. Toll-like receptor 4 knockout ameliorates neuroinflammation due to lung-brain interaction in mechanically ventilated mice.

    PubMed

    Chen, Ting; Chen, Chang; Zhang, Zongze; Zou, Yufeng; Peng, Mian; Wang, Yanlin

    2016-08-01

    Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system, and increasing evidence supports its role in inflammation, stress, and tissue injury, including injury to the lung and brain. We aimed to investigate the effects of TLR4 on neuroinflammation due to the lung-brain interaction in mechanically ventilated mice. Male wild-type (WT) C57BL/6 and TLR4 knockout (TLR4 KO) mice were divided into three groups: (1) control group (C): spontaneous breathing; (2) anesthesia group (A): spontaneous breathing under anesthesia; and (3) mechanical ventilation group (MV): 6h of MV under anesthesia. The behavioral responses of mice were tested with fear conditioning tests. The histological changes in the lung and brain were assessed using hematoxylin-eosin (HE) staining. The level of TLR4 mRNA in tissue was measured using reverse transcription-polymerase chain reaction (RT-PCR). The levels of inflammatory cytokines were measured with an enzyme-linked immunosorbent assay (ELISA). Microgliosis, astrocytosis, and the TLR4 immunoreactivity in the hippocampus were measured by double immunofluorescence. MV mice exhibited impaired cognition, and this impairment was less severe in TLR4 KO mice than in WT mice. In WT mice, MV increased TLR4 mRNA expression in the lung and brain. MV induced mild lung injury, which was prevented in TLR4 KO mice. MV mice exhibited increased levels of inflammatory cytokines, increased microglia and astrocyte activation. Microgliosis was alleviated in TLR4 KO mice. MV mice exhibited increased TLR4 immunoreactivity, which was expressed in microglia and astrocytes. These results demonstrate that TLR4 is involved in neuroinflammation due to the lung-brain interaction and that TLR4 KO ameliorates neuroinflammation due to lung-brain interaction after prolonged MV. In addition, Administration of a TLR4 antagonist (100μg/mice) to WT mice also significantly attenuated neuroinflammation of lung-brain interaction due to prolonged MV. TLR4 antagonism

  8. Dopamine dynamics associated with, and resulting from, schedule-induced alcohol self-administration: Analyses in dopamine transporter knockout mice

    PubMed Central

    Mittleman, Guy; Call, Stanford B.; Cockroft, Jody L.; Goldowitz, Dan; Matthews, Douglas B.; Blaha, Charles D.

    2011-01-01

    Preclinical and clinical evidence suggest an association between alcoholism and the primary regulator of extracellular dopamine concentrations, the dopamine transporter (DAT). However, the nature of this association is unclear. We determined if ten days of voluntary alcohol self-administration followed by withdrawal could directly alter DAT function, or if genetically-mediated changes in DAT function and/or availability could influence vulnerability to alcohol abuse. Heterozygous (DAT+/-) and homozygous mutant (DAT-/-) and wildtype (DAT+/+) mice were allowed to consume 5% alcohol in a schedule-induced polydipsia (SIP) task. In vivo fixed potential amperometry in anesthetized mice was used to (1) identify functional characteristics of mesoaccumbens dopamine neurons related to genotype, including dopamine autoreceptor (DAR) sensitivity, DAT efficiency, and DAT capacity, (2) determine if any of these characteristics correlated with alcohol drinking observed in DAT+/+ and DAT+/- animals, and (3) determine if SIP-alcohol self-administration altered DAR sensitivity, DAT efficiency, and DAT capacity by comparing these characteristics in wildtype (DAT+/+) mice that were SIP-alcohol naïve, with those that had undergone SIP-alcohol testing. DAT-/- mice consumed significantly less alcohol during testing and this behavioral difference was related to significant differences in DAR sensitivity, DAT efficiency, and DAT capacity. These functional characteristics were correlated to varying degrees with g/kg alcohol consumption in DAT+/+ and DAT+/- mice. DAR sensitivity was consistently reduced and DAT efficiency was enhanced in SIP-alcohol experienced DAT+/+ mice in comparison to naïve animals. These results indicate that DAR sensitivity is reduced by SIP-alcohol consumption and that DAT efficiency is modified by genotype as well as SIP-alcohol exposure. DAT capacity appeared to be strictly associated with SIP-alcohol consumption. PMID:21354763

  9. Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits.

    PubMed

    Paolone, Giovanna; Mallory, Caitlin S; Koshy Cherian, Ajeesh; Miller, Thomas R; Blakely, Randy D; Sarter, Martin

    2013-12-01

    Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/- mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/- mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/- mice. Additionally, CHT+/- mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/- mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/- cholinergic terminals. Further analyses of molecular and functional compensations in the CHT+/- model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders.

  10. Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits

    PubMed Central

    Cherian, Ajeesh Koshy; Miller, Thomas R.; Blakely, Randy D.; Sarter, Martin

    2013-01-01

    Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/− mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/− mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/− mice. Additionally, CHT+/− mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/− mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/− cholinergic terminals. Further analyses of molecular and functional compensations in the CHT +/− model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders. PMID:23958450

  11. Alterations of Amino Acids and Monoamine Metabolism in Male Fmr1 Knockout Mice: A Putative Animal Model of the Human Fragile X Mental Retardation Syndrome

    PubMed Central

    Gruss, Michael; Braun, Katharina

    2001-01-01

    The Fragile X syndrome, a common form of mental retardation in humans, is caused by silencing the fragile X mental retardation (FMR1) geneleading to the absence of the encoded fragile X mental retardation protein 1 (FMRP). We describe morphological and behavioral abnormalities for both affected humans and Fmr1 knockout mice, a putative animal model for the human Fragile X syndrome. The aim of the present study was to identify possible neurochemical abnormalities in Fmr1 knockout mice, with particular focus on neurotransmission. Significant region-specific differences: of basal neurotransmitter and metabolite levels were found between wildtype and Fmr1 knockout animals, predominantly in juveniles (post-natal days 28 to 31). Adults (postnatal days 209 to 221) showed only few abnormalities as compared with the wildtype. In juvenile knockout mice, aspartate and taurine were especially increased in cortical regions, striatum, hippocampus, cerebellum, and brainstem. In addition, juveniles showed an altered balance between excitatory and inhibitory amino acids in the caudal cortex, hippocampus, and brainstem. We detected very few differences in monoamine turnover in both age stages. The results presented here provide the first evidence that lack of FMRP expression in FMRP knockout mice is accompanied by age-dependent, region-specific alterations in neurotransmission. PMID:12018775